Stop the beat to see the rhythm: excitation-contraction uncoupling in cardiac research

Abstract

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite the many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. Compared to other uncoupling agents, blebbistatin is the most frequently used as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue.

Drivers of Sinoatrial Node Automaticity in Zebrafish: Comparison With Mechanisms of Mammalian Pacemaker Function

“…In isolated rabbit hearts, right ventricular and atrial tissue, and in rat ventricular myocytes, it was first shown that blebbistatin has no effect on SAN activity or on cardiomyocyte APD ( Fedorov et al, 2007 ). Since that time, studies using rabbit hearts have confirmed a lack of an effect of blebbistatin on APD, while later studies using rabbit and pig hearts have demonstrated an increase ( Swift et al, 2021 ). The increase in APD that we observed in the zebrafish atrium agrees with these later findings.…”

“…The increase in APD that we observed in the zebrafish atrium agrees with these later findings. It is unclear whether this increase in APD is due to non-specific interactions of blebbistatin with cardiac ion channels and/or transporters or is instead a secondary effect of the loss of contraction caused by excitation-contraction uncoupling, for instance due to a loss of mechano-electric coupling effects or to a decrease in sarcolemmal ATP-sensitive K + channel current caused by a decrease in metabolic demand ( Swift et al, 2021 ). The other electrophysiological findings in our study (decreased DD slope in the SAN and increased APD in the ventricle) suggest there may be direct effects of blebbistatin on ion channels and/or transporters, warranting further investigations to determine the specific mechanism(s) involved.…”

“…, conduction, refractoriness, APD restitution, arrhythmia threshold), as well as Ca 2+ handling ( e.g. , cytosolic Ca 2+ levels, Ca 2+ transient amplitude and duration), important for the function of the SAN and working myocardium ( Swift et al, 2021 ), which deserve consideration in future studies using zebrafish.…”

“…The other electrophysiological findings in our study (decreased DD slope in the SAN and increased APD in the ventricle) suggest there may be direct effects of blebbistatin on ion channels and/or transporters, warranting further investigations to determine the specific mechanism(s) involved. It is worth noting that blebbistatin may also affect other aspects of cardiac electrophysiology (e.g., conduction, refractoriness, APD restitution, arrhythmia threshold), as well as Ca 2+ handling (e.g., cytosolic Ca 2+ levels, Ca 2+ transient amplitude and duration), important for the function of the SAN and working myocardium (Swift et al, 2021), which deserve consideration in future studies using zebrafish.…”

Real-Time Optical Mapping of Contracting Cardiac Tissues With GPU-Accelerated Numerical Motion Tracking

“…Motion artifacts arise quickly during optical mapping even with the slightest motion if the measurements are performed in an ordinary fashion: extracting and analyzing the optical signals pixel by pixel without numerical motion tracking ( 15 , 16 ). As a result, over the past 30 years, the motion has been avoided at all costs in optical mapping studies ( 17 ). To overcome this limitation, optical mapping has more recently been combined with numerical motion tracking and stabilization techniques to track and measure fluorescence on the deforming tissue surface in a co-moving reference frame, which effectively inhibits motion artifacts ( 6 , 16 , 18 – 20 ).…”