Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart
et al. (2013) Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused
Mouse Heart. PLoS ONE 8(4): e60967. doi:10.1371/journal.pone.0060967
Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart
Danchen Gao 0 1
Li Zhang 0 1
Ranvir Dhillon 0 1
Ting-Ting Hong 0 1
Robin M. Shaw 0 1
Jianhua Zhu 0 1
Rajesh Mohanraj, UAE University, United Arab Emirates
0 Funding: This work was supported by National Institutes of Health [Grant R01HL094414] (RMS) (http://grants.nih.gov/grants/about_grants.htm). This work was supported by Science Technology Department of Zhejiang Province,China[2011R10069] (DCG) (http://www.zjkjt.gov.cn/html/node05/detail0502/2011/0502_ 22744.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
1 1 Department of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University , Hangzhou, Zhejiang , China , 2 Cardiovascular Research Institute, University of California San Francisco , San Francisco , California, United States of America, 3 Department of Medicine, Division of Cardiology, University of California San Francisco , San Francisco, California , United States of America
Background: Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury. Methods and Results: In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 mM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress. Conclusion: Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.
Heart failure is a major cause of morbidity and mortality in the
United States , of which diastolic heart failure (DHF) is an
important entity with rising prevalence. Of the 6 million patients
with heart failure, as many as half had diastolic dysfunction [2,3].
The one year mortality associated with hospitalization due to
diastolic dysfunction is between 22 and 29% . Myocardial
ischemia is a major contributor to DHF. Acute ischemia can result
in DHF due to rapid myocardial changes including edema,
calcium accumulation, and inflammation [5,6] and the severity of
diastolic dysfunction depends on the duration of ischemia .
Hearts subjected to chronic microvascular or untreatable chronic
ischemia also have diastolic dysfunction .
Current therapeutic approaches to DHF due to ischemia focus
on relieving the ischemia with reperfusion . Ironically,
ischemia/reperfusion (I/R) can result in direct myocardial injury
 and negatively affect diastolic function. In recent decades, the
mechanisms involved in I/R injury have started to be identified.
Cellular death and damage pathways involve subcellular
organelles such as mitochondria which are critical mediators due to their
ability to generate Adenosine triphosphate (ATP) and reactive
oxygen species (ROS). During ischemia, progressive ATP
depletion inhibits ion pump function which leads to intracellular
accumulation of calcium [10,11]. Also, reintroduction of oxygen
during reperfusion will regenerate ATP, however, it will also
damage the electron transport chain resulting in increased
mitochondrial generation of ROS [12,13]. Mitochondrial Ca2+
overload  and increased ROS result in opening of the
mitochondrial permeability transition pore (MPT) , which
initiates apoptosis and cell death by causing mitochondrial swelling
and rupture. Interestingly, inhibition of MPT is reported to reduce
infarct size .
More recently, the integrity and morphology of mitochondrial
network has been recognized as critical to cell fate. In a healthy
non-stressed intact cell, mitochondria consist of a continuous
mitochondrial reticulum, which undergoes constant fusion and
fission, two opposing processes controlled by local GTP gradients
and mitochondrial energetics . Dynamin-related GTPases
such as mitofusins (MFN1, MFN2) and the mitochondrial inner
membrane optic atrophy protein 1 (OPA1) isoforms are
profusion. Alternatively, scission requires the pro-fission multimers
containing mitochondrial fission protein 1 (FIS1), Mitochondrial
fission factor (MFF), and dynamin related protein 1 (Drp1). The
fine balance between mitochondria fusion and fission determines
cell fate . Cardiac I/R injury results in significant
mitochondrial fission, which induces apoptotic cell death. Inhibition of
mitochondrial fission mediated by Drp1 can limit infarct size in I/
R injury .
A critical yet unaddressed issue is whether mitochondrial
protection limits ischemia related diastolic dysfunction. Dynasore
is a cell-permeable small molecule that non-competitively inhibits
the GTPase activity of dynamin1, dynamin2 and Drp1 . We
found that low dose Dynasore significantly preserves lusitropy of
ex vivo perfused hearts subject to I/R injury. Dynasore also
increases cardiomyocyte survival, and decreases cellular ATP
consumption in stressed cardiomyocytes. In support of
mitochondrial protection, we found that low dose Dynasore is sufficient to
prevent oxidative stress induced mitochondrial fission in cultured
cells. Thus small molecule based Drp1 inhibition is a potential
therapeutic approach to ischemia related DHF.
Materials and Methods
Guidelines for Animal Research
This study was approved by the University of California San
Francisco Committee on the Use and Care of Animals (IACUC).
All procedures used in this study are in agreement with the
guidelines of the University of California San Francisco IACUC.
Animals were housed in UCSF facility of Laboratory Animal
Resource Center. All investigation conformed to the Guide for the
Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication no. 85-23, received
Unless otherwise stated, Dynasore (Figure 1) and all other
materials were purchased from Sigma Chemical (St. Louis, MO).
Male C6/Black mice (8,12 weeks; Charles River) were
anesthetized with isoflurane (flow 3%) and 100% O2 in an
anesthesia chamber and anti-coagulated with heparin (50 IU, i.p.).
After cervical dislocation, hearts were rapidly excised, mounted on
a Langendorff apparatus (ADInstrument, Colorado Springs, CO)
and perfused retrogradely [20,21] at a constant rate of 2.6 ml/min
with oxygenated Krebs-Henseleit buffer containing (mmol/L):
NaCl 118, NaHCO3 24, CaCL2.2H2O 2.5, KCL 4.7, KH2PO4
1.2, MgSO4-7H2O 1.2, Glucose 11, EDTA 0.5, adjusted to a pH
of 7.4 [22,23]. The apparatus was water-jacketed for temperature
control to maintain a core temperature of the heart at 37uC. The
buffer passed through a membranous lung made of SilasticTM
Medical Grade Tubing, which was gassed continuously with 95%
O2-5% CO2. Fine platinum electrodes were placed on the right
atrium and apex of the left ventricle to record the
electrocardiogram and heart rate throughout the experiment. A Millar
MIKRO-TIP catheter transducer (Millar Instruments, Houston,
TX) was inserted into the left ventricle from the left atrium to
measure left ventricular pressure. Left ventricular end diastolic
pressure (LVEDP), left ventricular end systolic pressure (LVESP)
and heart rate were monitored and recorded continuously using
PowerLab system (ADInstruments). Left ventricular developed
pressure (LVDP) was calculated by subtracting LVEDP from
LVESP. Hearts were paced at 360 bpm with bipolar electrodes
attached to the right atrium, using stimuli delivered from a
stimulator (ADInstruments Colorado Springs, CO).
After the initial 15 min stabilization, hearts were excluded from
further study if they exhibited one or more of the following
exclusion criteria: LVEDP higher than 20 mmHg; LVDP less than
50 mmHg; intrinsic heart rate less than 280 bpm or irregular; or
aortic regurgitation. The volume of the perfusate was reduced to
200 ml and allowed to recirculate. The hearts were then
randomized to one of the following two treatment groups:
Dynasore group (n = 8, added into the recirculating perfusate in
stepwise fashion to reach a final concentration of 1 mM within
120 min of recirculation) or DMSO control group (n = 8, added in
Figure 1. Cellular Targets of Dynasore. Dynasore is a specific small molecular GTPase inhibitor that targets Dynamin1 and Dynamin2 which are
responsible for pinching off endocytic vesicles, and Drp1 which is responsible for mitochondrial fission.
a similar manner of Dynasore). Hearts were then subjected to
30 min of global ischemia followed by 1 hour of reperfusion.
Pacing was initiated after stabilization except during ischemia and
was reinitiated 2 min after reperfusion.
Myocardial cTnI Release
Cardiac effluent was collected from apex at baseline, before
ischemia and during reperfusion. The samples were snap frozen
immediately in liquid nitrogen and stored at 280uC for later
analysis. Cardiac effluent samples from 5 hearts in each group
were then used to determine cardiac specific troponin I (cTnI)
concentration using a commercial cTnI ELISA kit (Life
Diagnostics, Inc., West Chester, PA).
Determination of Infarct Size
Propidium iodide (excitation, 535 nm; emission, 617 nm) was
used to determine infarct size according to a previously established
method [24,25] with modest modification. Briefly, at final 15 min
reperfusion period, 300 mg of propidium iodide (Sigma Chemical,
St. Louis, MO) was injected into the right atrium and allowed to
perfuse through the coronary vasculature. At the end of
reperfusion, hearts were then removed from the apparatus, sliced
perpendicularly to the long axis of the heart into 4,5
equalthickness transverse slices. The top and bottom surfaces of each
slice were imaged by widefield epifluorescence microscopy with a
Cy3 filter at an exposure of 500 ms per frame, and grayscale
images were captured using a sensitive CCD camera with white
pixels corresponding to PI positive signals. Total left ventricular
area and infarct area for each image were analyzed using ImageJ
software. Infarct size was calculated by total infarct area summed
from all the slices and expressed as percentage of the total left
Isolation and Culture of Adult Mouse Cardiomyocytes
Mouse ventricular myocytes were isolated from male adult C6/
Black mouse (8,12 weeks; Charles River) after dissociation with
(N = 8)
(N = 8)
(N = 8)
(N = 8)
(N = 8)
(N = 8)
*, ***indicate P,0.05, P,0.001 when compared between the two treatment groups.
LVEDP, left ventricular end diastolic pressure; LVESP, left ventricular end systolic pressure; BL, baseline.
collagenase II (2 mg/ml, Worthington, Lakewood, NJ) using a
previously described method [26,27]. After dissociation,
cardiomyocytes were plated on laminin-precoated 35 mm2 culture dishes
at a density of ,1,500/mm2 and maintained in a humidified
atmosphere of 5% CO2 at 37uC. After 1 hour of plating,
cardiomyocytes were replenished with fresh medium (serum
supplemented or depleted) and subjected to 2 hours of drug
treatment (Dynasore or vehicle) followed by oxidative stress
(30 mM H2O2 for 35 min). For ATP supplement experiments, the
cells were treated with 3 mM ATP for 30 min before exposure to
In vitro Cardiomyocyte Survival and Viability Assay
After exposure to 30 mM H2O2 for 35 min , cardiomyocyte
survival and viability were analyzed by trypan blue exclusion
(TBE) assay, which is a method to determine cell survival and
changes in cell morphology in experimental models [28,29]. In
brief, cardiomyocytes were stained with 0.04% (w/v) trypan blue
solution (Gibco, Invitrogen, Carlsbad, CA) at room temperature
for 7 min. When cell membranes are irreversibly damaged, the
anionic dye trypan blue is taken up by dead cells. Cardiomyocytes
were then visualized at 406 magnification by microscopy. For
each experiment, a total of 200 cardiomyocytes were analyzed
from 10 different fields/dish. Cells that excluded trypan blue
(TBEs) were considered to have survived. Healthy rod-shaped
myocytes (rods) were identified when the length/width ratio was
.3:1 . Contracted cells were defined when the length/width
ratio was ,3:1. Trypan blue-positive cells were identified when
the trypan blue was present irrespective of whether the cells were
rod-shaped or contracted [28,31]. Morphologic changes (viability)
were measured by determining the number of rods relative to all
TBEs (rods and contracted cells) of the 200 cardiomyocytes
analyzed. The percent survival and viability were calculated as
Total number of TBEs
Total number of myocytes(TBEs z nonTBEs)
Note total number of myocytes (TBEs+non-TBEs) = 200.
A luminescence assay (Promega, Madison, WI) was used to
quantify cardiomyocyte and Hela cell ATP content . Briefly,
after Dynasore treatment and H2O2 exposure, cardiomyocytes
were lysed and ATP content was measured in the cell lysates.
Meanwhile, in a separate set of wells following same experimental
protocol, surviving cardiomyocytes were counted using a TBE
assay. Cellular ATP per single live cardiomyocyte was then
calculated for each treatment condition. Similar procedures were
applied to cultured non-stressed Hela cells treated with control or
Live-cell Mitochondria Imaging with Spinning Disc
HeLa cells (ATCC, Manassas, VA) were maintained in DMEM
(Invitrogen) supplemented with 10% FBS (Invitrogen) and
100 mg/ml Normocin (Amaxa, Lonza Walkersville Inc,
Walkersville, MD). Cells were maintained in a humidified atmosphere of
5% CO2 at 37uC. Cells were seeded at a density of 7 6 104 cells/
cm2 and allowed to adhere overnight. Cells were then transduced
with Organelle LightsTM Mito-RFP *BacMam 1.0 (Invitrogen).
Twenty-four hours after transduction, cells were pretreated with
either control or 1 mM Dynasore for 1 hour before being exposed
to normal conditions or 200 mM H2O2 for 15 minutes. Before and
after exposure to H2O2, cells were imaged using a Nikon Ti
inverted microscope, Yokogowa CSU-X1 spinning disk confocal
unit with 568-nm DPSS laser source, and a high resolution Cool
SNAP HQ2 camera (Photometrics, Tucson, AZ). Images were
acquired at 400 ms exposure per frame and automatically
processed using a bas relief filter to highlight edges.
Statistical analysis was performed using GraphPad Prism 5. The
data are expressed as means 6 SEM. Difference between control
and experimental groups were determined using a one-way
analysis of variance (ANOVA) for multiple groups. Difference
between every two groups was determined using Bonferroni post
hoc test. For comparison between two groups with timed repeated
measurements, a two-way ANOVA (treatment and time are
considered as the two variances) was used. P,0.05 was considered
to be significant.
In Langendorff Perfused Mouse Hearts, Dynasore
Prevents Pathologic LVEDP Elevation Following I/R Injury
We investigated the effects of Dynasore on intracardiac
pressure during I/R injury. Using Langendorff perfusion, hearts
were subjected to no-flow global ischemia followed by
restoration of flow, with and without the presence of Dynasore during
the entire period. Results are in Figure 2A which contains
ventricular pressure tracings at baseline, during ischemia, and
post reperfusion from two representative hearts treated with
vehicle or 1 mM of Dynasore, which was determined as a safe,
complication free, cardiac protective dose based on a pilot study
testing the pharmacological dose response curve of Dynasore
Figure 7. Dynasore prevents oxidative stress-induced mitochondrial fission. Cultured human Hela cells were used for mitochondrial
morphology study. Top, Hela cells have elongated connected mitochondrial network (left), which was fragmented after oxidative stress (right).
Bottom, 1 mM Dynasore pretreatment prevents oxidative stress-induced mitochondrial fragmentation.
(0.110 mM). As indicated, 30 min of global ischemia caused a
significant elevation of LVEDP, which only had partial recovery
after reperfusion. In addition, global ischemia resulted in a
significantly drop of cardiac systolic function indicated by left
ventricular developed pressure (LVDP), which returned to its
pre-ischemia baseline after 60 min of reperfusion. The
concurrent increase of LVEDP with a drop of LVDP indicates that a
severe hypo-contractile state is initiated after ischemia and
persists into reperfusion. However, pretreatment with 1 mM
Dynasore prevented elevation of LVEDP without a change of
LVDP. The summarized ventricular pressure data from eight
independent experiments of each condition are presented in
Figure 2B (LVEDP), Figure 2C (LVDP) and Table 1. Note a
significant decrease of LVEDP in Dynasore treated hearts is
present during the entire ischemia and reperfusion period.
Dynasores beneficial effect on LVEDP and not LVDP indicates
it benefits lusitropy more than it does on inotropy.
In I/R Injured Mouse Hearts, Dynasore Decreases
Improved lusitropy implies less stress and ischemia related
damage to the myocardium. We investigated myocardial damage
with two separate direct assays: staining hearts for cardiomyocyte
death and measuring troponin release. Propidium iodide (PI), a
nuclear fluorescent dye, permeates through the damaged plasma
membrane of cardiomyocytes that are at the early stage of cell
death. As seen in the left panel of Figure 3A, the PI signal and
hence cell death in heart slices from non-treated hearts is
significantly higher. The PI positive area was traced in ImageJ
and presented as percentage of total ventricular area. Infarct size
calculated using this method indicates that, in control hearts,
30 min of ischemia followed by 1 hour of reperfusion results in
39.6% of PI positive infarct area. Perfusion with Dynasore
significantly reduces infarct size to 8.1% (80% reduction,
Myocardial death was further evaluated by measuring cTnI in
the cardiac efflux. As seen in Figure 3B, myocardial damage
induced by I/R injury causes cTnI release into cardiac effluent, an
event within an hour post reperfusion (clinical cTnI requires as
much as 6 hours to be detected . With Dynasore pretreatment,
the early cardiac efflux of cTnI was decreased 70% (at 30 minutes
of reperfusion), further confirming the protective effects of
Dynasore on cardiac muscle.
In Cultured Cardiomyocytes, Dynasore Increases Cell
Survival and Viability
The data in Figures 2 and 3 involved protocols of complete
no-flow ischemia with whole heart preparations. We were
interested if the protective effects of Dynasore extend to a more
traditional cellular cardiomyocyte assay with a more controlled
stress insult. Isolated adult mouse cardiomyocytes in culture
were subjected to oxidative stress by addition of hydrogen
peroxide in the presence or absence of serum. Healthy adult
cardiomyocytes have a typical elongated rod shape. However
oxidative stress significantly damage cell morphology resulting in
a well described contracted morphology . We observed
significant cardiomyocyte contraction with oxidative stress to
30 mM H2O2 (Figure 4). Note that Dynasore prevents
cardiomyocyte morphological changes induced by oxidative stress.
Cell survival and viability can further be assayed by TBE and
cell morphological changes, respectively (see Methods). As seen in
the bar graphs of Figure 4, as expected, oxidative stress causes a
significant reduction in both cell survival and viability. However
the presence of Dynasore in the culture medium results in
significantly improved survival and a major improvement in
viability. The beneficial effects of Dynasore on viability and
survival were dose dependent and are also more prominent in
serum free conditions (Figure 4B).
Dynasore Preserves Cellular ATP Content in Stressed
Dynasores beneficial effect on cell fate and the ameliorating
effect of serum (Figure 4) suggest that the mechanism of
Dynasores effect is related to energetics. This hypothesis was
tested by recording cellular ATP content in oxidative stressed
cardiomyocytes. To control for different amount of living
cardiomyocytes per culture well, net ATP content was normalized
to the amount of surviving cardiomyocytes counted per dish. As
seen in Figure 5A, as low as 1 mM dose of Dynasore increases
cellular ATP content in live cardiomyocytes. To confirm that
Dynasore induced higher levels of ATP are protective, we
performed an ATP rescue experiment and found, in Figure 5B,
that direct supplementation with exogenous ATP has similar effect
to Dyansore on cardiomyocyte viability subjected to oxidative
Next, we explored whether Dynasore simply preserves ATP
during stress or can actually generate ATP independent of a
metabolic insult. Since the process of dissociating and culturing
adult mouse cardiomyocytes is already a stress to these primary
cells, a typical cell line of Hela cells in standard cell culture
hemostasis was used in this analysis. As seen in Figure 6, low dose
Dynasore (13 mM) has no effect on cellular ATP content,
indicating that Dynasore may preserve ATP in stressed cells
rather than be responsible for ATP production. Note high dose
Dynasore (.10mM) increases cellular ATP content, possibly
indicating direct ATP production or, more likely, the energetic
benefit of limiting Dynamin GTPase dependent endocytosis at
Dynasore Inhibits Oxidative Stress-induced Mitochondrial
Given the reported inhibitory effects of Dynasore on Drp1
 and the observed effect on increasing cell survival and
viability (Figure 4) as well as preserving cellular ATP content
(Figure 5), we hypothesized that the cardioprotective effect of
Dynasore (Figure 2 and 3) is mediated by inhibition of Drp1
dependent mitochondrial fission. To test this hypothesis,
cultured cells were transduced with baculovirus expressing a
mitochondrial targeted fluorescent protein. Instead of adult
mouse cardiomyocytes, HeLa cells were used for this study
because the relatively flat HeLa cell morphology is permissive to
detailed high resolution mitochondrial imaging. The cardiac
atrial origin HL-1 cells and neonatal cardiomyocytes are not
used for this study due to their resistance to oxidative stress
, probably related to differential mitochondrial fusion-fission
dynamics. The morphology of mitochondria was examined by
spinning disc confocal microscopy before and after the exposure
to 200 mM H2O2 for 15 minutes. As seen in Figure 7, the
nonstressed mitochondria have an elongated and well-organized
reticulum network, whereas oxidative stress induces
mitochondrial fragmentation. Note the cellular morphology is also altered
after oxidative stress, resulting in contracted and smaller cells.
Consistent with the above hypothesis, pretreatment of the cells
with Dynasore prevented oxidative stress induced mitochondrial
fission (Figure 7, bottom row), retaining the original organized
We have found that the small dynamin-GTPase inhibitor
Dynasore protects mitochondria and significantly benefits cardiac
lusitropy in hearts subjected to I/R injury. The cardioprotective
effect is observed in both ex vivo perfused mouse heart preparations
and isolated cultured cardiomyocytes. Dynasore also, in dose
dependent fashion, increases cell survival in cultured primary adult
mouse cardiomyocytes exposed to oxidative stress. In the surviving
cells, Dynasore preserves cellular ATP content whereas adding
exogenous ATP provides a similar rescue in the absence of
Dynasore. Of note, the cardioprotective dose of Dynasore is
significantly lower than the dose used to effectively block
A Novel Cardiac Lusitropic Role of Dynasore
The small molecule Dynasore was identified as an endocytosis
inhibitor seven years ago . It was found to be a
noncompetitive inhibitor of the GTPase activity of dynamin1,
dynamin2, and the mitochondrial pro-fission dynamin isotype
Drp1 (Cartoon in Figure 1). By blocking plasma membrane
dynamin1 and dynamin2, Dynasore acts as a potent blocker of
dynamin-dependent coated vesicle formation, resulting in
stabilization of intermediates including U-shaped and O-shaped pits
. Since its discovery, Dynasore has been used as an effective
cardiac related endocytosis inhibitor due to its potency and limited
Since Dynasore inhibits Drp1 in vitro , it is interesting that
Dynasore significantly prevents LVEDP elevation during I/R
injury without affecting LVDP (Figure 2) indicating a novel
lusitropic effect. Diastolic dysfunction is usually associated with
prominently altered nucleotide levels  or ATP turnover and
catabolism . Therefore the beneficial lusitropic effect may be
mediated by Dynasores ability to preserve the ATP reserve in
stressed cardiomyocytes (Figures 5). Dynasore inhibits GTPase
activity at both the plasma membrane (Dynamin 1, 2) and the
mitochondria membrane (Drp1) . However, the current
lusitropic dose of Dynasore (1 mM, Figure 2) is significantly lower
than the previously reported inhibitory dose of Dynasore (IC50
,15 mM) on dynamin dependent endocytosis , indicating an
effect separate from dynamin inhibition at the plasma membrane.
The low non-endocytosis related dose of Dynasore that blocks
oxidative stress-induced mitochondrial fission (Figure 7) is most
likely due to Drp1 inhibition [18,38].
Interestingly, Dynasore improves diastolic function with acute
I/R injury, without affecting systolic function (Figure 2). It might
be that no-flow ischemia permits the accumulation of toxic
metabolites, such as low pH, which can limit inotropy independent
of myocardial energetics. However inotropy was unaffected by
Dynasore during the reperfusion period which should wash out
toxic metabolites, even while lusitropy was preserved (Figure 2).
Therefore lusitropy specificity could result from diastolic function
being a more sensitive indicator of cardiac ATP and other
mitochondrial dependent energy production.. In healthy
individuals, mild hypoxia results in diastolic dysfunction without affecting
systolic function . Diastolic sensitivity to hypoxia could be due
to a decline in high energy phosphate metabolism . Advanced
age is also a well established association with progressive diastolic
dysfunction. Hypoxia and age both result in irreversible damage to
mitochondrial genes involved in oxidative phosphorylation .
These mutations limit mitochondrial function and could
contribute to progressive diastolic dysfunction . It is possible that
rescue of oxidative phosphorylation could be beneficial to not just
acute diastolic dysfunction, but chronic diastolic dysfunction as
Mitochondrial Morphology and Cardiomyocyte Survival
In non-stressed conditions, mitochondria fusion prevails
resulting in elongated, tubular, and interconnected mitochondria
networks. During ischemia, the dynamic balance shifts from
fusion to fission, resulting in fragmented and discontinuous
mitochondria [18,43], as well as mitochondrial outer membrane
permeablization, release of apoptotic factors, and activation of
apoptosis. Apoptotic cell death is understood to be a major
contributing factor of I/R injury . Recent studies show that
alteration of mitochondrial morphology is significant in ischemic
hearts [18,43] and modulation of which can protect the heart
against I/R injury .
The apoptotic cell death that results from mitochondrial fission
and fragmentation is mediated by activation of a key
mitochondrial pro-fission protein Drp1 [38,45]. Recently, it was reported
that a dominant-negative mutant of Drp1 induces mitochondrial
elongation and pharmacological inhibition of Drp1 protects
against I/R injury in the heart . We found in human cells
that Dynasore significantly prevents stress induced mitochondrial
fragmentation and maintains a normal elongated mitochondrial
morphology (Figure 7). Drp1 is a known target of Dynasore, and it
follows that the mitochondrial protection of Dynasore is mediated
by inhibition of Drp1. Future studies could explore the effect of
Dynasore on activities of other mitochondrial fusion and fission
related regulators, such as MFN1, MFN2, OPA1, FIS1, MFF.
In conclusion, our study provides the first evidence that
Dynasore has a potent lusitropic effect during I/R injury. The
mechanism is mitochondrial protection and preservation of
oxidative phosphorylation. Pharmaceutical therapy that preserves
mitochondrial function may not just benefit myocardial survival,
but improve diastolic dysfunction as well.
We are grateful to Dr. James W Smyth for his critical scientific advice and
assistance with the cartoon images.
Designed the experiments: LZ TTH. Performed data analysis: LZ TTH
DCG. Supervised experimental design: JZ RMS. Technical support: RD.
Performed the experiments: DCG. Contributed reagents/materials/
analysis tools: JZ RMS. Wrote the paper: JZ RMS TTH DCG.
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