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

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 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. When compared with 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

Exposure to Secondhand Smoke and Arrhythmogenic Cardiac Alternans in a Mouse Model.

BackgroundEpidemiological evidence suggests that a majority of deaths attributed to secondhand smoke (SHS) exposure are cardiovascular related. However, to our knowledge, the impact of SHS on cardiac electrophysiology, [Formula: see text] handling, and arrhythmia risk has not been studied.ObjectivesThe purpose of this study was to investigate the impact of an environmentally relevant concentration of SHS on cardiac electrophysiology and indicators of arrhythmia.MethodsMale C57BL/6 mice were exposed to SHS [total suspended particles (THS): [Formula: see text], nicotine: [Formula: see text], carbon monoxide: [Formula: see text], or filtered air (FA) for 4, 8, or 12 wk ([Formula: see text]]. Hearts were excised and Langendorff perfused for dual optical mapping with voltage- and [Formula: see text]-sensitive dyes.ResultsAt slow pacing rates, SHS exposure did not alter baseline electrophysiological parameters. With increasing pacing frequency, action potential duration (APD), and intracellular [Formula: see text] alternans magnitude progressively increased in all groups. At 4 and 8 wk, there were no statistical differences in APD or [Formula: see text] alternans magnitude between SHS and FA groups. At 12 wk, both APD and [Formula: see text] alternans magnitude were significantly increased in the SHS compared to FA group ([Formula: see text]). SHS exposure did not impact the time constant of [Formula: see text] transient decay ([Formula: see text]) at any exposure time point. At 12 wk exposure, the recovery of [Formula: see text] transient amplitude with premature stimuli was slightly (but nonsignificantly) delayed in SHS compared to FA hearts, suggesting that [Formula: see text] release via ryanodine receptors may be impaired.ConclusionsIn male mice, chronic exposure to SHS at levels relevant to social situations in humans increased their susceptibility to cardiac alternans, a known precursor to ventricular arrhythmia. https://doi.org/10.1289/EHP3664

β-adrenergic inhibition prevents action potential and calcium handling changes during regional myocardial ischemia

β-adrenergic receptor (β-AR) blockers may be administered during acute myocardial infarction (MI), as they reduce energy demand through negative chronotropic and inotropic effects and prevent ischemia-induced arrhythmogenesis. However, the direct effects of β-AR blockers on ventricular electrophysiology and intracellular Ca2+ handling during ischemia remain unknown. Using optical mapping of transmembrane potential (with RH237) and sarcoplasmic reticulum (SR) Ca2+ (with the low-affinity indicator Fluo-5N AM), the effects of 15 min of regional ischemia were assessed in isolated rabbit hearts (n = 19). The impact of β-AR inhibition on isolated hearts was assessed by pre-treatment with 100 nM propranolol (Prop) prior to ischemia (n = 7). To control for chronotropy and inotropy, hearts were continuously paced at 3.3 Hz and contraction was inhibited with 20 μM blebbistatin. Untreated ischemic hearts displayed prototypical shortening of action potential duration (APD80) in the ischemic zone (IZ) compared to the non-ischemic zone (NI) at 10 and 15 min ischemia, whereas APD shortening was prevented with Prop. Untreated ischemic hearts also displayed significant changes in SR Ca2+ handling in the IZ, including prolongation of SR Ca2+ reuptake and SR Ca2+ alternans, which were prevented with Prop pre-treatment. At 5 min ischemia, Prop pre-treated hearts also showed larger SR Ca2+ release amplitude in the IZ compared to untreated hearts. These results suggest that even when controlling for chronotropic and inotropic effects, β-AR inhibition has a favorable effect during acute regional ischemia via direct effects on APD and Ca2+ handling

Functional Differences in Engineered Myocardium from Embryonic Stem Cell-Derived versus Neonatal Cardiomyocytes

Stem cell-derived cardiomyocytes represent unique tools for cell- and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated. We reasoned that physiological metrics of engineered cardiac tissues offer a means of comparison. We built laminar myocardium engineered from cardiomyocytes that were differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Tissues assembled from progenitor-derived myocytes and neonate myocytes demonstrated similar cytoskeletal architectures but different gap junction organization and electromechanical properties. Progenitor-derived myocardium had significantly less contractile stress and slower longitudinal conduction velocity than neonate-derived myocardium, indicating that the developmental state of the cardiomyocytes affects the electromechanical function of the resultant engineered tissue. These data suggest a need to establish performance metrics for future stem cell applications

A tissue-engineered jellyfish with biomimetic propulsion

Reverse engineering of biological form and function requires hierarchical design over several orders of space and time. Recent advances in the mechanistic understanding of biosynthetic compound materials, computer-aided design approaches in molecular synthetic biology and traditional soft robotics, and increasing aptitude in generating structural and chemical microenvironments that promote cellular self-organization have enhanced the ability to recapitulate such hierarchical architecture in engineered biological systems. Here we combined these capabilities in a systematic design strategy to reverse engineer a muscular pump. We report the construction of a freely swimming jellyfish from chemically dissociated rat tissue and silicone polymer as a proof of concept. The constructs, termed 'medusoids', were designed with computer simulations and experiments to match key determinants of jellyfish propulsion and feeding performance by quantitatively mimicking structural design, stroke kinematics and animal-fluid interactions. The combination of the engineering design algorithm with quantitative benchmarks of physiological performance suggests that our strategy is broadly applicable to reverse engineering of muscular organs or simple life forms that pump to survive

Different paths, same destination: divergent action potential responses produce conserved cardiac fight-or-flight response in mouse and rabbit hearts

Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff‐perfused rabbit and mouse hearts. Dual optical mapping with voltage‐ and Ca2+‐sensitive dyes allowed for analysis of spatio‐temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P < 0.05) followed by shortening (46.5 ± 9.1 ms at t = 60 s, P = NS vs. t = 0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by β‐adrenergic activation. Simulations with species‐specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight‐or‐flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly