Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.

High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes.

Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity. Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 µm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell. Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules. Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure.

Changes in cellular Ca2+ and Na+ regulation during the progression towards heart failure.

In adapting to disease and loss of tissue, the heart shows great phenotypic plasticity that involves changes to its structure, composition and electrophysiology. Together with parallel whole body cardiovascular adaptations, the initial decline in cardiac function resulting from the insult is compensated. However, in the long term, the heart muscle begins to fail and patients with this condition have a very poor prognosis, with many dying from disturbances of rhythm. The surviving myocytes of these hearts gain Na+ , which is positively inotropic because of alterations to Ca2+ fluxes mediated by the Na+ /Ca2+ exchange, but compromises Ca2+ -dependent energy metabolism in mitochondria. Uptake of Ca2+ into the sarcoplasmic reticulum (SR) is reduced because of diminished function of SR Ca2+ ATPases. The result of increased Ca2+ influx and reduced SR Ca2+ uptake is an increase in the diastolic cytosolic Ca2+ concentration, which promotes spontaneous SR Ca2+ release and induces delayed afterdepolarisations. Action potential duration prolongs because of increased late Na+ current and changes in expression and function of other ion channels and transporters increasing the probability of the formation of early afterdepolarisations. There is a reduction in T-tubule density and so the normal spatial arrangements required for efficient excitation-contraction coupling are compromised and lead to temporal delays in Ca2+ release from the SR. Therefore, the structural and electrophysiological responses that occur to provide compensation do so at the expense of (1) increasing the likelihood of arrhythmogenesis; (2) activating hypertrophic, apoptotic and Ca2+ signalling pathways; and (3) decreasing the efficiency of SR Ca2+ release.

GPER limits adverse changes to Ca2+ signalling and arrhythmogenic activity in ovariectomised guinea pig cardiomyocytes.

Background: The increased risk of post-menopausal women developing abnormalities of heart function emphasises the requirement to understand the effect of declining oestrogen levels on cardiac electrophysiology and structure, and investigate possible therapeutic targets, namely the G protein-coupled oestrogen receptor 1 (GPER). Methods: Female guinea pigs underwent sham or ovariectomy (OVx) surgeries. Cardiomyocytes were isolated 150-days post-operatively. Membrane structure was assessed using di-8-ANEPPs staining and scanning ion conductance microscopy. Imunnohistochemistry (IHC) determined the localisation of oestrogen receptors. The effect of GPER activation on excitation-contraction coupling mechanisms were assessed using electrophysiological and fluorescence techniques. Downstream signalling proteins were investigated by western blot. Results: IHC staining confirmed the presence of nuclear oestrogen receptors and GPER, the latter prominently localised to the peri-nuclear region and having a clear striated pattern elsewhere in the cells. Following OVx, GPER expression increased and its activation reduced Ca2+ transient amplitude (by 40%) and sarcomere shortening (by 32%). In these cells, GPER activation reduced abnormal spontaneous Ca2+ activity, shortened action potential duration and limited drug-induced early after-depolarisation formation. Conclusion: In an animal species with comparable steroidogenesis and cardiac physiology to humans, we show the expression and localisation of all three oestrogen receptors in cardiac myocytes. We found that following oestrogen withdrawal, GPER expression increased and its activation limited arrhythmogenic behaviours in this low oestrogen state, indicating a potential cardioprotective role of this receptor in post-menopausal women.

Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation-contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand-receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine-glycine-aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode's solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine-glycine-aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

Extracellular Vesicles from Human Cardiac Fibroblasts Modulate Calcium Cycling in Human Stem Cell-Derived Cardiomyocytes.

Cardiac fibroblasts regulate the development of the adult cardiomyocyte phenotype and cardiac remodeling in disease. We investigate the role that cardiac fibroblasts-secreted extracellular vesicles (EVs) have in the modulation of cardiomyocyte Ca2+ cycling-a fundamental mechanism in cardiomyocyte function universally altered during disease. EVs collected from cultured human cardiac ventricular fibroblasts were purified by centrifugation, ultrafiltration and size-exclusion chromatography. The presence of EVs and EV markers were identified by dot blot analysis and electron microscopy. Fibroblast-conditioned media contains liposomal particles with a characteristic EV phenotype. EV markers CD9, CD63 and CD81 were highly expressed in chromatography fractions that elute earlier (Fractions 1-15), with most soluble contaminating proteins in the later fractions collected (Fractions 16-30). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with fibroblast-secreted EVs and intracellular Ca2+ transients were analyzed. Fibroblast-secreted EVs abbreviate the Ca2+ transient time to peak and time to 50% decay versus serum-free controls. Thus, EVs from human cardiac fibroblasts represent a novel mediator of human fibroblast-cardiomyocyte interaction, increasing the efficiency of hiPSC-CM Ca2+ handling.

Moving in the right direction: elucidating the mechanisms of interaction between flecainide and the cardiac ryanodine receptor.

Flecainide is used to treat catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmia caused by disrupted cellular Ca2+ handling following ß-adrenergic stimulation. The clinical efficacy of flecainide in this context involves complex effects on multiple ion channels that may be influenced by the disease state. A compelling narrative has been constructed around flecainide's nonselective block of sarcoplasmic reticulum (SR) lumen-to-cytoplasm Ca2+ release through intracellular calcium release channels (RyR2). However, ion fluxes across the SR membrane during heart contraction are bidirectional, and here, we review experimental evidence that flecainide's principal action on RyR2 involves the partial block of ion flow in the cytoplasm-to-lumen direction (i.e., flecainide inhibits RyR2-mediated SR 'countercurrent'). Experimental approaches that could advance new knowledge on the mechanism of RyR2 block by flecainide are proposed. Some impediments to progress in this area, that must be overcome to enable the development of superior drugs to treat CPVT, are also considered.

OCT2013, an ischaemia-activated antiarrhythmic prodrug, devoid of the systemic side effects of lidocaine.

BACKGROUND AND PURPOSE: Sudden cardiac death (SCD) caused by acute myocardial ischaemia and ventricular fibrillation (VF) is an unmet therapeutic need. Lidocaine suppresses ischaemia-induced VF, but its utility is limited by side effects and a narrow therapeutic index. Here, we characterise OCT2013, a putative ischaemia-activated prodrug of lidocaine. EXPERIMENTAL APPROACH: The rat Langendorff-perfused isolated heart, anaesthetised rat and rat ventricular myocyte preparations were utilised in a series of blinded and randomised studies to investigate the antiarrhythmic effectiveness, adverse effects and mechanism of action of OCT2013, compared with lidocaine. KEY RESULTS: In isolated hearts, OCT2013 and lidocaine prevented ischaemia-induced VF equi-effectively, but OCT2013 did not share lidocaine's adverse effects (PR widening, bradycardia and negative inotropy). In anaesthetised rats, i.v. OCT2013 and lidocaine suppressed VF and increased survival equi-effectively; OCT2013 had no effect on cardiac output even at 64 mg·kg-1 i.v., whereas lidocaine reduced it even at 1 mg·kg-1 . In adult rat ventricular myocytes, OCT2013 had no effect on Ca2+ handling, whereas lidocaine impaired it. In paced isolated hearts, lidocaine caused rate-dependent conduction slowing and block, whereas OCT2013 was inactive. However, during regional ischaemia, OCT2013 and lidocaine equi-effectively hastened conduction block. Chromatography and MS analysis revealed that OCT2013, detectable in normoxic OCT2013-perfused hearts, became undetectable during global ischaemia, with lidocaine becoming detectable. CONCLUSIONS AND IMPLICATIONS: OCT2013 is inactive but is bio-reduced locally in ischaemic myocardium to lidocaine, acting as an ischaemia-activated and ischaemia-selective antiarrhythmic prodrug with a large therapeutic index, mimicking lidocaine's benefit without adversity.

The Effect of Estrogen on Intracellular Ca2+ and Na+ Regulation in Heart Failure.

Contradictory findings of estrogen supplementation in cardiac disease highlight the need to investigate the involvement of estrogen in the progression of heart failure in an animal model that lacks traditional comorbidities. Heart failure was induced by aortic constriction (AC) in female guinea pigs. Selected AC animals were ovariectomized (ACOV), and a group of these received 17ß-estradiol supplementation (ACOV+E). One hundred-fifty days post-AC surgery, left-ventricular myocytes were isolated, and their electrophysiology and Ca2+ and Na+ regulation were examined. Long-term absence of ovarian hormones exacerbates the decline in cardiac function during the progression to heart failure. Estrogen supplementation reverses these aggravating effects.

Approaches to High-Throughput Analysis of Cardiomyocyte Contractility.

The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiology and pathophysiology of the myocardium. However, the isolation of cardiomyocytes introduces various technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low-throughput experimental studies, in which single cells are studied over the course of a pharmacological or physiological intervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-HTS). We can assess the shortening of sarcomeres, cell length, Ca2+ handling, and cellular morphology of almost 4 cells per minute. This increase in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficient numbers of cells in each preparation can be assessed for multiple conditions reducing these batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods that compensate for the hierarchical effects of clustering within heart preparations and demonstrate a significant false-positive rate, which is potentially present in conventional studies. We demonstrate a more stringent way to perform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig, and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulness of the CC-HTS in such studies. We specifically focus on the effects of agents that directly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g., isolated muscle bundles, thin slices, perfused dual innervated isolated heart, and perfused ventricular wedge). All suffer from low throughput but can be regarded as providing independent data points in contrast to the clustering problems associated with isolated cell studies. The greater productivity and sampling power provided by CC-HTS may help to reestablish the utility of isolated cell studies, while preserving the unique insights provided by studying the contribution of the fundamental, cellular unit of myocardial contractility.

Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies.

AIMS: It is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests). METHODS AND RESULTS: Ca2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF. Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF. CONCLUSION: Ca2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses.

Age- and strain-related aberrant Ca2+ release is associated with sudden cardiac death in the ACTC E99K mouse model of hypertrophic cardiomyopathy.

Patients with hypertrophic cardiomyopathy, particularly young adults, can die from arrhythmia, but the mechanism underlying abnormal rhythm formation remains unknown. C57Bl6 × CBA/Ca mice carrying a cardiac actin ( ACTC) E99K (Glu99Lys) mutation reproduce many aspects of human hypertrophic cardiomyopathy, including increased myofilament Ca2+ sensitivity and sudden death in a proportion (up to 40%) of young (28-40 day old) animals. We studied the hearts of transgenic (TG; ACTC E99K) mice and their non-TG (NTG) littermates when they were in their vulnerable period (28-40 days old) and when they were adult (8-12 wk old). Ventricular myocytes were isolated from the hearts of TG and NTG mice at these two time points. We also examined the hearts of mice that died suddenly (SCD). SCD animals had approximately four times more collagen compared with age-matched NTG mice, yet myocyte cell size was normal. Young TG mice had double the collagen content of NTG mice. Contraction and Ca2+ transients were greater in cells from young TG mice compared with their NTG littermates but not in cells from adult mice (TG or NTG). Cells from young TG mice had a greater propensity for Ca2+ waves than NTG littermates, and, despite similar sarcoplasmic reticulum Ca2+ content, a proportion of these cells had larger Ca2+ spark mass. We found that the probability of SCD in young TG mice was increased when the mutation was expressed in animals with a CBA/Ca2+ background and almost eliminated in mice bred on a C57Bl6 background. The latter TG mice had normal cellular Ca2+ homeostasis. NEW & NOTEWORTHY Mice with the actin Glu99Lys hypertrophic cardiomyopathy mutation ( ACTC E99K) are prone to sudden cardiac death around 40 days, associated with increased Ca2+ transients, spark mass, and fibrosis. However, adult survivors have normal Ca2+ transients and spark density accompanied by hypertrophy. Penetrance of the sudden cardiac death phenotype depends on the genetic background of the mouse. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/calcium-regulation-in-e99k-mouse-heart/ .

Effect of ovariectomy on intracellular Ca2+ regulation in guinea pig cardiomyocytes.

This study addressed the hypothesis that long-term deficiency of ovarian hormones after ovariectomy (OVx) alters cellular Ca2+-handling mechanisms in the heart, resulting in the formation of a proarrhythmic substrate. It also tested whether estrogen supplementation to OVx animals reverses any alterations to cardiac Ca2+ handling and rescues proarrhythmic behavior. OVx or sham operations were performed on female guinea pigs using appropriate anesthetic and analgesic regimes. Pellets containing 17ß-estradiol (1 mg, 60-day release) were placed subcutaneously in selected OVx animals (OVx + E). Cardiac myocytes were enzymatically isolated, and electrophysiological measurements were conducted with a switch-clamp system. In fluo-4-loaded cells, Ca2+ transients were 20% larger, and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials, creating a larger "window" current. SR Ca2+ stores were 22% greater in the OVx group, and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations, and a greater percentage of these cells showed delayed afterdepolarizations after exposure to isoprenaline compared with sham myocytes. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx + E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pigs lead to changes in myocyte Ca2+-handling mechanisms that are considered proarrhythmogenic. 17ß-Estradiol replacement prevented these adverse effects.NEW & NOTEWORTHY Ovariectomized guinea pig cardiomyocytes have higher frequencies of Ca2+ waves, and isoprenaline-challenged cells display more early afterdepolarizations, delayed afterdepolarizations, and extra beats compared with sham myocytes. These alterations to Ca2+ regulation were not observed in myocytes from ovariectomized guinea pigs supplemented with 17ß-estradiol, suggesting that ovarian hormone deficiency modifies cardiac Ca2+ regulation, potentially creating proarrhythmic substrates.

Erratum: High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes: [J. Biophotonics 9, No. 3, 311-323 (2016)].

In the article by M.B. Sikkel et al. (doi: 10.1002/jbio.201500193), published in J. Biophotonics 9, 311-323 (2016), an error occurred in the computer code that was used to generate Figure 3. This erratum is published to correct Figure 3, the calculated value of tgeom and the experimentally determined value of toptics in the text of the article.

Restitution slope is principally determined by steady-state action potential duration.

AIMS: The steepness of the action potential duration (APD) restitution curve and local tissue refractoriness are both thought to play important roles in arrhythmogenesis. Despite this, there has been little recognition of the apparent association between steady-state APD and the slope of the restitution curve. The objective of this study was to test the hypothesis that restitution slope is determined by APD and to examine the relationship between restitution slope, refractoriness and susceptibility to VF. METHODS AND RESULTS: Experiments were conducted in isolated hearts and ventricular myocytes from adult guinea pigs and rabbits. Restitution curves were measured under control conditions and following intervention to prolong (clofilium, veratridine, bretylium, low [Ca]e, chronic transverse aortic constriction) or shorten (catecholamines, rapid pacing) ventricular APD. Despite markedly differing mechanisms of action, all interventions that prolonged the action potential led to a steepening of the restitution curve (and vice versa). Normalizing the restitution curve as a % of steady-state APD abolished the difference in restitution curves with all interventions. Effects on restitution were preserved when APD was modulated by current injection in myocytes pre-treated with the calcium chelator BAPTA-AM - to abolish the intracellular calcium transient. The non-linear relation between APD and the rate of repolarization of the action potential is shown to underpin the common influence of APD on the slope of the restitution curve. Susceptibility to VF was found to parallel changes in APD/refractoriness, rather than restitution slope. CONCLUSION(S): Steady-state APD is the principal determinant of the slope of the ventricular electrical restitution curve. In the absence of post-repolarization refractoriness, factors that prolong the action potential would be expected to steepen the restitution curve. However, concomitant changes in tissue refractoriness act to reduce susceptibility to sustained VF. Dependence on steady-state APD may contribute to the failure of restitution slope to predict sudden cardiac death.

Functional consequences of co-expressing connexin40 or connexin45 with connexin43 on intercellular electrical coupling.

The functional characteristics of the co-expression of connexin43, connexin40, and connexin45 proteins in human myocardium are thought to play an important role in governing normal propagation of the cardiac electrical impulse and in generating the myocardial substrate for some arrhythmias and conduction disturbances. A rat liver epithelial cell line, that endogenously expresses connexin43, was used to induce also expression of connexin40 or connexin45 after stable transfection using an inducible ecdysone system. Electrical coupling was estimated from measurement of the input resistance of transfected cells using an intracellular microelectrode to inject current and record changes to membrane potential. However, varied expression of the transfected connexin40 or connexin45 did not change electrical coupling, although connexin43/40 co-expression led to better coupling than connexin43/45 co-expression. Quantification of endogenous connexin43 expression, at both mRNA and protein levels, showed that it was altered in a manner dependent on the transfected connexin isotype. The data using rat liver epithelial cells indicate an increased electrical coupling upon expression of connexin40 and connexin43 but decreased coupling with connexin45 and connexin43 co-expression.

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor.

BACKGROUND AND PURPOSE: Flecainide is a use-dependent blocker of cardiac Na(+) channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na(+) channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca(2) (+) -release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na(+) and/or RyR2 channels. EXPERIMENTAL APPROACH: We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca(2) (+) sparks in intact adult rat cardiac myocytes. KEY RESULTS: Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca(2) (+) spark frequency. CONCLUSIONS AND IMPLICATIONS: Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca(2) (+) sparks is likely, by analogy with flecainide, to result from Na(+) channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na(+) and RyR2 channels.

High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes.

Oblique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure.