ARTICLE
https://doi.org/10.1038/s42003-022-03279-y
OPEN
Simultaneous triple-parametric optical mapping
of transmembrane potential, intracellular calcium
and NADH for cardiac physiology assessment
1234567890():,;
Sharon A. George
1,2,3 ✉, Zexu Lin1 & Igor R. Efimov
1,2,3 ✉
Investigation of the complex relationships and dependencies of multiple cellular processes
that govern cardiac physiology and pathophysiology requires simultaneous dynamic
assessment of multiple parameters. In this study, we introduce triple-parametric optical
mapping to simultaneously image metabolism, electrical excitation, and calcium signaling
from the same field of view and demonstrate its application in the field of drug testing and
cardiovascular research. We applied this metabolism-excitation-contraction coupling
(MECC) methodology to test the effects of blebbistatin, 4-aminopyridine and verapamil on
cardiac physiology. While blebbistatin and 4-aminopyridine alter multiple aspects of cardiac
function suggesting off-target effects, the effects of verapamil were on-target and it altered
only one of ten tested parameters. Triple-parametric optical mapping was also applied during
ischemia and reperfusion; and we identified that metabolic changes precede the effects of
ischemia on cardiac electrophysiology.
1 Department of Biomedical Engineering, The George Washington University, Washington, DC, USA. 2 Department of Biomedical Engineering, Northwestern
University, Chicago, IL, USA. 3These authors jointly supervised this work: Sharon A George, Igor R Efimov. ✉email: ;
igor.efi
[email protected]
COMMUNICATIONS BIOLOGY | (2022)5:319 | https://doi.org/10.1038/s42003-022-03279-y | www.nature.com/commsbio
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ARTICLE
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COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03279-y
ardiac physiology is profoundly complex, and the careful
coordination of numerous cell signaling pathways governs
every single heartbeat1,2. These physiological processes
that vary beat to beat and long term are usually sub-divided into
metabolic, electrical, and mechanical components governed by
metabolism-excitation-contraction coupling (MECC). Electrical
activity in the heart includes a sequence of opening and closing of
ion channels and pumps which cause cardiomyocyte depolarization
and repolarization. The resulting changes in the transmembrane
potential (Vm) in the cardiomyocytes are recorded as action
potentials using electrical or optical methods. The electrical excitation of the heart serves as the trigger of a mechanical contraction.
Excitation-contraction coupling or the translation of electrical excitation to mechanical contraction is controlled by cytosolic calcium
(Ca2+) ion concentration, which increases following electrical excitation and Ca2+ ion binds to a contractile protein in the cardiomyocyte, triggering contraction. Both the electrical and mechanical
processes of the heart require energy, which is provided in the form
of ATP generated by metabolic processes in the mitochondria. Thus,
all three components of cardiac function are intertwined into
MECC. As such, studying this complex MECC phenomenon
requires the ability to simultaneously assess these three facets of
cardiac function. In this study, we report a new approach to
simultaneously image Vm, Ca2+, and NADH (metabolic marker).
Optical mapping is a methodology that optically records cardiac
physiology with a high spatial and temporal resolution, either as
autofluorescence of endogenous biological substances or as fluorescence of specifically designed dyes3,4. Optical mapping of the heart
was first applied to record Vm and then NADH autofluorescence5,6.
Since then, optical mapping has also been applied to record Ca2+ 7.
Dual parameter optical mapping of Vm and NADH, as well as Vm
and Ca2+, have also been applied in assessing cardiac physiology8,9.
However, as described above, the three interdependent facets of
cardiac function will all need to be measured simultaneously to
develop a complete picture of cardiac physiological modulation by
drugs or disease. In this study, we report for the first time, a spatially
and temporally co-registered triple-parametric optical mapping
system that incorporates three cameras to simultaneously capture
NADH, Vm, and Ca2+ signals from the same field of view.
Preclinical safety and efficacy testing are crucial components of
the drug development process. This step is important in determining
dosing and toxicity which could include identifying off-target effects
of drugs before clinical trials and before they are approved for use in
patients. Cardiotoxicity is the primary cause (19%) of drug withdrawal from the market in the United States10 and the second
leading cause worldwide10,11, underscoring the need for efficient and
thorough cardiac methodologies of drug screening. Current preclinical drug testing primarily focuses on the effects of drugs on the
electrical activity (QT interval) and contractility of the heart10,12.
While this is an essential first step, it does not give a complete
picture of cardiac physiology modulation by drugs as it does not
consider the calcium handling or metabolic states of the cardiac
tissue. Unexpected off-target effects of the drugs being tested could
result in serious complications or fatality. In this study, we present a
novel approach to measure ten different important aspects of cardiac
MECC using triple-parametric optical mapping. We investigated the
effects of three different compounds (blebbistatin, 4-aminopyridine,
and verapamil) using triple-parametric optical mapping and present
the data in a ten-parameter panel (TPP). TPP graphs include
information on action potential upstroke, duration and conduction,
intracellular calcium release, and reuptake as well as the metabolic
state of the heart.
Triple-parametric optical mapping and TPP graphs could also
benefit the study of complex cardiac diseases such as ischemia
and reperfusion. Acute ischemic bouts are known to have multiple and severe effects on cardiac physiology. Ischemia has been
2
previously demonstrated to alter the electrical activity13–15, calcium handling16 as well as the metabolic6 functions of the heart.
However, the sequence and the interrelationship between these
three aspects of cardiac MECC have not been studied simultaneously before, due to the lack of appropriate methodology. In
this study, we also determined the simultaneous modulation of
multiple aspects of cardiac physiology by ischemia and their
restoration during reperfusion. Thus, applying triple-parametric
optical mapping to study MECC during disease progression could
provide valuable new targets for therapy.
Results
Triple-parametric optical mapping system was 3D printed and set up
as illustrated in Fig. 1a, b and the separation of signals of different
wavelengths is illustrated in Fig. 1c. All design files for 3D printed
hardware (in STL format) and data analysis software (Matlab) are
available under an open-source license at Github (https://github.c (...truncated)
