Cardiac Excitation-contraction Research Articles

Activation of G Protein-Coupled Estrogen Receptor (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway.

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-β-estradiol (E2, 10 nM) or G-1 (1 μM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 μM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

Activation of the cannabinoid type 2 (CB2) receptor improves cardiac contractile performance in fish, Brycon amazonicus

In addition to their well-known classical effects, cannabinoid CB1 and CB2 receptors have also been involvement in both deleterious and protective actions on the heart under various pathological conditions. While the potential therapeutic applications of the endocannabinoid system in the context of cardiovascular function are indeed a viable prospect, significant debate exists within the literature regarding whether CB1, CB2, or a combination of both receptors exert a favorable influence on cardiac function. Hence, the aim of this study was to investigate the effects of CB1 + CB2 or CB2 agonists on cardiac excitation-contraction (E-C) coupling, utilizing fish (Brycon amazonicus) as an experimental model. The CB2 agonist elicited marked positive inotropic and lusitropic responses in isolated ventricular myocardium, induced cyclic adenosine 3′,5′-monophosphate (cAMP) production, and upregulated critical Ca2+ handling proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Our current study demonstrated, for the first time, that CB2 receptor activation-induced effects improved the efficiency of Ca2+ cycling, excitation-contraction coupling (E-C coupling), and cardiac performance in under physiological conditions. Hence, CB2 receptors could be considered a potential therapeutic target for modulating cardiac contractile dysfunctions.

Abstract P2067: Loss Of Cardiomyocyte β2 Spectrin Promotes Cardiac Dyad Dysfunction And Accelerated Heart Failure

Background: The cardiac dyad is composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and its associated calcium (Ca 2+ ) release units, which are integral for cardiac excitation-contraction (E-C). Proper E-C coupling requires Ca 2+ influx via L-type Ca 2+ channels (LTCC) which triggers Ca 2+ release from adjacent ryanodine receptor 2 (RyR2) receptors located in jSR. Loss of the βII-spectrin scaffolding protein alters RyR2 localization; however, its contribution to cardiac dyad organization and E-C coupling remains unclear. Hypothesis: We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control (βII-flox) mice were generated and hearts were harvested for immunoblotting, qRT-PCR, and transmission electron microscopy (TEM). Cardiomyocytes were isolated and stained using proximity ligation assay (PLA) and visualized by confocal microscopy. To assess heart response to pressure overload, control and βII-cKO mice were subjected to transaortic constriction surgery followed by treatment with or without, diltiazem and monitored by echocardiography, and electrocardiography. Results: Cardiac dyad structure and components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKO hearts showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR and immunoblotting analysis revealed altered expression of dyad mRNAs and proteins. Preliminary PLA demonstrated reduced density of LTCC-RyR2 complexes. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF), which can be rescued by treatment with diltiazem. Conclusions: We demonstrate that βII-spectrin is required for cardiac dyad integrity, and that βII-spectrin-mediated HF can be attenuated by diltiazem.

Abstract 13200: Loss of Cardiomyocyte β2 Spectrin Promotes Cardiac Dyad Dysfunction and Accelerated Heart Failure

Background: The cardiac dyad and associated calcium (Ca 2+ ) release units (CRUs) are integral for cardiac excitation-contraction (E-C) coupling. The cardiac dyad is a multicomponent complex composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and CRU-associated proteins. Proper E-C coupling requires Ca 2+ influx via long-lasting-L-type Ca 2+ channels (Cav1.2), which triggers Ca 2+ release from adjacent ryanodine receptor 2 (RyR2) located in jSR. βII-spectrin loss alters RyR2 localization, but its contribution to cardiac dyad organization and E-C coupling remains unclear. Hypothesis: We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control mice (βII-flox) were generated and harvested for immunoblotting, qRT-PCR, and imaging by transmission electron microscopy (TEM). Mice hearts were also interrogated by echocardiography following transaortic constriction. Results: Cardiac dyad structure and CRU components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKOhearts by TEM showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR showed increased expression of calsequestrin 1 and 2, decreased phospholambam, Ncx and Cacna1c (major Cav1.2 subunit), while Ryr2 , Jph2 , Serca2a are unchanged. Protein expression of NCX was decreased, while expression of CAV1.2, RYR2, CASQ2, and SERCA2A remained unchanged. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF). Conclusions: This is the first demonstration that the βII-spectrin cytoskeletal protein is critically important for maintaining the structural integrity of the cardiac dyad. βII-spectrin loss and the resulting alterations in CRU structure, genes, and protein composition promoting accelerated HF.

Biological noise is a key determinant of the reproducibility and adaptability of cardiac pacemaking and EC coupling.

Each heartbeat begins with the generation of an action potential in pacemaking cells in the sinoatrial node. This signal triggers contraction of cardiac muscle through a process termed excitation-contraction (EC) coupling. EC coupling is initiated in dyadic structures of cardiac myocytes, where ryanodine receptors in the junctional sarcoplasmic reticulum come into close apposition with clusters of CaV1.2 channels in invaginations of the sarcolemma. Cooperative activation of CaV1.2 channels within these clusters causes a local increase in intracellular Ca2+ that activates the juxtaposed ryanodine receptors. A salient feature of healthy cardiac function is the reliable and precise beat-to-beat pacemaking and amplitude of Ca2+ transients during EC coupling. In this review, we discuss recent discoveries suggesting that the exquisite reproducibility of this system emerges, paradoxically, from high variability at subcellular, cellular, and network levels. This variability is attributable to stochastic fluctuations in ion channel trafficking, clustering, and gating, as well as dyadic structure, which increase intracellular Ca2+ variance during EC coupling. Although the effects of these large, local fluctuations in function and organization are sometimes negligible at the macroscopic level owing to spatial-temporal summation within and across cells in the tissue, recent work suggests that the "noisiness" of these intracellular Ca2+ events may either enhance or counterintuitively reduce variability in a context-dependent manner. Indeed, these noisy events may represent distinct regulatory features in the tuning of cardiac contractility. Collectively, these observations support the importance of incorporating experimentally determined values of Ca2+ variance in all EC coupling models. The high reproducibility of cardiac contraction is a paradoxical outcome of high Ca2+ signaling variability at subcellular, cellular, and network levels caused by stochastic fluctuations in multiple processes in time and space. This underlying stochasticity, which counterintuitively manifests as reliable, consistent Ca2+ transients during EC coupling, also allows for rapid changes in cardiac rhythmicity and contractility in health and disease.

Cardiac Alternans Occurs through the Synergy of Voltage- and Calcium-Dependent Mechanisms.

Cardiac alternans is characterized by alternating weak and strong beats of the heart. This signaling at the cellular level may appear as alternating long and short action potentials (APs) that occur in synchrony with alternating large and small calcium transients, respectively. Previous studies have suggested that alternans manifests itself through either a voltage dependent mechanism based upon action potential restitution or as a calcium dependent mechanism based on refractoriness of calcium release. We use a novel model of cardiac excitation-contraction (EC) coupling in the rat ventricular myocyte that includes 20,000 calcium release units (CRU) each with 49 ryanodine receptors (RyR2s) and 7 L-type calcium channels that are all stochastically gated. The model suggests that at the cellular level in the case of alternans produced by rapid pacing, the mechanism requires a synergy of voltage- and calcium-dependent mechanisms. The rapid pacing reduces AP duration and magnitude reducing the number of L-type calcium channels activating individual CRUs during each AP and thus increases the population of CRUs that can be recruited stochastically. Elevated myoplasmic and sarcoplasmic reticulum (SR) calcium, [Ca2+]myo and [Ca2+]SR respectively, increases ryanodine receptor open probability (Po) according to our model used in this simulation and this increased the probability of activating additional CRUs. A CRU that opens in one beat is less likely to open the subsequent beat due to refractoriness caused by incomplete refilling of the junctional sarcoplasmic reticulum (jSR). Furthermore, the model includes estimates of changes in Na+ fluxes and [Na+]i and thus provides insight into how changes in electrical activity, [Na+]i and sodium-calcium exchanger activity can modulate alternans. The model thus tracks critical elements that can account for rate-dependent changes in [Na+]i and [Ca2+]myo and how they contribute to the generation of Ca2+ signaling alternans in the heart.

Computer-based virtual laboratory simulations: LabHEART cardiac physiology practical.

Practical demonstration of cardiomyocyte function requires substantial preparation, a source of freshly isolated animal hearts, and specialized equipment. Even where such resources are available, it is not conducive for demonstration to any more than a few students at a time. These approaches are also not consistent with the 3R principle (replacement, reduction, and refinement) of ethical use of animals. We present an implementation of the LabHEART software, developed by Donald Bers and Jose Puglisi, for medical students. Prior to the activity, students had lectures covering the physiological and pharmacological aspects of cardiac excitation-contraction (EC) coupling. We used this problem-based activity to help students consolidate their knowledge and to allow a hands-on approach to explore the key features of EC coupling. Students simulate and measure action potentials, intracellular calcium changes, and cardiomyocyte contraction. They also apply drugs that target ion channels (e.g., nifedipine or tetrodotoxin) or sympathetic input (using isoproterenol) and explore changes to EC coupling. Furthermore, by modifying the biophysical parameters of key ion channels involved in the electrical activity of the heart, students also explore the effect of channelopathies such as long QT syndromes. We describe approaches to implement this activity in a flipped classroom format, with recorded lecture materials provided ahead of the practical to facilitate active learning. We also describe our experiences implementing this activity online. The content and difficulty of the activity can be altered to suit individual courses and is also amenable to promote peer-driven learning.

The architecture and function of cardiac dyads.

Cardiac excitation-contraction (EC) coupling, which links plasma membrane depolarization to activation of cardiomyocyte contraction, occurs at dyads, the nanoscopic microdomains formed by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). In a dyadic junction, EC coupling occurs through Ca2+-induced Ca2+ release. Membrane depolarization opens voltage-gated L-type Ca2+ channels (LTCCs) in the T-tubule. The resulting influx of extracellular Ca2+ into the dyadic cleft opens Ca2+ release channels known as ryanodine receptors (RYRs) in the jSR, leading to the rapid increase in cytosolic Ca2+ that triggers sarcomere contraction. The efficacy of LTCC-RYR communication greatly affects a myriad of downstream intracellular signaling events, and it is controlled by many factors, including T-tubule and jSR structure, spatial distribution of ion channels, and regulatory proteins that closely regulate the activities of channels within dyads. Alterations in dyad architecture and/or channel activity are seen in many types of heart disease. This review will focus on the current knowledge regarding cardiac dyad structure and function, their alterations in heart failure, and new approaches to study the composition and function of dyads.

Insights From Computational Modeling Into the Contribution of Mechano-Calcium Feedback on the Cardiac End-Systolic Force-Length Relationship.

In experimental studies on cardiac tissue, the end-systolic force-length relation (ESFLR) has been shown to depend on the mode of contraction: isometric or isotonic. The isometric ESFLR is derived from isometric contractions spanning a range of muscle lengths while the isotonic ESFLR is derived from shortening contractions across a range of afterloads. The ESFLR of isotonic contractions consistently lies below its isometric counterpart. Despite the passing of over a hundred years since the first insight by Otto Frank, the mechanism(s) underlying this protocol-dependent difference in the ESFLR remain incompletely explained. Here, we investigate the role of mechano-calcium feedback in accounting for the difference between these two ESFLRs. Previous studies have compared the dynamics of isotonic contractions to those of a single isometric contraction at a length that produces maximum force, without considering isometric contractions at shorter muscle lengths. We used a mathematical model of cardiac excitation-contraction to simulate isometric and force-length work-loop contractions (the latter being the 1D equivalent of the whole-heart pressure-volume loop), and compared Ca2+ transients produced under equivalent force conditions. We found that the duration of the simulated Ca2+ transient increases with decreasing sarcomere length for isometric contractions, and increases with decreasing afterload for work-loop contractions. At any given force, the Ca2+ transient for an isometric contraction is wider than that during a work-loop contraction. By driving simulated work-loops with wider Ca2+ transients generated from isometric contractions, we show that the duration of muscle shortening was prolonged, thereby shifting the work-loop ESFLR toward the isometric ESFLR. These observations are explained by an increase in the rate of binding of Ca2+ to troponin-C with increasing force. However, the leftward shift of the work-loop ESFLR does not superimpose on the isometric ESFLR, leading us to conclude that while mechano-calcium feedback does indeed contribute to the difference between the two ESFLRs, it does not completely account for it.

OCT3 gated intracellular β1AR activation enhances excitation‐contraction coupling in heart

Rationaleβ1AR‐adrenergic receptor (β1AR) plays a central role in sympathetic regulation of cardiac excitation‐contraction (EC) coupling. Stimulation of β1AR by catecholamines enhances cardiomyocyte contraction and Ca2+transient via the cAMP‐protein kinase A (PKA) pathway. Organic cation transporter 3 (OCT3) plays a role in cleaning up the extracellular catecholamines and transporting a portion of catecholamine into myocardium. Recent progress found that β1AR is also present at intracellular membranes. However, how the intracellular β1AR was activated, and the role of OCT3 in regulating intracellular β1AR signaling and EC‐coupling remains unknown.ObjectiveTo characterize the presence and activation of intracellular β1AR‐adrenergic receptors and their impact on cardiac EC‐couplingMethods and ResultsWe used a genetic deletion of OCT3 to inhibit the entrance of catecholamine into the cells. We used isolated myocytes to measure β1AR‐induced contractility and Ca2+transients. We used PKA activity reporters to detect local activation via intracellular β1AR. We found that catecholamine‐induced inotropic effects were diminished in OCT3‐knock out hearts. OCT3 deficiency significantly attenuated norepinephrine (NE) induced PKA activities at the sarcoplasmic reticulum, myocyte contractility, and Ca2+transient. Additional data show β1AR associates with sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) at the sarcoplasmic reticulum (SR). Inhibition of OCT3 blocked the transportation of NE, and attenuated β1AR‐induced PKA activity at the SR and EC‐coupling.ConclusionOur finding demonstrates a previously unrecognized intracellular β1AR‐mediated local PKA activity at SR that is gated by OCT3. Upon catecholamine stimulation, this SR‐localized β1AR signal plays a critical role in promoting E‐C coupling and cardiac contractile function. Thus, internal β1AR signaling may presents a novel therapeutic target in cardiac diseases treatment.Support or Funding InformationNIH HL113413VABX009200

Mechano-electric and mechano-chemo-transduction in cardiomyocytes.

Cardiac excitation-contraction (E-C) coupling is influenced by (at least) three dynamic systems that couple and feedback to one another (see Abstract Figure). Here we review the mechanical effects on cardiomyocytes that include mechano-electro-transduction (commonly referred to as mechano-electric coupling, MEC) and mechano-chemo-transduction (MCT) mechanisms at cell and molecular levels which couple to Ca2+ -electro and E-C coupling reviewed elsewhere. These feedback loops from muscle contraction and mechano-transduction to the Ca2+ homeodynamics and to the electrical excitation are essential for understanding the E-C coupling dynamic system and arrhythmogenesis in mechanically loaded hearts. This white paper comprises two parts, each reflecting key aspects from the 2018 UC Davis symposium: MEC (how mechanical load influences electrical dynamics) and MCT (how mechanical load alters cell signalling and Ca2+ dynamics). Of course, such separation is artificial since Ca2+ dynamics profoundly affect ion channels and electrogenic transporters and vice versa. In time, these dynamic systems and their interactions must become fully integrated, and that should be a goal for a comprehensive understanding of how mechanical load influences cell signalling, Ca2+ homeodynamics and electrical dynamics. In this white paper we emphasize current understanding, consensus, controversies and the pressing issues for future investigations. Space constraints make it impossible to cover all relevant articles in the field, so we will focus on the topics discussed at the symposium.

Abstract 192: Sex Dependent Differences in Cardiac Myocyte Excitation-Contraction Coupling After Chronic Stimulation of the Delta or Kappa Opioid Receptors During Normoxic and Hypoxic Conditions

Introduction: In 2016 the US government declared opioid misuse a public emergency. While most opioid related deaths can be attributed to overdose, recent data suggest a link between opioid use and cardiovascular disease. Further, epidemiological data suggest it may be sex dependent, with females (♀) being at a somewhat higher risk than males (♂). This is paradoxical because until menopause, ♀ have a lower risk of heart disease when compared to ♂. Healthy adult hearts express the δ and κ ORs, but not the μOR. Therefore, the overall goals of this study using a reductionist model were to: 1). To determine if there are sex specific differences in cardiac myocyte excitation-contraction coupling between ♂ and ♀ myocytes exposed to δ or κ OR agonists during normoxia/hypoxia. 2). Examine if 17β-Estradiol enhances or augments the effects of δ or κ OR stimulation. Methods: Myocytes were isolated from adult ♂ and ♀ Sprague-Dawley rats and cultured overnight in M199 HEPES media, or M199 HEPES media containing either 1 μM DADLE (δ agonist), 20 μM U50-488 (κ agonist), 100 nm 17β-Estradiol, 1μM DADLE + 100 nm 17β-Estradiol, or 20 μM U50-488 + 100 nm 17β-Estradiol. To simulate an infarct, a subset of the myocytes was incubated at 1%O 2 , 5% CO 2 for 4 hours + 10 nM isoproterenol followed by 60 min normoxia (21%O 2 , 5% CO 2 ) before recordings. Excitation-Contraction was measured using an IonOptix rig (IonOptix LLC, Milton MA) at 36±1° at 1 Hz pacing. Results: During normoxia, ♂ myocytes compared to ♀ myocytes had an increased peak contraction and peak Ca 2+ transient. The addition of 100 nM 17β-Estradiol did not affect peak contraction in either sex but decreased the Ca 2+ transient in ♂ myocytes while increasing it in ♀ myocytes. DADLE and U50-488 had no effect on ♀ myocyte contractility but decreased ♂ myocyte contractility by a minimum of 13% and a maximum of 26% with/without 100 nM 17β-Estradiol. ♀ myocytes were immune to the effects of hypoxia, even exhibiting a slight increase in their peak calcium compared to control conditions, arguably because of the addition of isoproterenol. ♂ myocytes on the other hand, had an overall decrease in excitation-contraction coupling during hypoxia which was made worse by OR stimulation. Conclusion: ♀ myocytes are immune to the detrimental effects of δ and κ OR stimulation.

Double Regulation of Cardiac Excitation-Contraction Coupling and Oxidant Stress by Pirfenidone

Pirfenidone (PFD) is well-known for improving cardiac performance in models of disease. Thus, here we have investigated its functional effects on cardiomyocytes. Cells were cultured under control culture conditions or the presence of PFD (1 mM, 1-2d). Subsequently, they were subjected to electrical stimulation to examine the levels of intracellular Ca2+ and contractility. Remarkably, PFD improved both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were also enhanced. Excitation-contraction coupling (ECC) was also investigated, under whole-cell patch-clamp. In keeping with a previous report, PFD elevated twofold the activity of CaV1.2. Besides, a similar increase in the magnitude of Ca2+ transients was also observed. Thus the gain of ECC was unaltered. Likewise, PFD did not alter the caffeine-sensitive Ca2+ store, indicating stimulation of Ca2+-induced Ca2+-release at constant sarcoplasmic reticulum Ca2+ load. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal during caffeine applications was 50% slower in PFD-treated cells (compared with controls), indicating that PFD downregulates the NCX activity. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidant agents (acetaldehyde and peroxide hydrogen). Conversely, the amount of nitric oxide (NO) was either, increased (in atrial myocytes) or unchanged (in ventricular myocytes). Western-blot analysis showed that endothelial nitric oxide synthase expression remains unaffected, while a dual regulation was observed for that of neuronal isoform (nNOS) which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. Thus, in atrial cardiomyocytes, an up-regulation of nNOS was enough to augment the synthesis of NO. Apart from improving our knowledge of the molecular mechanisms of PFD action, these findings may also help in explaining the corresponding cardioprotective effects.

Expression of calcium channel transcripts in the zebrafish heart: dominance of T-type channels.

Calcium channels are necessary for cardiac excitation-contraction (E-C) coupling, but Ca2+ channel composition of fish hearts is still largely unknown. To this end, we determined transcript expression of Ca2+ channels in the heart of zebrafish (Danio rerio), a popular model species. Altogether, 18 Ca2+ channel α-subunit genes were expressed in both atrium and ventricle. Transcripts for 7 L-type (Cav1.1a, Cav1.1b, Cav1.2, Cav1.3a, Cav1.3b, Cav1.4a, Cav1.4b), 5 T-type (Cav3.1, Cav3.2a, Cav3.2b, Cav3.3a, Cav3.3b) and 6 P/Q-, N- and R-type (Cav2.1a, Cav2.1b, Cav2.2a, Cav2.2b, Cav2.3a, Cav2.3b) Ca2+ channels were expressed. In the ventricle, T-type channels formed 54.9%, L-type channels 41.1% and P/Q-, N- and R-type channels 4.0% of the Ca2+ channel transcripts. In the atrium, the relative expression of T-type and L-type Ca2+ channel transcripts was 64.1% and 33.8%, respectively (others accounted for 2.1%). Thus, at the transcript level, T-type Ca2+ channels are prevalent in zebrafish atrium and ventricle. At the functional level, peak densities of ventricular T-type (ICaT) and L-type (ICaL) Ca2+ current were 6.3±0.8 and 7.7±0.8 pA pF-1, respectively. ICaT mediated a sizeable sarcolemmal Ca2+ influx into ventricular myocytes: the increment in total cellular Ca2+ content via ICaT was 41.2±7.3 µmoll-1, which was 31.7% of the combined Ca2+ influx (129 µmoll-1) via ICaT and ICaL (88.5±20.5 µmoll-1). The diversity of expressed Ca2+ channel genes in zebrafish heart is high, but dominated by the members of the T-type subfamily. The large ventricular ICaT is likely to play a significant role in E-C coupling.

Potassium currents in the heart: functional roles in repolarization, arrhythmia and therapeutics.

This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias (3-4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was 'K+ channels and regulation', and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen-Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state-of-the-art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.

Potassium channels in the heart: structure, function and regulation.

This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was 'K+ Channels and Regulation'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K+ channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage-gated K+ channel function, as well as the mechanisms involved in regulation of K+ channel gating, expression and membrane localization. Given the critical role for K+ channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K+ channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K+ channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K+ channels in cardiac electrophysiology, i.e. how K+ currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K+ channel-based therapeutics. A fundamental understanding of K+ channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.

Insight towards the identification of cytosolic Ca2+ -binding sites in ryanodine receptors from skeletal and cardiac muscle.

Ca2+ plays a critical role in several processes involved in skeletal and cardiac muscle contraction. One key step in cardiac excitation-contraction (E-C) coupling is the activation of the cardiac ryanodine receptor (RYR2) by cytosolic Ca2+ elevations. Although this process is not critical for skeletal E-C coupling, the activation and inhibition of the skeletal ryanodine receptor (RYR1) seem to be important for overall muscle function. The RYR1 and RYR2 channels fall within the large category of Ca2+ -binding proteins that harbour highly selective Ca2+ -binding sites to receive and translate the various Ca2+ signals into specific functional responses. However, little is known about the precise localization of these sites within the cytosolic assembly of both RYR isoforms, although several experimental lines of evidence have highlighted their EF-hand nature. EF-hand proteins share a common helix-loop-helix structural motif with highly conserved residues involved in Ca2+ coordination. The first step in predicting EF-hand positive regions is to compare the primary protein structure with the EF-hand motif by employing available bioinformatics tools. Although this simple method narrows down search regions, it does not provide solid evidence regarding which regions bind Ca2+ in both RYR isoforms. In this review, we seek to highlight the key findings and experimental approaches that should strengthen our future efforts to identify the cytosolic Ca2+ -binding sites responsible for activation and inhibition in the RYR1 channel, as much less work has been conducted on the RYR2 channel.

In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation–contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling.

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca(2+) sensitivity affect intracellular Ca(2+) homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na(+), Ca(2+) and K(+) channels and the Na(+)-Ca(2+) exchanger) and intracellular Ca(2+) handling proteins (e.g., ryanodine receptors and Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca(2+) changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca(2+) sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca(2+) sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca(2+) level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca(2+) sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca(2+) sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca(2+) handling, and myofilament Ca(2+) sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Simultaneous High-Precision Imaging of Local Calcium and Single Sarcomere Length in Rat Neonatal Cardiomyocytes via Expression of Yellow Cameleon-Nano140 in Z-Discs

In the present study, we developed a novel experimental system for simultaneous measurement of changes in the intracellular Ca2+ concentration ([Ca2+]i) at local areas of the T-tubules / Z-discs and single sarcomere length (SL) via expression of FRET-based Ca2+ indicator Yellow Cameleon (YC)-Nano140 in Z-discs (α-actinin) in rat neonatal cardiomyocytes. Under dual-view fluorescence microscopy, local [Ca2+]i was determined by the YFP/CFP fluorescence ratio and SL by the peak-to-peak distance of the YFP fluorescence profile (see Shintani et al., J Gen Physiol 2014 for SL nanometry: S.D., 12 nm in the present study). During spontaneous beating, although [Ca2+]i changed uniformly in a localized area (5 consecutive sarcomeres) of a myocyte, sarcomere shortening / lengthening was not synchronized, but rather occurred in an asynchronous fashion, suggesting that the length of each sarcomere is determined via tug-of-war forces of sarcomeres along the myofibril. Likewise, we successfully quantified amplifications of systolic [Ca2+]i and single sarcomere contraction upon β-adrenergic stimulation. Furthermore, α-actinin-YC-Nano140 enabled high-precision simultaneous measurements of local [Ca2+]i and single sarcomere contraction under electric field stimulation at 5 Hz. The present experimental system has a broad range of application possibilities for analyzing cardiac excitation-contraction (E-C) coupling in detail at the single sarcomere level. Here, we discuss 1) the technical aspects of the application of YC-Nano140 for the analysis of cardiac E-C coupling, and 2) mechanistic implications of single sarcomere dynamics in relation to local [Ca2+]i changes.