Myocyte membrane and microdomain modifications in diabetes: determinants of ischemic tolerance and cardioprotection
Russell et al. Cardiovasc Diabetol
Myocyte membrane and microdomain modifications in diabetes: determinants of ischemic tolerance and cardioprotection
Eugene F. Du Toit
Jason N. Peart
Hemal H. Patel
John P. Headrick 0
0 School of Medical Science, Griffith University , Southport, QLD 4217 , Australia
Cardiovascular disease, predominantly ischemic heart disease (IHD), is the leading cause of death in diabetes mellitus (DM). In addition to eliciting cardiomyopathy, DM induces a 'wicked triumvirate': (i) increasing the risk and incidence of IHD and myocardial ischemia; (ii) decreasing myocardial tolerance to ischemia-reperfusion (I-R) injury; and (iii) inhibiting or eliminating responses to cardioprotective stimuli. Changes in ischemic tolerance and cardioprotective signaling may contribute to substantially higher mortality and morbidity following ischemic insult in DM patients. Among the diverse mechanisms implicated in diabetic impairment of ischemic tolerance and cardioprotection, changes in sarcolemmal makeup may play an overarching role and are considered in detail in the current review. Observations predominantly in animal models reveal DM-dependent changes in membrane lipid composition (cholesterol and triglyceride accumulation, fatty acid saturation vs. reduced desaturation, phospholipid remodeling) that contribute to modulation of caveolar domains, gap junctions and T-tubules. These modifications influence sarcolemmal biophysical properties, receptor and phospholipid signaling, ion channel and transporter functions, contributing to contractile and electrophysiological dysfunction, cardiomyopathy, ischemic intolerance and suppression of protective signaling. A better understanding of these sarcolemmal abnormalities in types I and II DM (T1DM, T2DM) can inform approaches to limiting cardiomyopathy, associated IHD and their consequences. Key knowledge gaps include details of sarcolemmal changes in models of T2DM, temporal patterns of lipid, microdomain and T-tubule changes during disease development, and the precise impacts of these diverse sarcolemmal modifications. Importantly, exercise, dietary, pharmacological and gene approaches have potential for improving sarcolemmal makeup, and thus myocyte function and stress-resistance in this ubiquitous metabolic disorder.
Caveolae; Cardioprotection; Cholesterol; Diabetes; Fatty acids; Glucose transport; Infarction; Phospholipids
Diabetes impacts myocardial ischemic tolerance and cardioprotection
Clinical evidence indicates DM sensitizes human hearts
to I–R injury [
], which is generally consistent with
experimental findings in animal models, though
conflicting observations arise. Compounding the problem of
infarct intolerance, DM may also render hearts broadly
refractory to established cardioprotective stimuli that
include ischemic pre- and post-conditioning (direct
or remote) and protective G protein-coupled receptor
(GPCR) agonism, together with the anti-infarct effects
of ATP-gated K+ channel (KATP) openers, anesthetics,
phosphodiesterase-5 (PDE-5) inhibition and heat shock
]. Thus, while elusive cardioprotective
] are of particular value in the high-risk
DM population, implementation appears an even greater
challenge in this cohort. Prevalence of DM and
insulinresistance in those suffering IHD may in turn contribute
to poor translation of experimental cardioprotection in
these patients. Relatively few studies specifically address
the conundrum of I–R sensitivity and cardioprotective
insensitivity in DM [
]. Investigations to date implicate
a diversity of mechanisms extending beyond
fundamental alterations in glucose and lipid metabolism, including
associated glycation/glycosylation [
], oxidative stress
], abnormal survival kinase signaling [
exosome dysfunction , excessive
ubiquitin–proteasome system activity [
], suppression of sirtuin-1
], and changes in miRNA expression [
others. Considerable attention has focused on
mitochondrial dysfunction, including shifts in quality
control mechanisms (mitophagy, fission/fusion), as a point
of convergence in the complex pathogenesis of diabetic
10, 19, 20
]. However, the sarcolemma
is also a critical though under-appreciated nexus,
influencing DM progression and its impacts [
transcriptomic profiling indicates that the largest group
of diabetes-modified cardiac genes encode membrane/
plasma membrane components [
], consistent with
more recent studies identifying DM-dependent changes
in transcripts for membrane and structural proteins,
sarcolemmal receptors and ion channels [
for glucose and fatty acids, ion channels and
exchangers, and receptor systems governing insulin responses,
inflammation, mitochondrial quality control, and cell
stress, growth and death are all located within the
sarcolemma, while mitochondrial function is also sensitive
to sarcolemmal domains and proteins. Perturbations in
membrane composition and architecture may thus be
critical to the dysfunctional stress responses
characteristic of diabetic myocardium, together with other cardiac
outcomes including hypertrophy and contractile
dysfunction. We herein review the clinical and
experimental evidence of DM-dependent changes in myocardial
ischemic tolerance and cardioprotection, before focusing
specifically on sarcolemmal changes and their
contribution to the cardiac sequelae of DM.
Effects of DM in human myocardium
Diabetes induces a spectrum of abnormalities within the
myocardium and coronary vasculature. Diastolic
dysfunction, fibrosis and hypertrophy functionally and
structurally underpin diabetic cardiomyopathy [
changes are linked to reactive oxygen species (ROS)
generation, inflammation, mitochondrial dysfunction,
and abnormalities in molecular quality control,
including autophagy, fission/fusion, endoplasmic reticulum
(ER) stress and unfolded protein responses. Coronary
endothelial dysfunction and vascular remodeling
exaggerate atherosclerosis and impair vascular control and
coronary perfusion, potentially contributing to cardiac
dysfunction. These changes in myocardial and coronary
phenotypes (and underlying molecular mechanisms) may
participate in impairment of myocardial stress tolerance,
hormesis and protective signaling, which may in turn
further exacerbate these phenotypic changes.
Myocardial ischemic tolerance
The impacts of DM on myocardial ischemic tolerance
and infarction remain somewhat contentious. Certainly
DM worsens long-term outcomes from ischemic insult,
including increased incidence of heart failure and
allcause mortality [
]. There is some evidence these
poor outcomes may involve diabetic impairment of
myocardial reperfusion [
], consistent with
vascular dysfunction and reduced coronary reserve [
The contribution of worsened infarction to poor
postischemic prognosis awaits further clarification, with
some contrasting data acquired. Diabetes can
significantly increase infarct size as assessed via scintigraphy
] and magnetic resonance imaging (MRI) [
Insulin-treated DM patients also exhibit worsened
myocardial infarction, mortality, major adverse cardiac events
and thrombosis compared with untreated or non-DM
subjects, potentially reflecting negative impacts of more
complex and prolonged disease [
]. On the other
hand, some myocardial scintigraphic [
] and MRI
] report no significant differences in infarction
in DM vs. non-DM STEMI patients post angioplasty.
Other evidence strongly supports exaggerated
myocardial damage and cell death in DM patients: DM markedly
increases morbidity and mortality (up to 90%) following
cardioplegic arrest [
]; DM promotes pro-apoptotic
signaling, apoptosis and contractile dysfunction in
reperfused human myocardium [
]; and DM exaggerates
oxidative damage and anti-oxidant depletion [
transcriptional changes and pro-inflammatory signaling
]. Analysis of I–R injury in ex vivo tissue reveals
significantly impaired resistance of myocardium from T1
and T2DM patients, including increased apoptosis
(partially caspase- and PARP-dependent) and oncosis [
Anderson et al. [
] more recently provided evidence
that myocardium from DM patients has a greater
propensity for mitochondria-dependent cell death. There is
also evidence of exaggeration of post-ischemic
contractile dysfunction in DM: the studies of Hoogslag et al. [
and Dimitriu-Leen et al. [
] reveal worsened myocardial
longitudinal strain independently of infarct size,
supporting greater mechanical disruption in DM. Hyperglycemia
itself has been shown to increase infarct size and
mortality in infarct patients [
1, 28, 52–54
]. This may also involve
impaired reperfusion, though there is evidence
hyperglycemia exaggerates infarction by increasing the area at risk
]. Use of insulin and sulfonylureas to manage
hyperglycemia may additionally worsen ischemic injury,
morbidity and mortality [
34–36, 56, 57
Conversely, there is some limited evidence
myocyte ischemic tolerance might be increased in T1DM
patients, though for skeletal and not cardiac tissue [
This is consistent with some rodent studies in acute
T1DM models (see below). Nonetheless, the weight of
experimental evidence supports reduced myocardial
I–R tolerance in DM, encompassing exaggerated
apoptosis, oncosis and infarction, contractile dysfunction and
markers of oxidative damage. It remains unclear to what
extent poor post-ischemic prognosis reflects exaggerated
ischemic insult, impaired reperfusion, and increased
propensity to cell death. The roles of individual metabolic
disturbances (hyperglycemia, hyperinsulinemia,
insulin-resistance, dyslipidemia), coronary dysfunction and
compromised reflow, together with intrinsic myocardial
stress-resistance, thus require further detailed analysis.
Studies broadly support the desensitization or
elimination of diverse cardioprotective responses in DM
myocardium, though again this is not universal. There are
relatively few studies of diabetic impacts on
cardioprotective responses in human myocardium. Ishihara et al.
] reported that DM inhibits ischemic
preconditioning in anterior wall infarct patients, while Lee et al. [
present evidence of impaired preconditioning responses
in DM patients undergoing angioplasty. Galiñanes and
colleagues found that ex vivo myocardium from DM
patients was insensitive to ischemic preconditioning [
and subsequently identified loss of responsiveness not
only to preconditioning but to phenylephrine, adenosine
and diazoxide (implicating signal dysfunction proximal
to protein kinase C (PKC) and p38 mitogen-activated
protein kinase (MAPK) [
]. More recent studies support
desensitization of DM myocardium to hypoxic
preconditioning in association with impaired
phosphatidylinositol 3 kinase (PI3K) and Akt signaling [
], and failure
of ischemic preconditioning in myocardium from DM
]. On the other hand, some studies confirm
protective efficacies of anesthetic post-conditioning in
ex vivo myocardium [
] and of ischemic
preconditioning in vivo  in DM patients. Additionally, a
metaanalysis assessing influences of risk factor across ten trials
of post-conditioning in STEMI [
] verified significant
interactions with age and sex (reduced efficacy in older
and/or female patients) yet not with DM. The authors
concede analytical limitations may lead to an
under-estimation of the influences of co-morbidities such as DM. A
subsequent focused albeit smaller analysis also failed to
identify interaction between DM and post-conditioning
in STEMI patients [
], though also failed to detect the
sex and age effects revealed by Zhou et al., highlighting
limited power to detect effects in small sample sizes via
posteriori statistical analysis.
Complicating effects of anti‑hyperglycemia therapies
In addition to the underlying DM cardio-pathology, there
is evidence clinical approaches to managing
hyperglycemia may impair cardioprotective signaling and worsen
ischemic outcomes. Sulfonylurea use is associated with
greater ischemic injury and infarction in DM [
and inhibition of ischemic preconditioning in both
nonDM and DM patients [
] and ex vivo myocardium
from DM patients [
]. Glinide also impairs
preconditioning in DM patients [
]. These negative impacts
are consistent with their ability to inhibit KATP channels
implicated in transducing or mediating cardiac
In addition, insulin treatment has been linked to a
paradoxic worsening of complications, all-cause
mortality and cardiac outcomes in DM [
regarding potentially untoward effects of glycemic
control arose from epidemiological evidence of increased
mortality in insulin-treated vs. untreated T2DM patients
], together with observations of insulin effects
on cardiac events [
] and mortality in heart failure
complicated by T2DM . Evidence of worsened
outcomes with insulin and sulfonylureas over metformin
] suggests direct insulin- and KATP channel dependent
actions rather than simple glucose-lowering. However,
whether involving direct effects of insulin, influences of
acutely reduced glucose (or overt hypoglycemia)
following chronic hyperglycemia, or the fact insulin-treated
patients often exhibit greater comorbidities and
suffer more protracted disease, awaits further clarification.
There are potential mechanisms by which insulin might
worsen cardiovascular outcomes despite normalization
of glucose. For example, insulin can induce weight gain
which can exaggerate cardiovascular (and also cancer)
risks, while atherogenic and mitogenic effects may
accelerate atherosclerosis/IHD. Moreover, there is evidence
insulin treatment up-regulates pro-inflammatory tumor
necrosis factor α and interleukin-1 to a greater extent
in T1DM vs. healthy animals [
insulin-dependent NO generation may promote oxidative stress [
together with vascular damage through increased
circulatory pulsatility [
]. Hypoglycemia as a result of poor
glycemic control may also increase arrhythmogenesis,
cardiac events and mortality [
], though whether this
reflects a causal relationship is unclear, with other
studies reporting no association between hypoglycemia and
cardiac or all-cause mortality in T2DM [
there is evidence hyperglycemia can promote
compensatory mechanisms that protect against I–R injury,
including improvements in anti- vs. pro-oxidant balance
and protein integrity [
], which might be countered
by reductions in glucose levels. The hearts of diabetic
patients do appear desensitized to the injurious effects of
elevated glucose [
]. However, further work is needed in
disentangling these complexities.
Effects of DM in animal and in vitro models
There are some conflicting reports regarding impacts of
DM on myocardial infarction and cardioprotection in
animal models. Reviewed previously [
2, 84, 85
], studies in
different species and models report increases, no change,
or reductions in infarct size with DM. Similarly, despite
a substantial body of evidence supporting impaired
protection via pre- or post-conditioning and GPCR
agonists, some report preserved responses to similar stimuli
]. Reasons for these discrepancies are debated,
though disease progression and the presence of
dyslipidemia appear to be important. While infarct
enlargement is observed across species and models of T1DM
and T2DM , infarct reduction is predominantly
identified in rodent models of acute streptozotocin (STZ)
dependent hyperglycemia [
2, 86, 88–90
]. This may reflect
distinct impacts of acute (0–6 week) vs. established or
chronic disease. While some also report apparent
cardioprotection in models of T2DM [
], this may
similarly reflect distinct changes on early transition to T2DM
vs. established disease [
]. Presence or absence of
dyslipidemia may also be important, with some
evidence hypercholesterolemia has opposing effects on
infarct tolerance compared with hyperglycemia alone
. Mechanisms implicated in differing ischemic
tolerance in acute vs. chronic DM include shifts in PI3K/Akt
12, 94, 95
] and extracellular signal-regulated kinase 1/2
(ERK1/2) signaling [
], mitochondrial glucose oxidation
and malate-aspartate shuttle function [
capillary density, vascular endothelial growth factor (VEGF)
expression and endothelial nitric oxide synthase (eNOS)
]. Clinically, the negative impacts of chronic
disease are most relevant regarding infarction and
cardioprotection, with acute effects relevant only during
transition to disease and potentially on cessation of therapy.
Almost universally, observations support worsened
myocardial ischemic tolerance in models of chronic T1DM or
T2DM, with the weight of evidence supporting
associated failure in diverse cardioprotective responses.
T1DM and infarction
A range of studies report worsened infarction in
experimental models of T1DM [
] while some report no
effect on infarct tolerance [
6, 13, 101–112
protection against both infarction [
86, 88, 89, 113
contractile dysfunction [
]. However, as alluded to above,
a biphasic pattern may emerge in STZ-dependent rodent
models with evidence of early protection followed by
restoration or worsening of infarct tolerance beyond 6–8
weeks. Protection against infarction evident 1–4 weeks
after STZ challenge is lost from 8 weeks [
reduced ischemic tolerance may emerge by 20 weeks
] (in association with impaired ERK1/2
phosphoactivation). Ma et al. [
] report that protection against
infarction and caspase-3 activation in T1DM rats is
transient, apparent at 2 weeks and lost by 6 weeks, in
association with transient changes in capillary density, VEGF
expression, Akt phosphorylation, and eNOS expression.
Similarly, early protection against arrhythmogenesis at
2 weeks (with improved maintenance of Na+, Ca2+, K+
and Mg2+) transitions to worsened outcomes after 8
weeks in T1DM rats [
]. Acute hyperglycemia itself
has been shown to worsen myocardial infarction [
], exert no effect [
], or less
commonly to reduce infarction [
]. Reasons for these
disparate observations are unclear, and together with
the basis of apparently opposing effects of early vs. late
hyperglycemia in rat models of T1DM, warrant further
T1DM and cardioprotection
Beyond a transient intrinsic protection in the early stages
of STZ-induced hyperglycemia [
2, 86, 88–90
report inhibition or complete loss of cardioprotective
responses in rodent models of T1DM [
Protective ‘conditioning’ responses negated or inhibited
include ischemic pre- [
110, 116, 133
102, 104, 106, 108, 134, 135
], delayed protection
with ischemic preconditioning [
], and remote post-conditioning [
Protective responses to pharmacological stimuli including
anesthetic post-conditioning [
101, 104, 111
], opioid [
103, 107, 112
] and adenosine GPCR
], and adiponectin [
] and cytokine [
receptor activation are also lost in T1DM. Przyklenk and
] present evidence post-conditioning may
actually exaggerate injury in the context of T1DM. Acute
hyperglycemia also inhibits cardioprotective responses,
blocking ischemic pre- [
] and post-conditioning
, remote ischemic perconditioning [
] and post-conditioning [
with glucose-insulin-potassium (GIK) protection .
Nonetheless, there are some reports of preserved
protection in models of T1DM, including exercise [
and ischemic preconditioning [
], while Potier et al.
] identify a specific shift to protective efficacy of B2
bradykinin receptors in T2DM hearts (vs. B1 receptors
in non-DM tissue). Atorvastatin is also reportedly
cardioprotective in T1DM rats [
], involving a glycogen
synthase kinase 3β (GSK3β) dependent protection linked
to heat shock factor 1 and heat shock protein 70 (HSP70).
T2DM and infarction
Elements of T2DM individually modify infarct tolerance
and cardioprotective signaling, including dyslipidemia
], insulin-resistance and hyperglycemia [
]. Studies identify exaggerated infarction
and contractile dysfunction in different models of T2DM
]. Nonetheless, there are also reports of
unchanged infarct tolerance [
87, 126, 134, 147–150
reduced infarction in models of T2DM. The latter
reductions are observed 5 days post STZ injection in high-fat
fed rats (a protection negated by hypercholesterolemia)
], and at 16 weeks in Zucker diabetic fatty (ZDF) and
lean Goto-Kakizaki (GK) T2DM rats [
]. As for
T1DM, disease progression appears to be key, with
evidence of a transient protection during disease onset that
is lost with T2DM progression in GK rats , while
infarct-intolerance also emerges with chronic T2DM in
ZDF rats [
]. These latter studies link the evolution of
infarct tolerance with established T2DM to shifts in Akt
signaling, suppression of malate-aspartate shuttle
proteins and impaired post-ischemic recovery of glucose
T2DM and cardioprotection
Diverse cardioprotective responses are impaired or
negated in models of T2DM, including loss of ischemic
] and post-conditioning [
134, 142, 145
Heinduced pre- and post-conditioning [
], and protection
via anesthetic [
], erythropoietin [
, and β3-adrenergic receptor activation [
Interestingly, and consistent with membrane dysfunction,
T2DM abolishes the cardioprotective potential of human
and rat exosomes [
]. Exosomes are ~ 100 nm lipid
bilayer vesicle derivatives of endosomes that play a role in
transmitting protective signals between cells and tissues
]. Cardioprotection may involve exosomal
HSP70dependent activation of myocyte toll like receptor 4
and reperfusion injury salvage kinase (RISK) signaling
]. Failure of exosomes to induce protection in DM
appears to involve abnormal vesicle structure/function
rather than impaired protective signaling since exosomes
from healthy donors are protective [
]. While exosome
size was unaltered, contents of CD81 and HSP70 were
increased in DM. Given evidence of exosome
involvement in endogenous protection, this dysfunction may
contribute to impairment of both conditioning responses
and intrinsic ischemic tolerance.
Conversely, there are reports of preserved
cardioprotective responses in T2DM, including efficacy of far red/
near infrared light [
], sphingosine-1-phosphate [
peroxisome proliferator-activated receptor γ (PPARγ)
], post-ischemic glutamate [
] and H2S
]. Exercise may also retain efficacy,
improving ischemic tolerance in the hearts of T2DM
(GK) rats [
], and glycemic state and ischemic
tolerance in obese mice subjected to 20 weeks of high-fat
feeding . Those cardioprotective modalities
consistently preserved in different models of DM demand
further focused study as potentially efficacious therapeutic
While somewhat contentious, studies of human and
animal myocardium generally support detrimental effects
of both T1 and T2DM on myocardial ischemic tolerance
and cardioprotection (Table 1). Mechanistic
interrogation supports a complex pathogenesis, including
signaling dysfunction (e.g. impaired PI3K/Akt signaling) [
12–14, 60, 90, 94
], and abnormalities in mitochondrial
function and quality control [
10, 19, 20
ubiquitin–proteasome system activity [
], and gene and miRNA expression .
Influencing many of these potential effector mechanisms,
the sarcolemma plays an overarching role in governing
ischemic tolerance and cardioprotection. Cardiac
sarcolemmal changes arise in DM (see Table 2), reflecting
altered lipid metabolism and incorporation,
modification of resident lipids and proteins, and significant
structural and functional remodeling of caveolae [
] and gap junctions [
further below, such changes modify the fundamental
biophysical properties of the membrane, glucose and
fatty acid utilization, ion channel function, propensity
to membrane disruption, and signaling via the insulin
receptor (InsR) and receptors governing cardiac stress,
growth and death responses.
Sarcolemmal changes in DM
Though research has largely focused on intracellular and
metabolic determinants of cardiac stress responses in
DM, the sarcolemma plays a key role in governing these
and other changes and warrants further research
]. The sarcolemma represents the myocytes
structural bounds, and is the primary environmental and
inter-cellular interface; a scaffold for ion channel,
receptor, transport and mechano-transduction complexes,
and medium for detection of intra- and extra-cellular
stressors. It is thus intimately involved in receptor
signaling, ion homeostasis, substrate delivery, inflammatory
and immune function, and detection and transduction
of physico-chemical changes. As the site of glucose and
fatty acid uptake and InsR signaling, the sarcolemma and
its microdomains are a fundamental substrate for the
8 week ⇓ ⇓APN
12 week ⇓ ⇓β3-AR
Mouse—ob/ob 8–10 week old ⇓ ⇓IPreC
Mouse—db/db 10–12 week old ⇓ ⇓IPostC
12–14 week old ⇔ ⇓IPostC
Unreported ⇔ ⇔Infra-red light
12 week old Not tested ⇔H2S PreC
Rat—STZ/HFD 6 week ⇔ ⇔S1P
Rat—HFD 4 week ⇔ ⇔Erythropoietin
8 week ⇑ ⇓Sevoflurane
Rat—ZDF 12 week old ⇔ ⇔Glutamate
16 week old ⇑ ⇓IPreC
Rat—ZO 10–12 week old ⇓ ⇓IPreC, ⇓Diazoxide, ⇓HePreC
Rat—GK 12 week old ⇔ ⇓PPAR
Rat—OLETF 25–30 week old ⇓ ⇓Erythropoietin
Rat—mtFHH 12–14 week old ⇔ ⇓Isoflurane
HFD high fat diet, ZDF Zucker diabetic fatty, ZO Zucker obese, GK Goto-Kakizaki, OLETF Otsuka Long-Evans-Tokushima fatty, mtFHH T2D crossbreed with mtDNA
from fawn hooded hypertensive rats, IPreC ischaemic preconditioning, IPostC ischaemic postconditioning, HOPreC hyperoxic preconditioning, HePreC helium
preconditioning, LPreC ischaemic late preconditioning, RPreC remote preconditioning, S1P sphingosine-1-phosphate, APN adiponectin, β3-AR β3-adrenergic receptor,
Changes (up or down) in levels of myocardial or sarcolemmal lipids in models of T1DM are summarized. Sarcolemmal lipid changes are not well defined in models
of T2DM. Changes in specific saturated and unsaturated fatty acids species are indicated, with shortened numerical descriptions reflecting numbers of carbons and
double bonds (e.g. palmitic acid, 16:0; stearic acid, 18:0; linoleic acid, 18:2; docosahexaenoic acid, 22:6)
CGP choline glycerophospholipids, EGP ethanolamine glycerophospholipids, IGP inositol glycerophospholipids, Chol cholesterol, CL cardiolipin, FAs fatty acids,
FFA free fatty acid, LPC lysophosphatidylcholine, LPL lysophospholipid, NEFA non-esterified fatty acid, PC phosphatidylcholine, PE phosphatidylethanolamine,
PI phosphatidylinositol, PL phospholipid, PMC plasmenylcholine, PME plasmenylethanolamine, PS phosphatidylserine, SGP serine glycerophospholipids, SM
sphingomyelin, TRI triglyceride
metabolic dysregulation in DM. Molecular modification
and disruption of the sarcolemma can thus contribute to
multiple aspects of myocardial dysfunction and
pathology in DM.
Structurally the sarcolemma is a dynamic fluid bilayer
of phospholipids, comprising complex assemblies of
proteins, cholesterol and other lipids (Fig. 1a). Within this
lipid sea float organized clusters of sphingolipids and
cholesterol that form distinct microdomains known as
lipid rafts. An important sub-set of these rafts, the
caveolae are small invaginations (50–100 nm in diameter) that
appear particularly relevant in DM and its cardiac
158, 159, 163
]. Among other functions these ‘little
caves’ serve as structural and regulatory platforms for
receptor, ion channel and transporter proteins [
participate in mechanotransduction, protection against
disruption and regulation of membrane repair [
and govern cardioprotective signaling [
rafts can also serve as redox signaling platforms that
recruit and assemble nicotinamide adenine dinucleotide
(NADPH) oxidase subunits and related proteins [
]. The functional properties of the sarcolemma and
its microdomains are governed by molecular
composition, which is sensitive to diet, physical activity, genetic
makeup and disease, and is significantly disturbed in DM
(Fig. 1b, Table 2).
Changes in sarcolemmal lipid profiles and function in DM
The biophysical properties of the membrane determine
resident protein conformation, mobility and function.
Fluidity or viscosity governs molecular motion and
interactions within this lipid bilayer, thereby influencing the
functionality of receptors, transporters and ion channels
]. Fluidity is determined by lipid makeup, including:
the tightness of acyl-chain assembly on phospholipid
molecules; degree of phospholipid saturation; and local
ratios of cholesterol, lipids and proteins. Membranes
rich in cholesterol and tightly packed acyl chains possess
greater rigidity, impacting movement and interaction of
receptors and other molecules. Changes in sarcolemmal
(See figure on previous page.)
Fig. 1 a Sarcolemmal makeup and caveolar domains. Planar lipid rafts are more ordered elements of the sarcolemma, containing greater
sphingolipid and cholesterol levels and forming signaling microdomain platforms. A subset of rafts, caveolae, localize signaling integral to ischemic
tolerance and cardioprotection, including NOS, GPCRs, RTKs and coupled effector molecules. Caveolins are critical to caveolae formation and function
and protective signaling. b Modulation of caveolae/caveolins and related cardioprotective signaling in DM. Diabetes may exaggerate mitochondrial
dysfunction and associated death, while individual elements of DM may disrupt caveolar control and caveolin expression: (i)
hyperglycemiadependent PKCβ2 activation may suppress caveolin-3 expression/localization; (ii) saturated fats (e.g. palmitate) may displace or depress caveolin-3.
Disruption of caveolar control and caveolins will limit protective signaling to mitochondria, including caveolin-3 translocation/modulation. Potential
determinants of caveolin-3 expression and caveolar function include PKCβ2, saturated fats vs. n-3 PUFAs, AC (adenylate cyclase) and FAK (focal
adhesion kinase) signaling, myocardin activity and physical activity
biophysics as a result of altered lipid metabolism do
appear causally important in DM [
]: InsR signaling is
inhibited by reductions in membrane fluidity, and both
glucose transporter type 4 (GLUT-4) transport to the
] and glucose uptake [
parallel membrane fluidity changes. Analysis of hepatic cells
supports causal involvement of
sphingomyelin-dependent lipid microdomain changes in insulin-resistance and
T2DM . Inflammation, important in DM and
cardiovascular dysfunction, is also promoted by
abnormalities in membrane phospholipid and polyunsaturated fatty
acid (PUFA) composition [
]. Positive feedbacks
arise, whereby effects of DM and dyslipidemia on
sarcolemmal signaling evoke further lipid accumulation and
membrane dysfunction. For example, up-regulation of G
protein-coupled receptor kinase 2 (GRK-2) with DM or
high-fat feeding inhibits GPCR and InsR signaling,
promoting further lipid accumulation, insulin-resistance and
], with recent evidence these changes
in GRK-2 also inhibits cardioprotective opioid receptor
]. The fundamental biophysical properties
of the membrane can thus strongly influence the
development and pathological impacts of DM across cell types.
Initial studies in DM identified reduced erythrocytes
membrane fluidity [
], a change evident even in
newly formed cells  and subsequently confirmed
via different approaches in multiple cell types [
], including cardiomyocytes [
]. Reduced fluidity
is broadly consistent with increased membrane content
of lipids promoting rigidity, including cholesterol,
sphingomyelin and saturated fatty acids [
] (Table 2). It
is nonetheless interesting to note that erythrocytes
normally possess relatively little cholesterol and lack
caveolins. Specific myocardial analyses support increased
levels of cholesterol and fatty acid saturation vs.
desaturation and differential changes in phospholipids and
PUFAs, though studies are limited to models of T1DM/
In early work, Denton and Randle [
] found a
twofold increase in myocardial glycerides (predominantly
triglycerides) in alloxan-induced T1DM in rats
without significant changes in phospholipid content, though
sarcolemmal fractions were not specifically examined.
Increased cardiac triglyceride were confirmed in T1DM
rat hearts, together with elevations in free fatty acid levels
]. Early study of phospholipid content revealed
reductions in sarcolemmal cardiolipin and
phosphatidylethanolamine (PE) vs. elevated lysophosphatidylcholine
levels in T1DM hearts, while phosphatidylinositol (PI)
and phosphatidylserine (PS) were unchanged . Later
analysis of phospholipid makeup in hearts from T1DM
rats revealed phospholipid depletion of n-6 arachidonic
acid (AA; C20:4), docosatetraenoic acid (C22:4) and
docosapentaenoic acid (C22:5) species, whereas contents of
n-6 linoleic (C18:2) and dihomo-γ-linolenic acids (C20:3)
and n-3 eicosapentaenoic acid (C20:5) were increased
]. Subsequent studies confirm phospholipid
depletion of AA and also palmitic acid (16:0) vs. enrichment
with linoleic and dihomo-γ-linolenic acids [
] (Table 2), though Black et al.  found no change
in phospholipid AA content in T1DM rat hearts (while
phospholipid stearic acid and palmitate levels fell). Han
et al. [
] reported three major sarcolemmal changes in
T1DM rats: a reduced ratio of saturated:unsaturated PE
species; increased PI and plasmenylethanolamine; and
remodeling of triacylglycerol species. More recent
analysis in alloxan-induced T1DM in rats confirms increased
cardiac cholesterol, free fatty acids, triglycerides and lipid
saturation, reduced de-saturation and n-3 and n-6 PUFA
levels, and differential changes in phospholipids
including increased PE, sphingomyelin and lysophospholipid
vs. reduced phosphatidylcholine (PC) and PI + PS [
]. Collectively, these studies support cholesterol,
triglyceride and free fatty acid accumulation, increased
saturation vs. desaturation, and remodeling of the major
choline and ethanolamine phospholipids, with loss of AA
and accumulation of linoleic acid and dihomo-γ-linolenic
species (Table 2).
Shifts in cholesterol and fatty acid saturation are
important to changes in membrane biophysics. Membrane
fluidity is particularly dependent upon cholesterol content,
which is consistently increased in models of T1DM [
], in association with reduced fluidity and Ca2+ influx
]. Indeed, membrane cholesterol changes are
likely to contribute in multiple cardiovascular disorders
]. Cholesterol molecules provide structural support,
function as molecular ‘glue’ for lipid raft assembly,
promote curvature of the membrane [
], and are
important to caveolae formation [
]. Control of cholesterol
is therefore essential to maintenance of membrane
architecture, fluidity and microdomain formation. Shifts in the
degree of fatty acid saturation also influence fluidity, with
DM consistently increasing cardiac fatty acid levels and
saturation vs. desaturation [
]. Changes in
phospholipid profiles additionally alter fundamental
biophysical properties , together with protein activity [
], recruitment of signal proteins , propensity for
] and production of lipid second messengers
]. Sarcolemmal phospholipids may also undergo
relevant post-translation modification in DM, for
example altered N-methylation  may alter cardiac
sarcolemmal Ca2+ fluxes [
Polyunsaturated fatty acids exert complex effects on
membrane structure and function [
]. For example,
n-3 PUFAs such as eicosapentaenoic (EPA) and
docosahexaenoic acid (DHA) remodel cholesterol-enriched
lipid microdomains, with evidence their incorporation
increases molecular order (despite their high disorder).
Differences in lipid microdomain interactions of EPA
and DHA may lead to differential changes in
]. Both in vitro [
] and in vivo studies
] indicate n-3 PUFAs incorporate into
membrane fractions corresponding to rafts, generally within
the most abundant and DM-sensitive phospholipids (PE
and PC), and magnetic resonance studies show
differential changes in membrane structure when n-3 PUFAs
are incorporated into PE vs. PC, the phospholipid
fractions predominantly modified in DM [
193, 195, 198,
]. Lipid raft incorporation of n-3 PUFAs is
accompanied by reduced levels of the highly disordered n-6
PUFA AA , consistent with declining sarcolemmal
AA species in DM [
193, 198, 199
]. Several studies show
n-3 PUFAs can lower raft cholesterol levels, which may
underlie effects on protein lateral organization and
]. Impacts on cholesterol may include
shunting from raft to detergent-soluble membrane fractions
in some cell types [
], potentially reflecting the
poor affinity of n-3 PUFAs for cholesterol .
However, in some cell types reductions in raft cholesterol are
not matched by changes in the detergent-soluble
], which may be related to the ability of n-3
PUFA to promote internalization of lipid microdomains
(including raft cholesterol) [
]. An area of interest
has been the influence of PUFAs on ion channel
function, electrical stability and arrhythmogenesis, though
mechanisms underlying such effects are yet to be fully
Membrane dynamics are also influenced by glycation
and associated free radical production [
Treatment with the anti-glycation and anti-oxidant
compound resorcylidene aminoguanidine (RAG) reverses
DM-dependent reductions in cell membrane fluidity
. Profoundly reduced sensitivity to Ca2+ overload in
myocardium from DM rats is also inhibited [
additionally highlighting the importance of fluidity to ion
homeostasis in DM hearts. Roles of such
post-translational changes are discussed in more detail further below.
Unfortunately, studies of sarcolemmal changes in DM
have focused to date on models of T1DM, with little to
no information regarding changes in T2DM (Table 2).
Moreover, investigations have yet to detail temporal
patterns of sarcolemmal change during DM development and
progression. It would be of great value to undertake such
time-course analyses across what appears the critical range
for variable shifts in ischemic tolerance (i.e. no effects or
improved tolerance from weeks 1–6; reduced tolerance at
later times), permitting correlation of membrane makeup
and ischemic tolerance changes. Membrane lipid analyses
have been undertaken within yet not across these differing
time periods (Table 2), with broadly similar lipid changes
reported at 2–6 weeks [
195, 201, 202
] and 8–9 weeks [
]. No distinguishing feature is evident in later
membrane profiles, though there is only a single study
at ≥ 12 weeks [
]. There is also no information on the
time-course of changes in caveolae and caveolin proteins,
another knowledge gap deserving attention.
Remodeling of sarcolemmal microdomains
Membrane proteins and signaling are compartmentalized
between specialized microdomains rich in cholesterol
and sphingolipids vs. other membrane regions [
Though a simplified model given the true complexity of
the plasma membrane [
], sarcolemmal assemblies of
lipids and proteins may be divided into either lipid rafts
or planar platforms (loosely corresponding to detergent
insoluble membrane fractions) and non-raft domains
(corresponding to detergent soluble membrane). These
domains differ markedly in their ion channel, transporter,
receptor and signaling protein profiles [
membrane composition can thus disrupt signaling, ion
movement and substrate transport through differential
changes in lipid raft/caveolar vs. non-raft domains.
However, the compartmentation of proteins between raft and
non-raft regions remains a controversial topic, reflecting
in part varying outcomes with different membrane
Lipid raft vs. non‑raft proteins
These distinct regions may be differentially modified
in DM, although studies of the cardiac sarcolemma are
limited. For example T2DM db/db mice exhibit ~
tenfold elevations in the area of raft clusters in aortic
endothelium, confirming that increased cellular lipid
content can drive raft cluster formation [
horizontal and vertical clustering of rafts is observed,
increasing the height of these aggregates [
changes will modify resident protein integration and
function. Sequestration of specific ion channel,
receptor and transporter proteins within raft microdomains
is an important means of compartmentalizing and
specifying downstream signal transduction by the
sarcolemma. For example, caveolar localization ensures
signaling specificity of cardiomyocyte β-adrenoceptors,
limiting non-selective effects on sarcoplasmic reticulum
(SR) and myofilament function [
]. A sub-population
of L-type Ca2+ channels (LTCCs) has also been
identified in caveolae domains that appears critical in
regulating β-adrenoceptor [
] and hypertrophic calcineurin/
nuclear factor of activated T-cells (NFAT) signaling
]. Evidence also supports functional localization of
LTCCs to caveolae in human and rodent atrial myocytes,
with a caveolae-targeted LTCC antagonist inhibiting
Ca2+ fluxes [
]. In contrast, a recent report concludes
that neither a caveolae targeted LTCC activator or
inhibitor modifies function or hypertrophic responses in
murine hearts [
Regulation of other important channels and pumps
may be dependent upon caveolae domains. For
example, despite evidence cardiac Na+-K+-ATPase
localizes to non-raft regions [
], there is also evidence
for caveolae/caveolin association and control [
Almost half of the Na+-K+-ATPase α1-subunit and
nearly all of the glycosylated β1-subunit reportedly
localizes to cardiac caveolae [
], and caveolin-1
deletion inhibits interactions between Na+-K+-ATPase,
caveolin-3 and PI3K in cardiomyocytes [
inhibition of sarcolemmal Na+-K+-ATPase sub-unit
expression and activity in STZ-dependent T1DM
] may reflect a caveolae specific response.
Such an effect is consistent with predicted outcomes
of cholesterol accumulation [
]. Indeed, high dietary
cholesterol also reduces Na+-K+-ATPase [
cholesterol may render sarcolemmal penetration of
the ATPase complex energetically unfavorable, while
reducing surface charge density is chemically
]. Similarly, the cardiac Na+/Ca2+ exchanger is
suppressed in DM [
], and despite some evidence it
does not localize to rafts or caveolae [
], there is also
evidence the exchanger interacts with caveolin-3 in
sarcolemmal vesicles [
], and its activity is depressed
with cholesterol depletion [
]. Further work may
clarify the impacts of DM on cardiac raft and non-raft
ion channels and pumps.
Caveolar membrane microdomains
Evidence accumulated over the past decade highlights
a particular importance of caveolae in protection of
myocardium against metabolic (ischemia, hypoxia) and
mechanical stressors [
], together with
perturbations and potential involvement in cardiac disease [
]. Few studies have examined effects of DM on
caveolar structure and density, although constituent caveolin
proteins are significantly modified [
caveolar localization of signaling molecules altered [
]. Evidence implicates abnormal caveolar control
in the development and cardiac-specific effects of DM
Caveolae have at least four major functions: (i) as
signaling platforms in the membrane, for example for
receptor tyrosine kinases (RTKs) including the InsR [
], eNOS [
], other signaling proteins [
and ion channels [
]; (ii) regulating fatty acid
] and glucose handling ; (iii)
participating in mechanotransduction and acting as membrane
‘reservoirs’ to limit damage with mechanical stress [
]; and (iv) functioning as membrane transport
vesicles, budding from the membrane in response to
specific cues and participating in membrane repair .
Abnormalities within these regulatory domains will thus
influence ion and substrate movement, protective
signaling and myocyte responses to mechanical perturbation,
impairing cardiac responses to both pathologic insult and
As for lipid rafts in general, caveolae formation and
function are dependent upon lipid composition,
particularly cholesterol and sphingolipid content [
228, 261, 262
A key distinguishing feature is the presence of
cholesterol-associated caveolin proteins, involved in stabilizing
the physical architecture of these flasks and regulating
signaling and transport processes [
]. Depletion of
membrane cholesterol [
] or caveolins [
caveolae formation and negates myocardial responses to
diverse protective stimuli [
depletion also disrupts the Z-band localization of caveolin-3
in cardiomyocytes, and alters cytoskeletal architecture
. Highly abundant PS and phosphatidylinositol
(4,5)-bisphosphate [PIP2] may also be important,
concentrating within caveolae and functionally
compartmentalizing lipid pools [
]. Caveolae depletion
with caveolin-1 knockout or depletion leads to
re-organization of plasma membrane PS domains [
consistently down-regulates pathways of lipid metabolism
across cells [
]. There is evidence the caveolin
scaffolding domain—a 20-amino acid sequence initially
implicated in controlling signal molecules—has an intrinsic
capacity to concentrate local cholesterol, PS and PIP2
]. Effectively enriching caveolar oligomers in PS and
PIP2, this process is proposed as a means of attracting
membrane-sensing cavin proteins to initiate a cascade of
further caveolin, PIP2 and PS recruitment to membrane
]. Cholesterol concentration around caveolin
oligomers may modify biophysical properties to favor
membrane bending by the cavin-caveolin coat complex.
These inter-dependencies provide a basis for the
sensitivity of caveolae formation and function to cholesterol and
phospholipids, the levels of which are perturbed in DM
195, 196, 199–202
]. Given evidence of cholesterol
accumulation in the sarcolemma of DM hearts, a scenario of
both caveolar disruption via caveolin suppression and
reduced membrane fluidity via cholesterol accumulation
could arise. How accumulated cholesterol is distributed
between microdomains in DM is not clear, however it is
possible fluidity within depleted caveolae populations is
Caveolar “coat proteins”—the caveolins and cavin families
While there are few analyses of cardiac caveolar
architecture and density in DM, significant changes in
constituent caveolins and cavins are observed and likely disrupt
caveolae function and formation. Hyperglycemia may
suppress myocardial caveolin-3 in a PKCβ2 dependent
] and H9c2 cardiomyoblast caveolin-3 in
an oxidant-dependent manner [
hyperinsulinemia also depresses caveolar caveolin-3 in H9c2 cells
]. Moreover, saturated fats reduce cardiac caveolin-3
], as does aging [
], whereas PUFA
supplementation can up-regulate caveolin-3 expression [
Caveolin-1, in contrast, may be significantly up-regulated in
The caveolin proteins are primary structural and
regulatory elements of caveolae [
], though also play
important non-caveolar roles [
]. For example,
sequestration of active caspase-3 by extra-caveolar caveolins
may underlie protective effects of β-receptor antagonism
in DM hearts [
]. Three caveolin isoforms have been
identified with differing functions and tissue
158, 164, 206, 264
]. All are expressed in the central
nervous system, with ubiquitous caveolin-1 most highly
expressed in endothelium, fibroblasts and
pneumocytes, where it appears structurally supported by
caveolin-2 hetero-oligomerization. In contrast, caveolin-3 is
highly specific to striated muscle and plays crucial roles
in cardiac stress sensing/responses and cardioprotection
167–170, 256, 259, 264
]. Caveolins preferentially arrange
in homo-oligomers of 2 to ~ 16 monomers, forming
caveolar assembly units, and may require cholesterol
for effective insertion into the membrane . A
common feature of all isoforms are scaffold domains where
signal molecules including G proteins, PKC and eNOS
are proposed to physically interact. However, the basis of
regulatory molecular interactions with caveolins remains
to be defined [
]. Noted above, these domains also
appear important in locally concentrating cholesterol
and phospholipids [
]. Caveolin-3 is not only
essential to myocardial caveolae formation but is particularly
important to stress tolerance and cardioprotection.
] and mitochondrial [
] stress responses
are strongly caveolin-3 dependent, as is cardiac
protection via ischemic and anesthetic preconditioning [
] and opioid GPCRs . Caveolin-3 also influences
cholesterol transport [
], ion handling [
235, 283, 284
GLUT4 and glucose metabolism [
252, 285, 286
hypertrophic remodeling [
Despite these key roles, the control of myocardial
caveolin expression remains to be detailed, though studies in
other cells support transcriptional regulation by
myocardin. A member of a family of transcriptional co-activators
responsive to stress, myocardin up-regulates caveolins and
caveolae in smooth muscle cells [
]. Specific cardiac
studies are lacking, however human expression data
support a close association between Myocd and Cav1 gene
levels across tissues, including heart [
control of caveolin-1 and -2 and cavin-2 appears
independent of serum response factor whereas control of cavin-1 is
dependent on this transcription factor, providing for
differential control of cavin-1 vs. caveolins [
myocardin may be up-regulated by hyperinsulinemia [
and in other muscle cell types myocardin is up-regulated
by oxidative stress sensitive miR-145 [
emerging evidence reveals new roles for myocardins in
glucose and lipid homeostasis (including via caveolins) [
The few studies analyzing myocardial caveolins
in T1DM have employed relatively acute models
(0–6 weeks), and report a hyperglycemic depression of
] that may contribute to
diastolic dysfunction [
], impaired GLUT4
] and I–R intolerance [
]. Nonetheless, the
acuteness of STZ-induced hyperglycemia and variable
ischemic tolerance in these T1DM models raise
questions regarding relevance: paradoxical cardioprotection
in the initial weeks in rat T1DM models [
2, 84, 293
] is not
relevant to the ischemic intolerance observed in chronic
disease and T2DM. Hyperglycemia also acutely depresses
caveolin-3 expression in cardiac myoblasts [
hyperinsulinemia suppresses caveolar levels of caveolin-3
in H9c2 myoblasts, which may dysregulate
Akt-dependent InsR signaling [
]. No study has comprehensively
assessed mechanistic involvement of caveolin-3 in the
cardiac sequelae of T2DM, with only a single report of an
insignificant fall in cardiac Cav3 mRNA in the non-obese
GK rat model [
Inhibitory effects of saturated fatty acids [
and glucose  on cardiac caveolin-3 expression and
caveolin-dependent eNOS signaling present
plausible mechanisms for reduced cardioprotection in DM.
Impaired PI3K/Akt/NOS signaling is characteristic in
DM myocardium [
12, 90, 94, 95, 146, 250
], and these
signal elements cluster in caveolae [
] where eNOS
is regulated by caveolin-1 and -3 [
250, 275, 296–298
Akt signaling is promoted by caveolin-3 [
Studies in rodent models indicate that DM
dysregulation of RISK signaling, including PI3K/Akt and
glycogen synthase kinase-3β, underlies impaired protection
via cytokine receptors [
], GPCRs [
] and progestin
and adiponectin receptors [
]; and RISK-dependent
pre- and post-conditioning responses are also inhibited
in DM [
2, 116, 117, 130
]. Inhibition of Akt signaling and
ischemic tolerance in T1DM has been linked to
caveolin-3 depletion , as has disruption of adiponectin
receptor cardioprotection [
]. Recent work also
implicates oxidant-mediated dysregulation of caveolin-3/
eNOS signaling in the ischemic intolerance in T1DM
]. An increase in caveolin-1, reported by
Penumathsa et al. [
] in hearts of T1DM rats and Bucci
et al. [
] in aortic tissue, may also inhibit protective
signaling, suppressing eNOS activity [
promoting dephosphorylation of sarcolemma-associated
Akt . In support of this, Ajmani et al. [
that a ‘caveolin inhibitor’ and sodium nitrite both restore
preconditioning in T1DM rat hearts, however
significant limitations include multiple non-specific biological
actions of the inhibitor employed, and failure to measure
caveolin-1 expression or establish diabetic inhibition of
Less is known regarding potential roles of more
recently identified cavin proteins [
]. These coat
proteins homo- and heteroligomerize (independently of
membrane and caveolins) to form specific caveolar
subcomplexes, and are involved in orchestrating the
cellspecific formation, caveolin/cavin incorporation and
structural modeling of caveolae [
]. They may also
be released intracellularly with different stressors/stimuli
to regulate gene expression and non-caveolar processes.
Depletion of cavin-1 (with attendant loss of caveolae)
results in elevations in circulating triglycerides, glucose
intolerance and hyperinsulinemia [
], and inhibits
cardiac ischemic tolerance and stretch responses while
exaggerating cellular permeability (potentially via NOS
]. Perturbation of the caveolar system
via caveolin-1 depletion or knockout also dysregulates
cardiac stress responses [
]. Whether these gene
deletion effects reflect distinct roles and influences of
cavins and caveolins, or highlight the broader importance
of caveolae is presently unclear. However, differences
do emerge in the cardiac effects of cavin-1 vs.
caveolin knockout . Intriguingly, effects of cavin-1 and
caveolin-1 knockout suggest the diastolic dysfunction
in DM could involve disruption of sarcolemmal
caveolae: caveolar depletion in both cavin-1 [
] knockout hearts is associated with significant
diastolic dysfunction or stiffening. Caveolae provide an
effective membrane reserve to accommodate physical
deformation or stretch [
], potentially influencing the
compliance of cardiac cells. Although diabetic diastolic
dysfunction is attributed to fibrosis/hypertrophy [
sarcolemmal makeup and specifically caveolae and
associated signaling may contribute to this dysfunction [
Changes in sarcolemmal caveolae influence substrate
Caveolar domains are important in glucose and lipid
transport, and InsR receptor signaling [
Myocardial glucose transport via GLUT4 is spatially confined
to caveolar domains [
], where InsRs are also
]. Cardiac insulin-resistance and impaired
GLUT4 expression and transport in T2DM [
involve disruption of caveolae and caveolin-3 with DM
] and high-fat feeding [
Penumathsa et al.  report reduced expression and
association of GLUT4 and caveolin-3 in lipid-rafts of
T1DM rat hearts.
Activation of the InsR normally leads to a cascade
of Akt phospho-activation and phosphorylation of the
Rab-GTPase activating TBC1D4/AS160 protein, a distal
effector maintaining GLUT4 vesicles within an inactive
intracellular pool [
]. This initiates pathways mediating
docking and diffusion of GLUT4 vesicles at the plasma
]. This path not only increases GLUT4
exocytosis but can limit endocytosis to re-distribute
plasma membrane GLUT4. However, in cardiac [
and skeletal myocytes [
] insulin does not influence
endocytosis. Nonetheless, GLUT4 endocytosis in
skeletal myoblasts is sensitive to energy state (inhibited by
mitochondrial uncoupling), and both
clathrin-dependent and clathrin/caveolae-independent (yet
cholesteroldependent) endocytosis paths are involved [
energy-sensitive endocytosis reveals non-caveolae effects
of cholesterol, for example promoting negative
membrane curvature [
]. This not only further highlights
the importance of membrane cholesterol, but shows that
distinct membrane changes may independently modify
GLUT4 exocytosis and GLUT4 endocytosis.
Not only is GLUT4 movement influenced by
caveolae and caveolins, but signaling via the InsR is strongly
dependent upon these raft elements. Yamamoto et al.
] first demonstrated positive control of InsR
signaling via caveolin-1 and -3, including evidence of direct
caveolin interaction with the InsR kinase domain to
promote insulin receptor substrate 1 phosphorylation.
Caveolin-3, caveolin-1 and the InsR all interact in cardiac
myoblasts, and caveolin-3 depletion renders myocytes
insulin-resistant while caveolin-3 haplo-insufficiency
increases susceptibility to fatty acid induced
]. Disruption of caveolae or caveolin-3
expression in DM [
] is thus predicted to
limit cardiac InsR signaling, although a parallel elevation
in caveolin-1 as reported in a rat T1DM model [
modulate such effects. Supporting the value of targeting
caveolins, insulin-resistance in obese and DM mice is
reversed by hepatic overexpression of caveolin-3, which
substantially enhances InsR signaling [
Nonetheless, basal glucose metabolism appears largely unaltered
in hearts lacking either caveolin-3 [
] or caveolin-1
], and thus also devoid of caveolae, although
skeletal muscle insulin-resistance arises in both models [
]. While suggesting distinct caveolin/caveolar control
of substrate metabolism in cardiac vs. skeletal muscle,
cardiac InsR signaling and insulin-resistance have yet to
be detailed in these knockout models. Lifelong absence
of both caveolae and caveolins in these models may also
limit their relevance to more moderate and progressive
changes in DM. Other analyses confirm that reductions
in caveolin-3 inhibit insulin-stimulated glucose uptake in
cardiac myoblasts and myocytes , and that
hyperinsulinemia in cardiac myoblasts reduces caveolar
levels of caveolin-3 and insulin-dependent phospho-Akt
]. Insulin-dependent myocardial glucose uptake is
thus predicted to be impaired with reductions in
caveolin-3 expression in DM hearts, though this has yet to be
Ubiquitously expressed caveolin-1 may additionally
modulate InsR signaling in DM, and cardiac
expression is reportedly increased in T1DM rat hearts [
Caveolin-1 is also induced by micro-RNAs
up-regulated in obesity (miR103, miR107), and their
overexpression induces insulin-resistance in an entirely
caveolin-1 dependent manner [
]. However, changes
in caveolin-1 are not universal in obesity, some dietary
interventions may also augment caveolin-1 [
distinct from caveolin-3, cardiac expression of
caveolin-1 appears repressed with medium-chain
triglyceride but not palmitate supplementation [
work is needed to clarify effects of caveolin-3 and -1
on insulin-dependent glucose uptake and metabolism
in myocardium and cardiac myocytes, identifying
specific roles of the caveolins themselves vs. caveolae as
regulatory platforms, and the effects of moderate and
acute vs. prolonged changes in expression (modeling
changes in DM, and avoiding limitations of lifelong
Fatty acid uptake
Fatty acid transport is also compartmentalized within
lipid rafts and caveolae [
], with the regulatory InsR
]. Accumulation of long-chain fatty acid
metabolites is important in development of myocardial
], with more prolonged changes
involved in later development of heart failure. The major
cardiac fatty acid transporters CD36 and fatty acid
binding protein (FABP) normally relocate to the sarcolemma
from intracellular stores in response to insulin or
]. Active CD36 specifically localizes to
lipid rafts and caveolae where fatty acid uptake activity
is promoted, while inactive intracellular CD36 is
associated with non-raft fractions [
]. Overexpression of
CD36 enhances skeletal muscle fatty acid oxidation and
decreases plasma lipids [
], while deletion impairs
cardiac fatty acid uptake, though this may be metabolically
compensated by increased glucose oxidation [
Sarcolemmal CD36 not only governs uptake but targets fatty
acids to specific metabolic sites including mitochondria
], and plays roles in promoting 5′-AMP activated
protein kinase (AMPK) signaling, regulating Ca2+
signaling and levels, and acting as co-receptor for toll-like
]. Permanent sarcolemmal relocation of
transporters in obesity and DM thus greatly promotes
cardiac lipid and lipid metabolite accumulation to impair
insulin signaling and glucose utilization [
critical sites of control, sarcolemmal CD36 and FABP are
important therapeutic targets for countering myocardial
insulin-resistance and cardiomyopathy.
Changes to caveolae and caveolin-1 and -3 in DM are
predicted to impact CD36-dependent uptake given
functionally relevant caveolar localization and caveolin
control. Hearts from caveolin-3 haplo-insufficient mice do
express less CD36 in line with differing caveolin-3 levels,
though a twofold rise in caveolin-1 suggests potentially
complicating adaptation [
]. Lipid raft targeting of
CD36 may involve interaction with caveolin-1 based on
effects in non-muscle cells [
], and cardiac lipids and
fatty acid uptake are also reduced with caveolin-1
]. Diabetic up-regulation of caveolin-1 [
could thus promote lipid uptake, though myocardial
CD36 and caveolin-1 are not always linked: for
example, cardioprotective isoflurane increases caveolin-1 and
] while reducing caveolar CD36 levels [
Although lifelong absence of caveolin-3 does not reduce
cardiac fatty acid uptake [
], a halving of cholesterol
levels and a 40–50% increase in triglycerides confirm
major perturbations of fatty acid handling. Importantly,
and as noted above, this model reflects a complex
phenotype encompassing lifelong absence of caveolin-3 and
caveolae (thus caveolae-localized transporters), which
likely disrupts potential caveolin-1 control.
Membrane cholesterol—beyond fluidity and caveolar
Changes in membrane cholesterol do not only influence
membrane fluidity [
], curvature [
], and caveolar
223, 262, 267
], but also govern T-tubule
system integrity and excitation–contraction (E–C) coupling
], contractile function [
], glucose and
fatty acid transport [
320, 331, 333–336
functionality of membrane ion channels, receptors and transporters
]. These diverse effects of cholesterol on
sarcolemmal architecture and the functionality of associated
proteins may contribute to impairment of
cardioprotection and ischemic tolerance with hypercholesterolemia
138, 140, 340
] and are relevant to the DM myocardium.
For example, there is evidence that increased
membrane cholesterol is key to impaired GLUT4 traffic
in insulin-resistance and T2DM, though studies have
focused on skeletal muscle given its contribution to
systemic insulin sensitivity and glucose homeostasis:
glucose-intolerant animal models and humans
accumulate cholesterol in skeletal muscle membranes [
]; high-fat diets also increase skeletal muscle
cholesterol ; DM also increases cardiac cholesterol levels
]; cholesterol depletion with methyl-β-cyclodextrin
reversibly and dose-dependently increases plasma
membrane GLUT4 incorporation in myotubes [
cholesterol depletion improves glucose homeostasis in
high-fat fed animals, together with insulin-dependent
GLUT4 translocation and glucose uptake in muscle
]. The cholesterol depleting agent chromium also
improves glycemic control in T2DM patients [
activates GLUT4 trafficking and insulin-stimulated
glucose transport in a cholesterol- and AMPK-dependent
]. This is consistent with evidence AMPK
improves insulin-stimulated GLUT4 control by lowering
membrane cholesterol [
]. These observations support
regulation of insulin-stimulated GLUT4 translocation
via tissue cholesterol content, and suggest cholesterol
removal may be useful in countering myocyte
insulinresistance, although cardiac studies are lacking.
Additional to indirect influences on protein
confirmation and function, cholesterol recognition/interaction
amino acid consensus (CRAC) and more recently CARC
(similar to CRAC, with an opposite orientation—hence
“CARC”) domains have been identified in
transmembrane proteins, including receptors regulating cellular
stress responses [
]. Sometimes located within
the same transmembrane segment, these CRAC and
CARC domains can directly interact with cholesterol in
the cytoplasmic leaflet of the plasma membrane.
Modulating multiple ion channels [
] and receptors
], the cardiac significance of
sarcolemmal cholesterol:protein interactions awaits further
study, particularly in the context of DM and metabolic
Potential influences of DM on cardiac phospholipid
Membrane lipids not only serve structural roles but are
substrates in cell signaling (Fig. 2). Sarcolemmal
phospholipids are targeted by three primary phospholipase
groups to generate lipid signaling molecules:
phospholipases A2, C and D (PLA2, PLC and PLD, respectively).
Phospholipid signaling is implicated in cardiac
hypertrophy/cardiomyopathy and is perturbed in cardiovascular
disease states including DM [
]. Changes observed in
sarcolemmal glycerol-phospholipid species in DM rat
hearts likely contribute to membrane and contractile
195, 199, 202
]. In terms of ischemic tolerance,
phospholipases are implicated both in mediating and
protecting against ischemia–reperfusion injury [
]. This may reflect isoform specific effects,
including protection via PLCγ1 and injury via PLCδ1. Shifts in
membrane phospholipase signaling may thus contribute
to alterations in both infarct tolerance and
cardioprotection in DM.
Cardiac PLC activities are reduced in STZ-induced
T1DM rats, and basal and phosphatidic acid induced
IP3 generation are reduced in cardiomyocytes from DM
]. In contrast, increased PLC activity is
implicated in exaggerated α1-adrenergic receptor mediated
inotropy with acute (3 day) hyperglycemia [
outcomes with acute vs. chronic DM may be relevant to
patterns of early protection and later ischemic
intolerance in models of STZ dependent T1DM [
]. A reduced
rather than increased PLC activity may contribute to
abnormalities in contractility and α1-adrenergic receptor
responses with more protracted disease [
PLC generation of 1,2-diacylglycerol (DAG) may impact
other cellular processes [
], although myocardial
Fig. 2 Sarcolemmal phospholipid signaling via phospholipases. AA
arachidonic acid, DAG 1,2-diacylglycerol, IP3 inositol
1,4,5-triphosphate, PA phosphatidic acid, PC phosphatidylcholine, PIP2
phosphatidylinositol-4,5-bisphosphate, PLA2 phospholipase A2, PLC
phospholipase C, PLD phospholipase D
DAG levels are increased with more acute
STZ-dependent T1DM and in autoimmune DM (biobreeding) rats
], which could destabilize the membrane [
Phospholipase D activity reportedly declines in DM
], which may limit PA generation
and thereby PLC activation. Alterations in AA content
of sarcolemmal phospholipids could also reflect
dysfunctional PLC signaling [
]. Relatively little is known
regarding cardiac PLA2 signaling in DM, however
cardiac membrane associated PLA2 activity is increased in
rat models of DM [
]. The local environment of
caveolae is also important to phospholipid signaling. Up
to half of cellular PIP2 is located in caveolin-enriched
membrane fractions [
268, 361, 362
], and this pool is
specifically sensitive to GPCR and RTK activation [
a cholesterol-dependent manner [
]. For example, the
α1 adrenergic receptor (AR) and its Gq effector protein
are caveolae localized in adult cardiomyocytes [
and analysis in neonatal cells possessing both caveolar
and non-caveolar PIP2 fractions supports select caveolar
depletion upon α1-AR stimulation . Coupled with
localized PLC-dependent hydrolysis to DAG, this
compartmentation provides for select regulation of caveolar
populations of PIP2- and DAG-sensitive ion channels
and exchangers [
]. Shifts in caveolar makeup and
localized phospholipids thus have capacity to selectively
disturb cardiomyocyte receptor signaling and ion
Critical to cellular growth, substrate metabolism,
stress responses and cardioprotection, PI3K
isoforms catalyze production of the 3-phosphorylated
phosphoinositides phosphatidylinositol 3-phosphate,
phosphatidylinositol (3,4)-bisphosphate, and
phosphatidylinositol (3,4,5)-triphosphate. While membrane
phospholipid pools are modified in DM, it is unclear whether
changes are sufficient to influence PI3K signaling.
Certainly, dysfunctional PI3K/Akt signaling is implicated
in altered InsR control and impaired cardioprotection,
among other cardiac changes.
Membrane glycation, glycosylation, palmitoylation
and oxidation in DM
Glycation and enzymatic glycosylation are major
factors in the cardiac abnormalities arising in DM [
is oxidative stress [
]. Palmitoylation is also an
important determinant of sarcolemmal protein function
] and is highly relevant in metabolic disorders
such as DM [
], however, diabetic perturbations
have been largely studied in non-cardiac tissues. Other
modifications may also be relevant in DM, for
example reductions in phospholipid N-methylation [
depress Na+-dependent Ca2+ uptake [
] and enhance
ATP-dependent Ca2+ efflux [
] in the cardiac
sarcolemma of STZ-dependent T1DM models. Such effects
may be mechanistically relevant to paradoxical resistance
to external Ca2+ overload in DM hearts [
Advanced glycation end‑products (AGEs) and the receptor
for AGE (RAGE)
Chronic hyperglycemia promotes glycation, the
nonenzymatic covalent bonding of carbohydrates to
proteins and lipids. Glycation products in turn can form
cross-linked structures known as AGEs. These
modified proteins/lipids activate cell surface RAGE to trigger
ROS generation, activation of nuclear factor
kappa-lightchain-enhancer of activated B cells (NFκB) and
proinflammatory cytokine production. Positive feedback
between NFκB and RAGE expression exaggerates ROS
and cytokine generation. These processes are implicated
in vascular dysfunction in DM, and have been shown
to contribute to myocardial changes and dysfunction
]. Targeting AGE accumulation has also been
shown to improve myocardial ischemic tolerance in
different models of DM. For example, cardioprotection in
rat T1DM models with natural xanthonoid and
] and anti-hyperglycemic glitazones [
appear to involve inhibition of the AGE-RAGE axis and
AGE accumulation. However, cytoplasmic AGE
accumulation is typically documented in animal and human
tissues, and specific sarcolemmal targets of glycation have
not been investigated in detail.
Diabetes increases fluxes through accessory paths of
glucose metabolism, including the hexosamine
biosynthetic pathway (HBP) that produces the sugar donor
for enzyme-mediated β-O-linked-N-acetylglucosamine
(O-GlcNAc) modification of proteins or lipids. Studies
confirm that increased protein O-GlcNAc levels
contribute to the cardiac abnormalities of DM. This modulation
is complex, however, with O-GlcNAc mediating both
beneficial and detrimental effects [
elevations in O-GlcNAc may provide cytoprotection [
with acutely increased O-GlcNAc prior to ischemia or in
reperfusion reducing infarction and dysfunction [
Inhibition of O-linked β-N-acetylglucosamine transferase
(OGT) can also inhibit cardioprotection [
inhibition of protein O-GlcNAcase (OGA) may improve
cardiac ischemic tolerance [
]. Such effects might be
relevant to observations of acute protection early in
STZdependent hyperglycemia. Indeed, Jensen et al. [
present evidence O-GlcNAc signaling participates in remote
ischemic preconditioning and activates
cardioprotection in DM myocardium from T2DM patients (based on
functional I–R tolerance of atrial trabeculae).
Other evidence indicates chronic elevations in
O-GlcNAc are detrimental to the heart.
Hyperglycemia mediated HBP activation increases cardiomyoblast
], and inhibitors of OGA have been shown
to improve I–R tolerance in DM hearts, potentially via
preserved integrity of O-GlcNAc associated Z-line
protein structures [
]. Activation of the HBP and protein
O-GlcNAcylation modulates hypertrophic and cell
signaling pathways in T2DM [
]. Increased protein
O-GlcNAcylation in non-DM cardiomyocytes also decreased
hypertrophic signaling responses, while HBP inhibition
partly restored hypertrophic signaling in DM
cardiomyocytes. Cardiac beclin-1 and Bcl-2 have also been recently
identified as targets for O-GlcNAcylation [
blunted autophagy in cardiomyocytes from T2DM db/db
mice partly reversed by inhibiting the HBP.
Ramirez-Correa et al. [
] present evidence that Z-line localization of
O-GlcNAc and OGT and A-band localization of OGA is
disrupted, consistent with changes in human DM hearts.
Their data indicate subcellular redistribution of OGT and
OGA rather than changes in overall activities are
responsible for altered O-GlcNAcylation in DM. On the other
hand, Dassanayaka et al. [
] show O-GlcNAcylation is
not involved in inhibition of mitochondrial metabolism
in hyperglycemic cardiomyocytes.
There is only limited evidence for glycosylation
modifications of plasma membrane proteins. In coronary
endothelium OGA expression is decreased and OGT
expression and O-GlcNAcylation increased with DM
], with CX40 identified as a potential target of
O-GlcNAcylation regulating cell function. Effects of
glucosamine and OGT blockade on post-ischemic Ca2+ levels
also implicate modulation of sarcolemmal channels [
Further studies of cardiac sarcolemmal targets of
O-GlcNAcylation are required to clarify the role of this process
in membrane changes and dysfunction in T1 and T2DM.
Reversible S-palmitoylation (thioester attachment of
palmitic acid to cysteine) is an important protein
‘sorting’ signal, governing trafficking and membrane
]. Palmitoylation enhances membrane
affinity of many proteins to facilitate membrane
]. N-myristoylation (amide bond attachment of
myristoyl group to N-terminal glycine residues) may also
facilitate protein localization to membrane palmitoylases
]. Within the membrane, palmitoylated proteins
have high affinities for cholesterol and sphingolipid-rich
domains , which is important in targeting proteins
to membrane raft regions [
]. Some GPCRs are
palmitoylated down-stream of the 7th transmembrane domain
], which may be required for efficient plasma
membrane delivery [
]. Palmitoylation may regulate
internalization of some GPCRs and promote trafficking
of internalized proteins to the plasma membrane. Ion
channel and exchanger functions are also modified with
palmitoylation. For example, the cardiac Na+/K+-ATPase
is targeted by palmitoylation, though functional
outcomes await detailed study . The inactivation of the
Na+/Ca2+ exchanger is also strongly dependent on
Recent data support induction of endocytosis via
membrane protein palmitoylation. Massive endocytosis
(MEND) is an adapter-independent form of
endocytosis that can rapidly internalize up to 70% of the plasma
membrane in response to stressors such as Ca2+
]. Increased plasma membrane palmitoylation
promotes MEND in response to mitochondrial stress
], likely due to clustering of palmitoylated proteins in
lipid-ordered domains as a result of palmitoyl chain
affinity for the ordered lipid environment [
]. Reilly et al.
] show that elevations in palmitoylated Na+/Ca2+
exchanger 1 protein in the plasma membrane accelerates
MEND, mirroring effects of palmitoylated
] and suggesting palmitoylated proteins
promote formation of lipid-protein domains to trigger
endocytosis. Since acyl groups of palmitoylated
proteins insert more readily between the phospholipid head
groups of curved rather than planar membrane regions
, palmitoylated proteins will cluster in invaginated
lipid-ordered domains that may include caveolae [
the curved domains formed in endocytosis, and
potentially curved junctions between T-tubule and surface
sarcolemma. Clustering of palmitoylated membrane
proteins with large cytoplasmic domains (e.g.
Na+/K+ATPase, Na+/Ca2+ exchanger) may itself promote
membrane curvature [
]. Unfortunately, despite such effects
and the importance of palmitoylation to sarcolemmal
protein trafficking and function, few studies have
examined potential roles of altered palmitoylation in the
cardiac abnormalities of DM.
It is well established that oxidative stress is involved in
development and progression of DM and its
organ-specific complications [
10, 403, 404
], and shifts in cardiac
stress responses may involve oxidative modification of
sarcolemmal elements. Oxidative stress may underlie
changes in caveolae and caveolins: Su et al. [
the anti-oxidant N-acetylcysteine (NAC) limits changes
in caveolin-3 together with phosphorylated eNOS known
to localize to caveolae. Diabetes reduces association of
caveolin-3 and eNOS in cardiomyocytes, an effect
countered by antioxidant treatment. Protective effects of
NAC on hyperglycemic and hypoxic cell injury were also
abolished by knockdown of either caveolin-3 or eNOS,
supporting the notion hyperglycemic inhibition of eNOS
results from impaired caveolin-3 expression. Membrane
lipid metabolism also contributes to oxidative stress:
lipoxygenases oxidatively metabolize AA released from
the plasma membrane following PC hydrolysis,
generating ROS in the process. Hyperglycemia-induced
activation of 12/15-lipoxygenase is associated with cardiac
oxidative stress and DM cardiomyopathy [
beyond largely indirect evidence (e.g. preventing
caveolar changes with anti-oxidant intervention), there is
relatively little information available regarding the specific
sarcolemmal targets of oxidative modification in DM
hearts, and their roles in associated ischemic intolerance.
As for glycation/glycosylation and palmitoylation, further
studies are needed to clarify modifications to
sarcolemmal proteins in T1 and T2DM, and their roles in altered
Remodeling of the T‑tubule system in DM
Despite limited studies, and none in human
cardiomyocytes, evidence supports significant remodeling of
T-tubules in DM. Studies in skeletal muscle confirm
the T-tubule system is a functionally important target
governing glucose handling [
336, 405, 406
]. In heart,
McGrath et al. [
] report a pronounced fall in
functionally intact SR/T-tubular junctions together with an
increased T-tubule area (longitudinal rather than
transverse orientation) in rat models of T2DM. A subsequent
study in db/db mice identified a fall in T-tubule density
in this model of T2DM [
]. Despite differing
morphological outcomes, both studies highlight diabetic
disruption of T-tubule organization and functionality, likely
perturbing E–C coupling and contractile function. For
example, the synchrony of cardiomyocyte Ca2+ release
(influencing contractile function and
arrhythmogenesis) depends on T-tubule integrity, and
disorganization underlies cardiac dyssynchrony in different settings
]. Disruption of T-tubule structure and function
may thus mediate the reduction in synchrony observed in
Changes specifically within the T-tubule system may
also be important in altered substrate handling. Magnetic
resonance spectroscopic [
] and biochemical analyses
] confirm defective GLUT4 translocation in muscle
of T2DM patients, while studies in animal models
confirm impaired translocation in skeletal [
] and cardiac
]. Dissociation of T-tubules has been
shown to reduce basal and abolish insulin-dependent
glucose transport in skeletal muscle , confirming
a critical role in glucose metabolism and homeostasis.
Since the majority of GLUT4 translocation occurs
specifically within T-tubules [
] and cholesterol-rich
microdomains , T-tubule disruption and changes in
cholesterol will modify insulin-stimulated GLUT4
exocytosis in DM.
Changes in both membrane cholesterol and caveolae/
caveolins may contribute to the T-tubule dysfunction in
DM. In skeletal myocytes, cholesterol is more
concentrated within T-tubules compared to surface membrane
], which may contribute to lower
fluidity in the lipid phase of T-tubules compared with most
cell membranes [
]. While data are lacking for
cardiomyocytes, similar compartmentation in T-tubules is
likely. Cardiac Ca2+ levels and contractility are sensitive
to membrane cholesterol [
], and Zhu et al. 
recently confirmed cholesterols importance to
cardiomyocyte T-tubule stability and E–C coupling, an effect
apparently independent of caveolin-3/caveolae. The
integrity of intercalated disks and intercellular
communication were also sensitive to cholesterol. Caveolae and
caveolins are also important in T-tubule development
and maintenance of functional integrity [
the co-localization and interaction between
junctophilin-2 and caveolin-3 in dyadic structures to establish
efficient, synchronous EC coupling. Depletion of caveolin-3
could contribute to loss of dyadic integrity, and
junctophilin/caveolin-3 interactions are known to be
suppressed in cardiomyopathy [
Changes in gap junctions and sarcolemmal ion channels
Abnormal conduction and arrhythmogenesis is evident
in both DM patients [
] and animal models of
T1 and T2DM [
]. Together with changes to the
T-tubule system, shifts in gap junctions  and
sarcolemmal ion (Ca2+, Na+, K+) channels will disrupt
electrophysiology in DM, and influence cardiac responses
to I–R. Specialized gap junction pores provide effective
electrical coupling of adjacent cardiomyocytes, and are
critical not only to conduction and electrical stability but
responses to ischemia and cardioprotective stimuli [
Principle connexin (CX) protein components are altered
in DM, including evidence of modified expression and
phosphorylation. The latter post-translational changes
are functionally important: PKC inhibits cardiac gap
junction conductance [
] via CX-43
]; dephosphorylation of gap junction
elements results in their uncoupling [
] and lateralization
]; and excess phosphorylation of CX-43 by PKCε
may promote proteolysis to deplete junction channels in
DM myocardium .
In cultured myocytes CX-43 expression is suppressed
by hyperglycemia [
], potentially involving
PKCdependent miR-1/206 expression [
]; and by the
AGE-RAGE system, potentially involving PKC and ERK
]. In STZ-dependent T1DM in rats an
increased SA nodal expression of CX-43 (and -40 and
-45) is associated with nodal conduction delay [
while ventricular expression is reportedly unaltered
], reduced [
] or increased [
]. Olsen et al.
 observe reduced lateralization of CX-43 in hearts
from ZDF rats exhibiting reduced conduction velocity.
Phosphorylation of atrial and ventricular CX-43 declines
in models of DM [
439, 442, 443
], potentially as a result
of impaired PKCε expression [
], though these
investigators also report increased PKCε mediated CX-43
phosphorylation in DM myocardium, which may
promote proteolytic degradation [
]. The extent of
cardiac CX-43 phosphorylation reportedly declines with
progression of DM while protein nitration increases
]. Zhu et al. [
] also recently found that cholesterol
depletion not only destabilized cardiomyocyte T-tubules,
but disrupted the integrity of intercalated disks and
intercellular communication. Gap junction function and
inter-cellular communication may therefore be
influenced by sarcolemmal cholesterol changes in DM.
Supporting mechanistic involvement of gap junction changes
in the myocardial abnormalities of DM, benefit with
exercise in T2DM db/db mice is attributed to
restoration of CX-43 networks [
], and beneficial effects of
n-3 PUFA feeding on DM cardiomyopathy are linked to
increased CX-43 expression and phosphorylation
(associated with up-regulated PKCε) [
]. In contrast, it has
also been reported that moderate exercise reduces
ventricular CX-43 phosphorylation [
Sarcolemmal ion channels fundamental to E–C
coupling and relevant to I–R injury are modified in DM
myocardium, including changes in Ca2+ channels, levels
and contractile responses [
], K+ currents and
], and Na+ pumps [
may participate in enhanced arrhythmogenesis and risk
of sudden cardiac death [
423, 425, 427, 452
membrane lipids and biophysical properties in DM will
influence ion channel function, and changes in channel
transcription and expression patterns also arise. There
is evidence of increased transcription of Ca2+ channels
(Cacna1c, Cacna1g, Cacnb1) and Gja4 (CX-37), and
differential changes in K+ channels (Kcnj11 up, Kcnb1
down) in GK T2DM rat hearts [
]. Sucrose feeding
induces K+ channels (Kcnj2, Kcnj8) and Gja1 (CX-43)
and Gja4 in non-DM rats [
]. This group also reports
up-regulation of ventricular Cacna1h, Scn1b and Hcn2
vs. down-regulation of Hcn4, Kcna2, Kcna4 and Kcnj2 in
this model [
], and up-regulation of genes encoding
cardiac LTCC proteins (Cacna1c, Cacna1g, Cacna1h and
Cacna2d1) in association with prolongation of Ca2+
transients in the ZDF rat model of T2DM [
These transcriptional changes do translate to altered
channel expression, with shifts in Ca2+, K+ and Na+
channels all potentially contributing to
electrophysiological perturbations in DM hearts. Abnormal Ca2+
currents in cardiomyocytes from T1DM Akita mice involve
reduced sarcolemmal levels of the LTCC, potentially
related to impaired PI3K control [
]. The decline in
sarcolemmal Ca2+ permeability in T2DM db/db mice is
associated with reduced expression of the pore-forming
α1C subunit of the LTCC [
]. Though less well studied,
cardiac T-type Ca2+ channel expression/function may
also be modified in DM given caveolar localization and
sensitivity to caveolin-3 [
], and evidence of changes
in other cell types with chronic DM [
]. Reductions in
cardiomyocyte K+ current density in models of T1DM
also involve defective channel expression (potentially
involving AMPK signaling) [
], and action potential
prolongation in Otsuka-Long-Evans-Tokushima Fatty
rats is linked to down-regulation of endocardial Kv4.2
(voltage-gated K+ channel subfamily D) and
transmural KChIP2 (K+ channel interacting protein)
]. Impaired insulin signaling has been shown
to reduce the amplitude of the transient outward K+
current fast component in cardiomyocytes in
association with reduced Kv4.2 and KChIP2 expression [
Broadened ventricular repolarization and reduced
‘repolarization reserve’ in alloxan-induced T1DM in dogs may
also involve impaired K+ currents as a result of reduced
Kv4.3 (voltage-gated K+ channel subfamily D) and MinK
(voltage-gated K+ channel sub-family E subunit)
expression, while Kv1.4 (voltage-gated K+ channel subfamily A),
KChIP2 and KvLQT1 (voltage-gated K+ channel
subfamily D) were increased [
]. Such changes may
significantly predispose to sudden cardiac death.
Depressed INa may additionally play a role in altered
electrical activity in DM cardiomyocytes, with less Na+
influx during contraction linked to reduced expression of
both the Na+/K+-ATPase and Na+/Ca2+ exchanger [
This is consistent with sarcolemmal Na+-K+ pump
inhibition in other models of T1DM [
]. Changes in Ca2+,
K+, and Na+ channels are not only likely to increase
susceptibility to arrhythmias in I–R, but may well modulate
cell death processes. Moreover, expression of subunits
for the sarcolemmal KATP channel implicated in multiple
cardioprotective responses [
] is also disrupted in DM,
with SUR2A and Kir6.2 decreased both in myocytes from
T1DM rats and isolated myocytes subjected to
]. Such a change will not only desensitize
cardiac myocytes to KATP openers, but impair transduction
of cardioprotective signaling. The impacts of these varied
channel expression changes in DM will be exacerbated
by membrane lipid changes and structural modifications
to the sarcolemma, including shifts in microdomains
in which select channels cluster, and T-tubule and
Potential ‘membrane‑targeted’ therapies?
Based on the array of detrimental sarcolemmal changes
evident in DM, a number of therapeutic approaches
present themselves, including modifications to diet and
physical activity, cholesterol manipulation, and
modulation of caveolins and caveolar biology.
Targeting caveolae and caveolins
Given evidence of abnormal caveolin-3 expression in
models of DM, this caveolar protein has appeal as a
therapeutic target [
158, 159, 169
anti-diabetic effects of hepatic caveolin-3 gene transfer supports
the therapeutic potential of caveolin-3 in DM [
Although the regulation of cardiac caveolin-3 expression
is not well understood, there is evidence from
non-cardiac cells for transcriptional control by myogenin, ID2,
miR-22 and myocardin [
]. Myocardins are
important to formation of caveolae, and in glucose and lipid
]. Whether transcriptional control
of caveolins might be targetable is not clear. However,
hyperinsulinemia does up-regulate myocardin in cardiac
], which additional to modulating
hypertrophy could up-regulate caveolins and caveolae [
Conversely, insulin-resistance may reduce myocardin
expression and thereby caveolins and caveolae. Aortic
myocardin is substantially induced in GK (T2DM) rats,
which appears to involve a miR-145 dependent response
to oxidative stress [
Interestingly, cardiac caveolin-3 may be differentially
modifiable via dietary saturated fat [
] and PUFA [
], and hyperglycemic depression of caveolin-3 may
also be PKCβ2-dependent, providing a potential
pharmacological target. Lei et al.  show that inhibition
or knockdown of PKCβ2 counters hyperglycemic
depression of caveolin-3 in hearts and myocytes, and improves
cardiac Akt phosphorylation and diastolic function. This
group subsequently showed that PKCβ2 inhibition also
improved cardiac I–R tolerance together with caveolin-3
levels and control of Akt signaling in STZ-dependent
T1DM rats [
]. Supplementation with the anti-oxidant
NAC also attenuates PKCβ2 expression and hypertrophy
] while enhancing ischemic tolerance [
STZdependent T1DM rats. Caveolin-3 levels were not
measured, though a reduction in PKCβ2 activity is predicted
to improve caveolin-3 based on other work [
Other potential targets include adenylyl cyclase (AC)
and focal adhesion kinase (FAK) signaling paths: in vitro
studies suggest AC can repress caveolin-3 in cardiac
] while FAK up-regulates caveolin-3 in
skeletal myoblasts [
]. No data are available regarding
cardiac FAK signaling in DM, however, FAK may be
activated in hyperglycemic conditions [
], and FAK
induction in hypertrophied skeletal muscle is exaggerated in
T1DM rats [
]. In skeletal myotubes FAK also appears
important in insulin-dependent GLUT4 translocation
and glucose uptake [
]. Adenylate cyclase itself appears
functionally unaltered in DM hearts, while adrenergic
receptor mediated control is impaired [
vascular AC expression/function may be altered in DM
]. Inhibition of cardiac AC5 activity does protect
against cardiac abnormalities in T2DM and obesity [
Targeting caveolin-3 expression via acute gene therapy
with adeno-associated virus (AAV) for Cav3 improves
I–R and Ca2+ tolerance, preserves mitochondrial
stability and reduces reactive oxygen species [
addition, cardiac specific caveolin-3 overexpression enhances
functional outcomes post-I–R and reduces infarct size
(similar to effects of ischemic preconditioning), which
may be due to improved mitochondrial Ca2+ tolerance
and respiratory rates with reduced ROS generation [
Fridolfsson et al. [
] identified that increased O2
consumption in caveolin-3 overexpressing hearts improved
energy production without a parallel increase in ROS
generation. Further experiments targeting caveolin-3
to mitochondria confirmed improved mitochondrial
stability during Ca2+ challenge, and delayed
mitochondrial depolarization and improved respiratory complex
activity associated with enhanced ischemic tolerance.
Conversely, deletion results in mitochondrial
] and hypertrophy [
]. How caveolin-3
migrates to/communicates with mitochondria and
subsequently promotes mitochondrial and ischemic tolerance
remains to be further detailed.
Cholesterol lowering therapies
Reducing membrane cholesterol has capacity to improve
fluidity and counter some sarcolemmal
abnormalities evident in DM hearts. On the other hand, whether
reductions in cholesterol might adversely impact
caveolae, caveolins and T-tubules is unclear. Certainly, statins
are of value in DM, with low-dose treatment significantly
reducing cardiovascular events in T2DM patients [
However, while pleiotropic effects of statins include
‘anti-diabetic’ actions such as reduced inflammation in
T2DM patients [
], they may also include induction of
insulin-resistance and promotion of DM [
said, such effects appear modest relative to the benefits
of statins, and may only be a factor in those at particular
risk of new onset DM . Experimental studies show
statins do protect against myocardial ischemic injury in
hearts from DM and healthy animals, though again this
reflects pleiotropic effects of the drugs independent of
cholesterol lowering [
Modifiable diet and physical activity have long been
appreciated as major determinants of DM severity and
complications. Dietary modification can alter
sarcolemmal makeup and function, and inflammatory, glycation/
glycosylation and oxidative processes in the heart and
vessels. For example, homeostatic control of
inflammation is mediated by eicosanoids (prostaglandins,
leukotrienes, thromboxanes) whose generation is dependent
on the n-6 PUFA AA [
]. Shifts in saturated vs.
unsaturated fat intake can modify fundamental membrane
properties together with caveolar components, while
limitations in caloric intake may profoundly influence the
DM phenotype and promote protective outcomes.
Unsaturated vs. saturated fats
Mammalian species are unable to produce n-3 PUFAs,
thus must acquire these essential fatty acids via the diet.
Edible seeds such as flaxseed and chia seeds are rich
sources of the 18C n-3 PUFA α-linolenic acid, while
longer chain n-3 PUFAs (EPA, DHA) can be synthesized
from α-linolenic acid or consumption of fish oils. Once
acquired, n-3 PUFAs can integrate into the sarcolemma
to displace membrane AA: dietary n-3 PUFA
incorporation in myocardium and myocytes occurs at the expense
of n-6 PUFAs [
]. Consumption of n-3 PUFAs thus
reduces inflammation via disrupting production of
AAderived eicosanoids [
]. However, it is worth
noting that AA-derived eicosanoids (including prostaglandin
E2) exhibit both pro- and anti-inflammatory capabilities.
Dietary α-linolenic acid is cardioprotective in a rat
model of T2D [
], with 4 weeks of α-linolenic acid
supplementation improving ischemic tolerance
including enhanced functional outcomes and reductions in
infarction and markers of cell death (whereas no
protection was evident in non-DM rats). Cardioprotection was
linked to anti-inflammatory (reduced tumor necrosis
factor-α and interleukin-6) and anti-oxidative (reduced
superoxide and enhanced anti-oxidant capacity) actions,
possibly involving PI3K/Akt signaling [
Insulinresistance, glucose intolerance, dyslipidemia and cardiac
lipid accumulation after 3–6 months of a high-sugar diet
are also reversed by transition to a chia seed-rich diet
]. Consumption of n-3 PUFAs improves sarcolemmal
functions, critical to the management of DM
cardiomyopathy. For example, consumption of fish oils: enhances
EPA and DHA in cardiac membranes while reducing
]; prevents translocation of CD36, limiting
fatty acid uptake and lipid storage ; and counters
abnormal membrane fluidity in T1DM mice [
vegetarian diet improvement in linoleic acid content is also
associated with improved insulin sensitivity in subjects
with T2DM [
Diets containing high ratios of
PUFA/mono-unsaturated fatty acid (MUFA) improve insulin-binding and
glucose uptake in adipose cells from healthy and T1DM rats
]. Membranous phospholipid content is also altered,
with enhanced PUFA and reduced MUFA (though no
effect on total saturated phospholipids) [
Interestingly, even at very high insulin levels (1000 ng/mL),
cells from T1DM rats fed low PUFA/MUFA diets bind
less insulin than those fed high PUFA/MUFA diets and
exposed to lower insulin levels. This suggests that insulin
has greater affinity for cells with more unsaturated
membranes, which may be particularly useful in management
of insulin-resistant T2DM.
Enriched n-3 PUFA diets also modify ion exchange and
action potential duration, which may limit cardiac
propensity to I–R injury and arrhythmias. Isolated myocytes
from rabbits fed fish oil for 3 weeks exhibit increased
sarcolemmal EPA and DHA (vs. decreased MUFAs) and
20% shorter action potentials compared with myocytes
from animals on a n-9 MUFA-rich diet [
of myocytes to EPA and DHA shortened action
potentials in cells from n-9 MUFA and not n-3 PUFA fed
rabbits. These findings indicate action potential shortening
likely stems from altered membrane lipid composition
and not direct ligand-like interaction with ion channels
]. Other studies report inhibitory effects of PUFAs
on sarcolemmal K+ [
] and Ca2+ channels [
the Na+/H+ exchanger [
], potentially limiting
pathological Ca2+ overload in myocardial cells.
Dietary fats also influence caveolin expression and
thus caveolae. A palmitate enriched diet significantly
depresses cardiac caveolin-3 [
], whereas a
flaxseedenriched diet reverses reductions in cardiac caveolin-3
in cardiomyopathic hamsters [
], and prevents
reductions in skeletal muscle caveolin-3 in a model of muscular
dystrophy (also repairing sarcolemmal damage, reducing
inflammation and cell death) [
]. Effectiveness of such
diet intervention in a DM animal model awaits testing.
In addition to n-3 PUFA supplementation, improved
cardiac function in DM may be achievable through calorie
Caloric limitation and time‑restricted feeding
Calorie restriction or intermittent fasting may
provide significant benefit in DM, and such interventions
modify membrane composition in murine myocardium
]. Though prolonged caloric limitation is a
wellestablished protective stimulus, effects of brief or
moderate fasting await detailed study in DM animals. Severe
CR for 11 days generates unique I–R tolerance [
and 24–72 h of fasting enhances cardiac I–R tolerance
and mitochondrial viability in non-DM hearts [
]. There are surprisingly few studies of caloric
limitation in DM. A 30% limitation in calories for ≥ 2 months
improves glucose homeostasis and markers of systemic
or cardiac oxidative-stress in rodent models of T2DM
]. A similar CR regime fails to influence I–R
tolerance in models of T2DM and metabolic syndrome,
though benefit via ischemic preconditioning was restored
]. Contrasting reported protection with fasting, one
recent study suggests 18 h of fasting actually worsens
ischemic tolerance in T2DM and also non-DM rat hearts
], potentially linked to enhanced glucose vs. fatty
acid metabolism. Another recent study [
] also found
that loss of sevoflurane preconditioning with a high
calorie western diet was unaltered with 4 week of control diet
(though an apparently detrimental impact of sevoflurane
with the western diet was countered).
Whether ischemic tolerance with caloric limitation
involves membrane changes in either DM or non-DM
hearts remains to be established. However, modest (12 h)
fasting does induce membrane remodeling via a
reduction in acyl chains, predominately lost from C22:6 (DHA)
]. While effects of CR on myocardial caveolar
domains are unknown, it does prevent age-related
reductions in caveolin-1 in liver tissue [
], and repression of
caveolin-1 in breast tissue is mediated by a micro-RNA
(miR-203) that is induced with CR [
Circadian biology is extremely important in the
influences of fat and calorie intake on obesity and associated
metabolic disturbances, and restricted feeding times
rather than calorie intakes can be highly beneficial in
cardiometabolic disorders [
]. The timing of food intake
appears a key determinant of circadian rhythm,
particularly in metabolic organs, and the impacts of high-fat
feeding on body weight, insulin levels, glucose tolerance,
inflammation and hepatic steatosis can be effectively
countered by time-restricted feeding without caloric
]. Conversely, short-term feeding at the
wrong time of day can desynchronize peripheral clocks
and induce obesity and metabolic disorder [
Timerestricted feeding also counters cardiac aging changes in
the Drosophila model [
], however effects on
myocardial ischemic tolerance, or the cardiomyopathy and
sarcolemmal changes in DM, have yet to be tested.
Exercise in DM—membrane involvement?
Physical activity and VO2 have been identified as perhaps
the most important factors governing chronic disease
risk, particularly CVD and DM [
]. Up to 50% of
coronary artery disease can be prevented by 30 min of
moderate exercise daily (assessed in middle-aged women)
], and as little as 3 weeks of exercise can reduce
the clinical impact of metabolic syndrome (a
combination of coronary heart disease, hypertension and T2DM)
by 50% [
]. Not only substantially reducing
risk/incidence, exercise can be applied ‘therapeutically’ in existing
disease states to alleviate symptoms and counter
progression. Broadly beneficial systemic effects render physical
activity an effective therapy in disorders including cancer
], depression [
] and cardiovascular disease [
Exercise induces obvious metabolic advantages,
improving tissue and whole body VO2/oxidative capacity and
vascularity, cardiac functional reserve and efficiency,
insulin signaling and sensitivity, glucose and fat handling,
anti-oxidant status, inflammation and immune function
]. Analyses confirm benefits of physical
activity in patients with T2DM, though questions regarding
effective exercise prescription remain [
only reducing the incidence of infarction, exercise also
boosts myocardial tolerance to infarction  and may
improve or restore conventional protective responses in
models of stress, disease and aging [
]. Effects on the
DM heart revolve around improved glucose and fatty
acid metabolism, mitochondrial function and
oxidative stress, however sarcolemmal abnormalities are also
influenced. Exercise does modify fatty acid composition
of phospholipids and triglycerides in cardiac and skeletal
], and beneficial remodeling of plasma
membrane lipids is reported in other cell types .
Sarcolemmal effects in DM are less well defined.
Studies confirm exercise-dependent improvements
in cardiac function, survival signaling and ischemic
tolerance in models of T1DM [
] and T2DM [
]. While improvements in substrate metabolism are
broadly implicated, Pons et al. [
cardioprotection in ob/ob mice independent of hyperglycemia,
hypercholesterolemia, hyperinsulinemia, fat mass or
body weight. Schrauwen-Hinderling et al. [
that 12 week endurance/strength training improves
systemic insulin sensitivity and cardiac function in T2DM
patients without modifying cardiac lipid content. Altered
myocardial O-GlcNAcylation may participate, with
evidence swimming in T1DM rats increases OGA
activity and reduces cardiac protein O-GlcNAcylation [
However, this also reduces O-GlcNAcylation in
nonDM hearts [
]. Indeed, Medford et al. [
] show as
little as 15 min of exercise can alter myocardial
O-GlcNAcylation. Exercise protection in models of DM has
been linked to normalization of nitro-oxidative stress
and eNOS control [
], and improvements in PPARγ
coactivator-1α and Akt signaling [
], both effects that
may arise via restoration of sarcolemmal caveolae and
caveolin control of eNOS [
250, 275, 296–298
] and Akt
]. Studies in non-DM  and
DM hearts [
] do support up-regulation of caveolin-3,
though the contribution of this change to exercise
cardioprotection awaits analysis. Indirectly supporting
targeting of sarcolemmal elements, da Silva et al. [
that altered Ca2+ transients (and mitochondrial uptake)
in T1DM rat hearts are countered by swimming, which
also enhanced benefit via insulin.
More directly supporting improved sarcolemmal
makeup, Hesari et al. [
] report that exercise reduces
CX-43 phosphorylation in hearts from T1DM rats, and
Veeranki et al. [
] demonstrate beneficial effects of
exercise on CX-43 levels and gap-junction function in
db/db mice, associated with preservation of
mitochondrial function. This is consistent with evidence exercise
modulates sarcolemmal determinants of signaling and
E–C coupling in T2DM rats, including transcriptional
up-regulation of caveolin-3 and CX-43, and differential
changes in K+ channels (Hcn2, Kcnk3) [
unpublished findings support up-regulation of cardiac
caveolin-3 and protection against I–R with swim training in
mice, coupled with powerful anti-inflammatory effects of
exercise (data not shown).
Conclusions and perspectives
A diversity of mechanisms are involved in the cardiac
and coronary abnormalities arising in DM, and
evolution of DM cardiomyopathy. However, the sarcolemma
is a nexus for many fundamental mechanistic elements
and sequelae of DM. The ability of the sarcolemma to
withstand rupture is fundamentally important to cell
survival and stress tolerance and is governed by
molecular makeup and caveolar membrane ‘reserve’. The
sarcolemma is also the seat of glucose and fatty acid transport
and InsR control, and therefore fundamentally
participates in the pathogenesis of DM complications.
Furthermore, the functionality of ion channels and cell surface
receptors is determined by membrane makeup. Diabetes
impacts sarcolemmal architecture, remodeling T-tubules,
caveolar domains and gap junctions, disrupting E–C
coupling and promoting injury and arrhythmogenesis in
I–R. Specific molecular changes include increased
cholesterol and fatty acid saturation vs. reduced
desaturation, and differential shifts in phospholipids and PUFAs.
Caveolar proteins are a particularly important target in
DM, with evidence for caveolin-3 depletion and
caveolae dysfunction in dysregulation of GLUT4 and CD36
function, survival kinase and eNOS signaling.
Importantly, the sarcolemma is malleable, responsive to dietary
modification, physical activity and other interventions. A
further unraveling of the roles of sarcolemmal changes in
DM and its cardiac complications thus has potential to
inform approaches to managing these disorders,
improving ischemic tolerance and developing cardioprotective
therapies for the DM population. This requires further
focused investigation of sarcolemmal changes in animal
models and particularly in sufferers of T1 and T2DM,
though the latter presents a significant experimental
Drafted manuscript: JR, JPH. Critical analysis: EFD, JNP, HHP. Approval of final
manuscript: JR, JPH, EFD, JNP, HHP. All authors read and approved the final
1 Menzies Health Institute Queensland, Griffith University, Southport, QLD,
Australia. 2 VA San Diego Healthcare System and Department of
Anesthesiology, University of California San Diego, San Diego, USA. 3 School of Medical
Science, Griffith University, Southport, QLD 4217, Australia.
The authors declare that they have no competing interests.
Availability of data and materials
Ethics approval and consent to participate
JR was supported by a doctoral scholarship award from the Australian
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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