Search

Pathogenic variants in the type I ryanodine receptor (RYR1) result in a wide range of muscle disorders referred to as RYR1‐related myopathies (RYR1‐RM). We developed the first RYR1‐RM mouse model resulting from co‐inheritance of two different RYR1 missense alleles (Ryr1TM/SC‐ΔL mice). Ryr1TM/SC‐ΔL mice exhibit a severe, early onset myopathy characterized by decreased body/muscle mass, muscle weakness, hypotrophy, reduced RYR1 expression, and unexpectedly, incomplete postnatal lethality with a plateau survival of ~50% at 12 weeks of age. Ryr1TM/SC‐ΔL mice display reduced respiratory function, locomotor activity, and in vivo muscle strength. Extensor digitorum longus muscles from Ryr1TM/SC‐ΔL mice exhibit decreased cross‐sectional area of type IIb and type IIx fibers, as well as a reduction in number of type IIb fibers. Ex vivo functional analyses revealed reduced Ca2+ release and specific force production during electrically‐evoked twitch stimulation. In spite of a ~threefold reduction in RYR1 expression in single muscle fibers from Ryr1TM/SC‐ΔL mice at 4 weeks and 12 weeks of age, RYR1 Ca2+ leak was not different from that of fibers from control mice at either age. Proteomic analyses revealed alterations in protein synthesis, folding, and degradation pathways in the muscle of 4‐ and 12‐week‐old Ryr1TM/SC‐ΔL mice, while proteins involved in the extracellular matrix, dystrophin‐associated glycoprotein complex, and fatty acid metabolism were upregulated in Ryr1TM/SC‐ΔL mice that survive to 12 weeks of age. These findings suggest that adaptations that optimize RYR1 expression/Ca2+ leak balance, sarcolemmal stability, and fatty acid biosynthesis provide Ryr1TM/SC‐ΔL mice with an increased survival advantage during postnatal development. [ABSTRACT FROM AUTHOR]

During exercise or stress, the sympathetic system stimulates cardiac contractility via β‐adrenergic receptor (β‐AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation–contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type‐2‐specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock‐in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2‐S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β‐ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during β‐AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2‐S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2‐Ser2808 and RyR2‐S2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β‐AR‐mediated sensitization of RyRs. Key points: Phosphorylation of cardiac sarcoplasmic reticulum Ca2+‐release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function.Ablation of both the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs.The intra‐sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2‐double‐knock‐in cells.The RyR2 from double‐knock‐in cells exhibits increased Ca2+ sensitivity.Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel.Phosphorylation of Ser2030 might be sufficient for a β‐adrenergic response. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications Botulinum neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action would significantly ameliorate its pharmacological effects with beneficial outcomes for clinical use.Here, we combined BoNT/A with two fast‐acting inhibitors of excitation–contraction coupling inhibitors (ECCI), either the μ‐conotoxin CnIIIC or dantrolene, and tested the effect of their co‐injection on a model of hind‐limb paralysis in rodents using behavioural, biochemical, imaging and electrophysiological assays.The BoNT/A‐ECCI combinations accelerated the onset of muscle relaxation. Surprisingly, they also potentiated the peak effect and extended the duration of the three BoNT/A commercial preparations OnabotulinumtoxinA, AbobotulinumtoxinA and IncobotulinumtoxinA. ECCI co‐injection increased the number of BoNT/A molecules entering motoneuron terminals, which induced a faster and greater cleavage of SNAP‐25 during the onset and peak phases, and prolonged the attenuation of nerve‐muscle neurotransmission during the recovery phase. We estimate that ECCI co‐injection yields a threefold potentiation in BoNT/A pharmacological activity.Overall, our results show that the pharmacological activity of BoNT/A can be combined and synergized with other bioactive molecules and uncover a novel strategy to enhance the neuromuscular effects of BoNT/A without altering the neurotoxin moiety or intrinsic activity, thus maintaining its exceptional safety profile. [ABSTRACT FROM AUTHOR]

Key points Patients with myotonia congenita suffer from slowed muscle relaxation caused by hyperexcitability. The diaphragm is only mildly affected in myotonia congenita; discovery of the mechanism underlying its resistance to myotonia could identify novel therapeutic targets. Intracellular recordings from two mouse models of myotonia congenita revealed the diaphragm had less myotonia than either the extensor digitorum longus (EDL) or the soleus muscles. A mechanism contributing to resistance of the diaphragm to myotonia was reduced depolarization of the interspike membrane potential during repetitive firing of action potentials, a process driven by build‐up of K+ in small invaginations of muscle membrane known as t‐tubules. We explored differences between diaphragm and EDL that might underlie reduction of K+ build‐up in diaphragm t‐tubules. Smaller size of diaphragm fibres, which promotes diffusion of K+ out of t‐tubules, was identified as a contributor. Intracellular recording revealed slower repolarization of action potentials in diaphragm suggesting reduced Kv conductance. Higher resting membrane conductance was identified suggesting increased Kir conductance. Computer simulation found that a reduction of Kv conductance had little effect on K+ build‐up whereas increased Kir conductance lessened build‐up, although the effect was modest. Our data and computer simulation suggest opening of K+ channels during action potentials has little effect on K+ build‐up whereas opening of K+ channels during the interspike interval slightly lessens K+ build‐up. We conclude that activation of K+ channels may lessen myotonia by opposing depolarization to action potential threshold without worsening K+ build‐up in t‐tubules. In mouse models of the muscle disease myotonia congenita, the diaphragm has much less myotonia (muscle stiffness) than the extensor digitorum longus or soleus muscles. Identifying why the diaphragm is resistant to myotonia may help in developing novel therapy. We found the reason the diaphragm has less myotonia is that it is less prone to depolarization caused by K+ build‐up in t‐tubules during repetitive firing of action potentials. Smaller fibre size contributes to resistance to K+ build‐up with differences in K+ currents playing little role. Our data suggest drugs that open K+ channels may be effective in treating myotonia as they may lessen excitability without worsening K+ build‐up in t‐tubules. [ABSTRACT FROM AUTHOR]

Muscular efficiency during exercise has been used to interrogate aspects of human muscle energetics, including mitochondrial coupling and biomechanical efficiencies. Typically, assessments of muscular efficiency have involved graded exercises. Results of previous studies have been interpreted to indicate a decline in exercise efficiency with aging owing to decreased mitochondrial function. However, discrepancies in variables such as exercise stage duration, cycling cadence, and treadmill walking mechanics may have affected interpretations of results. Furthermore, recent data from our lab examining the ATP to oxygen ratio (P:O) in mitochondrial preparations isolated from NIA mouse skeletal muscle showed no change with aging. Thus, we hypothesized that delta efficiency (Δ€) during steady-rate cycling exercise would not be altered in older healthy subjects compared with young counterparts regardless of biological sex or training status. Young (21–35 yr) and older (60–80 yr) men (n = 21) and women (n = 20) underwent continual, progressive leg cycle ergometer tests pedaling at 60 RPM for three stages (35, 60, 85 W) lasting 4 min. Δ€was calculated as: (Δ work accomplished/Δ energy expended). Overall, cycling efficiencies were not significantly different in older compared with young subjects. Similarly, trained subjects did not exhibit significantly different exercise efficiencies compared to untrained. Moreover, there were no differences between men and women. Hence, our results obtained on healthy young and older subjects are interpreted to mean that previous reports of decreased efficiency in older individuals were attributable to metabolic or biomechanical comorbidities, not aging per se. NEW & NOTEWORTHY: Muscular power is reduced, but the efficiency of movement is unaltered in healthy aging. [ABSTRACT FROM AUTHOR]

Pulmonary artery hypertension (PAH) is characterised by increased pulmonary vascular resistance (PVR) resulting in elevated pressure in the pulmonary artery supplying the pulmonary circulation. Disease of the right ventricle (RV) often manifests as a result of PAH placing excessive pressure on the right side of the heart. Although a relatively rare disease in humans, the impact of sustained PAH is severe, with poor outcomes even in treated individuals. As PAH develops, the blood flow is restricted through the pulmonary arteries and the right ventricle hypertrophies due to the increased strain of pumping blood through the pulmonary circulation. With time, RV hypertrophy progresses to right heart failure, impacting the supply of blood to the left ventricle and systemic circulation. Although right heart failure can currently be treated, it cannot be cured. There is therefore a need for more research into the physiological changes that cause the heart to fail under pressure overload. This review aims to evaluate the monocrotaline (MCT) rat model of PAH as a means of studying the cellular mechanisms associated with the development of RV hypertrophy and right heart failure. [ABSTRACT FROM AUTHOR]

The mantle muscle of common cuttlefish, Sepia officinalis, is responsible both for high‐magnitude and rapid movements for locomotion, as well as sustained ventilation, which require specific metabolic, electrophysiological, and structural organization. Young cuttlefish have a highly oxidative phenotype and a rapid growth rate. Here, we show high rates of oxygen consumption and protein synthesis in juveniles, and these rates decay exponentially over the first few weeks of growth. This is associated with considerable citrate synthase activity (relative to larger cuttlefish) but a lack of glucose metabolism based on zero uptake of glucose by isolated muscle sheets and minimal activity of hexokinase (similar to larger animals). In contrast to glucose metabolism in the heart, glucose metabolism in these muscle sheets was not stimulated by extracellular taurine. Previous research revealed an unusual ion channel complement in mantle myocytes, the most notable feature of which is the lack of a Na+ current during depolarization. Because this adaptation is not consistent across the coleoid clade, we investigated excitation–contraction coupling. Here, mantle energetics and contractility, including the individual components of the total Ca2+ flux driving contraction, were studied. Results indicate that the majority of Ca2+ current underlying contractile stress development capacity in cuttlefish juveniles is not mediated by dihydropyridine‐sensitive L‐type channels, in contrast to their adult counterparts, and the sarcoplasmic reticulum contributes little to routine contractility. We had previously noted an influence of physiological levels of taurine in limiting cardiac contractility but found no analogous sensitivity in mantle muscle. Finally, transmission electron microscopy of subcellular architecture revealed the presence of sarcoplasmic tubular aggregates, suggesting that oxidative inhibition of sarcoplasmic reticulum function limits its role in this life stage. [ABSTRACT FROM AUTHOR]

A transformation is underway in precision and patient‐specific medicine. Rapid progress has been enabled by multiple new technologies including induced pluripotent stem cell‐derived cardiac myocytes (iPSC‐CMs). Here, we delve into these advancements and their future promise, focusing on the efficiency of reprogramming techniques, the fidelity of differentiation into the cardiac lineage, the functional characterization of the resulting cardiac myocytes, and the many applications of in silico models to understand general and patient‐specific mechanisms controlling excitation–contraction coupling in health and disease. Furthermore, we explore the current and potential applications of iPSC‐CMs in both research and clinical settings, underscoring the far‐reaching implications of this rapidly evolving field. [ABSTRACT FROM AUTHOR]

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell–cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation–contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (−25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions. [ABSTRACT FROM AUTHOR]

Bridging integrator 1 (BIN1) is a ubiquitous BIN–amphiphysin–Rvs (BAR) domain protein with tissue-specific isoforms and functions that is implicated in various diseases such as myopathies, heart failure, Alzheimer's disease, and cancer. In skeletal muscle, the inclusion of a phosphatidylinositol binding domain enables BIN1 to elongate transverse membrane tubules (T-tubules) linked to the sarcoplasmic reticulum. In cardiac muscle, BIN1 forms T-tubule microdomains and recruits the essential components of excitation–contraction coupling via the cytoskeleton. The tubulation properties of BIN1 are balanced by myotubularin 1 and dynamin 2 during tubulogenesis in cardiac and skeletal muscles. Modulation of the gene encoding either of these is a potential therapy for BIN1-linked myopathies. Amphiphysin-2 is a ubiquitously expressed protein also known as bridging integrator 1 (BIN1), playing a critical role in membrane remodeling, trafficking, and cytoskeleton dynamics in a wide range of tissues. Mutations in the gene encoding BIN1 cause centronuclear myopathies (CNM), and recent evidence has implicated BIN1 in heart failure, underlining its crucial role in both skeletal and cardiac muscle. Furthermore, altered expression of BIN1 is linked to an increased risk of late-onset Alzheimer's disease and several types of cancer, including breast, colon, prostate, and lung cancers. Recently, the first proof-of-concept for potential therapeutic strategies modulating BIN1 were obtained for muscle diseases. In this review article, we discuss the similarities and differences in BIN1's functions in cardiac and skeletal muscle, along with its associated diseases and potential therapies. [ABSTRACT FROM AUTHOR]

Abstract L-type calcium channels (LTCCs), the largest subfamily of voltage-gated calcium channels (VGCCs), are the main channels for Ca2+ influx during extracellular excitation. LTCCs are widely present in excitable cells, especially cardiac and cardiovascular smooth muscle cells, and participate in various Ca2+-dependent processes. LTCCs have been considered as worthy drug target for cardiovascular, neurological and psychological diseases for decades. Natural products from Traditional Chinese medicine (TCM) have shown the potential as new drugs for the treatment of LTCCs related diseases. In this review, the basic structure, function of LTCCs, and the related human diseases caused by structural or functional abnormalities of LTCCs, and the natural LTCCs antagonist and their potential usages were summarized.

We have recently identified a pool of intracellular β1 adrenergic receptors (β1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular β1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized β1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local β1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-β1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-β1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

L-type calcium channels (LTCCs), the largest subfamily of voltage-gated calcium channels (VGCCs), are the main channels for Ca2+ influx during extracellular excitation. LTCCs are widely present in excitable cells, especially cardiac and cardiovascular smooth muscle cells, and participate in various Ca2+-dependent processes. LTCCs have been considered as worthy drug target for cardiovascular, neurological and psychological diseases for decades. Natural products from Traditional Chinese medicine (TCM) have shown the potential as new drugs for the treatment of LTCCs related diseases. In this review, the basic structure, function of LTCCs, and the related human diseases caused by structural or functional abnormalities of LTCCs, and the natural LTCCs antagonist and their potential usages were summarized. [ABSTRACT FROM AUTHOR]

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca2+ levels, and of the heart rate. [ABSTRACT FROM AUTHOR]

Background: Observational studies of intermittent hemodialysis therapy have reported that the excess decrease in K+ concentration in plasma (KP) during treatment is associated with the destabilization of cardiac function. Elucidating the mechanism by which the decrease in KP impairs myocardial excitation is indispensable for a deeper understanding of prescription design. Methods: In this study, by using an electrophysiological mathematical model, we investigated the relationship between KP dynamics and cardiomyocyte excitability for the first time. Results: The excess decrease in KP during treatment destabilized cardiomyocyte excitability through the following events: (1) a decrease in KP led to the prolongation of the depolarization phase of ventricular cells due to the reduced potassium efflux rate of the Kr channel, temporarily enhancing contraction force; (2) an excess decrease in KP activated the transport of K+ and Na+ through the funny channel in sinoatrial nodal cells, disrupting automaticity; (3) the excess decrease in KP also resulted in a significant decrease in the resting membrane potential of ventricular cells, causing contractile dysfunction. Avoiding an excess decrease in KP during treatment contributed to the maintenance of cardiomyocyte excitability. Conclusions: The results of these mathematical analyses showed that it is necessary to implement personal prescription or optimal control of K+ concentration in dialysis fluid based on predialysis KP from the perspective of regulatory science in dialysis treatment. [ABSTRACT FROM AUTHOR]

In skeletal muscle, the Ca2+ release flux elicited by a voltage clamp pulse rises to an early peak that inactivates rapidly to a much lower steady level. Using a double pulse protocol the fast inactivation follows an arithmetic rule: if the conditioning depolarization is less than or equal to the test depolarization, then decay (peak minus steady level) in the conditioning release is approximately equal to suppression (unconditioned minus conditioned peak) of the test release. This is due to quantal activation by voltage, analogous to the quantal activation of IP3 receptor channels. Two mechanisms are possible. One is the existence of subsets of channels with different sensitivities to voltage. The other is that the clusters of Ca2+-gated Ryanodine Receptor (RyR) β in the parajunctional terminal cisternae might constitute the quantal units. These Ca2+-gated channels are activated by the release of Ca2+ through the voltage-gated RyR α channels. If the RyR β were at the basis of quantal release, it should be modified by strong inhibition of the primary voltage-gated release. This was attained in two ways, by sarcoplasmic reticulum (SR) Ca2+ depletion and by voltage-dependent inactivation. Both procedures reduced global Ca2+ release flux, but SR Ca2+ depletion reduced the single RyR current as well. The effect of both interventions on the quantal properties of Ca2+ release in frog skeletal muscle fibers were studied under voltage clamp. The quantal properties of release were preserved regardless of the inhibitory maneuver applied. These findings put a limit on the role of the Ca2+-activated component of release in generating quantal activation. [ABSTRACT FROM AUTHOR]

Cardiac arrhythmias cause significant morbidity and mortality and pose a major public health problem. They arise from disruptions in the normally orderly propagation of cardiac electrophysiological activation and recovery through successive cardiomyocytes in the heart. They reflect abnormalities in automaticity, initiation, conduction, or recovery in cardiomyocyte excitation. The latter properties are dependent on surface membrane electrophysiological mechanisms underlying the cardiac action potential. Their disruption results from spatial or temporal instabilities and heterogeneities in the generation and propagation of cellular excitation. These arise from abnormal function in their underlying surface membrane, ion channels, and transporters, as well as the interactions between them. The latter, in turn, form common regulatory targets for the hierarchical network of diverse signaling mechanisms reviewed here. In addition to direct molecular-level pharmacological or physiological actions on these surface membrane biomolecules, accessory, adhesion, signal transduction, and cytoskeletal anchoring proteins modify both their properties and localization. At the cellular level of excitation-contraction coupling processes, Ca2+ homeostatic and phosphorylation processes affect channel activity and membrane excitability directly or through intermediate signaling. Systems-level autonomic cellular signaling exerts both acute channel and longer-term actions on channel expression. Further upstream intermediaries from metabolic changes modulate the channels both themselves and through modifying Ca2+ homeostasis. Finally, longer-term organ-level inflammatory and structural changes, such as fibrotic and hypertrophic remodeling, similarly can influence all these physiological processes with potential pro-arrhythmic consequences. These normal physiological processes may target either individual or groups of ionic channel species and alter with particular pathological conditions. They are also potentially alterable by direct pharmacological action, or effects on longerterm targets modifying protein or cofactor structure, expression, or localization. Their participating specific biomolecules, often clarified in experimental genetically modified models, thus constitute potential therapeutic targets. The insights clarified by the physiological and pharmacological framework outlined here provide a basis for a recent modernized drug classification. Together, they offer a translational framework for current drug understanding. This would facilitate future mechanistically directed therapeutic advances, for which a number of examples are considered here. The latter are potentially useful for treating cardiac, in particular arrhythmic, disease. [ABSTRACT FROM AUTHOR]

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during β-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+] i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+] i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during β-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies. [Display omitted] • FKBP12.6 expression in normal mouse heart is very low: 0.16 ± 0.02 nmol/g of left ventricle. • Moderate overexpression of FKBP12.6 has a protective role against arrhythmias. • High overexpression of FKPB12.6 activates calcineurin and provoke dilated hypertrophy. • High level of FKBP12.6 is arrhythmogenic. • The effects of FKBP12.6 are unrelated to RyR2 phosphorylation at Ser2808. [ABSTRACT FROM AUTHOR]

The article focuses on recent developments in understanding skeletal muscle physiology, highlighting advancements in the field. Topics discussed include new insights into muscle fiber types and their functional significance, the role of satellite cells in muscle repair and regeneration, and emerging strategies for enhancing muscle health and performance.

T‐tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well‐co‐ordinated excitation–contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all‐optical approach to stimulate and record trans‐membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT‐generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho‐functional changes reduce the electrical connectivity between SS and TT membranes. Key points: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T‐tubular system) that propagate action potentials to the core of the cell, causing uniform excitation–contraction coupling across the cell.In the present study, we investigated whether the T‐tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma.For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains.Our findings demonstrate that T‐tubules are intrinsically excitable, revealing distinct characteristics of self‐generated T‐tubular action potentials.This active electrical capability would protect cells from voltage drops potentially occurring within the T‐tubular network. [ABSTRACT FROM AUTHOR]

Abstract Of the ions involved in myocardial function, Ca2+ is the most important. Ca2+ is crucial to the process that allows myocardium to repeatedly contract and relax in a well-organized fashion; it is the process called excitation–contraction coupling. In order, therefore, for accurate comprehension of the physiology of the heart, it is fundamentally important to understand the detailed mechanism by which the intracellular Ca2+ concentration is regulated to elicit excitation–contraction coupling. Aequorin was discovered by Shimomura, Johnson and Saiga in 1962. By taking advantage of the fact that aequorin emits blue light when it binds to Ca2+ within the physiologically relevant concentration range, in the 1970s and 1980s, physiologists microinjected it into myocardial preparations. By doing so, they proved that Ca2+ transients occur upon membrane depolarization, and tension development (i.e., actomyosin interaction) subsequently follows, dramatically advancing the research on cardiac excitation–contraction coupling.

Of the ions involved in myocardial function, Ca2+ is the most important. Ca2+ is crucial to the process that allows myocardium to repeatedly contract and relax in a well-organized fashion; it is the process called excitation–contraction coupling. In order, therefore, for accurate comprehension of the physiology of the heart, it is fundamentally important to understand the detailed mechanism by which the intracellular Ca2+ concentration is regulated to elicit excitation–contraction coupling. Aequorin was discovered by Shimomura, Johnson and Saiga in 1962. By taking advantage of the fact that aequorin emits blue light when it binds to Ca2+ within the physiologically relevant concentration range, in the 1970s and 1980s, physiologists microinjected it into myocardial preparations. By doing so, they proved that Ca2+ transients occur upon membrane depolarization, and tension development (i.e., actomyosin interaction) subsequently follows, dramatically advancing the research on cardiac excitation–contraction coupling. [ABSTRACT FROM AUTHOR]

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation–contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF. [ABSTRACT FROM AUTHOR]

Heating cardiomyocytes to 38-42°C induces hyperthermal sarcomeric oscillations (HSOs), which combine chaotic instability and homeostatic stability. These properties are likely important for achieving periodic and rapid ventricular expansion during the diastole phase of the heartbeat. Compared with spontaneous oscillatory contractions in cardiomyocytes, which are sarcomeric oscillations induced in the presence of a constant calcium concentration, we found that calcium concentration fluctuations cause chaotic instability during HSOs. We believe that the experimental fact that sarcomeres, autonomously oscillating, exhibit such instability due to the action of calcium concentration changes is important for understanding the physiological function of sarcomeres. Therefore, we have named this chaotic sarcomere instability that appears under conditions involving changes in calcium concentration as Sarcomere Chaos with Changes in Calcium Concentration (S4C). Interestingly, sarcomere instability that could be considered S4C has also been observed in the relaxation dynamics of EC coupling. Unlike ADP-SPOCs and Cell-SPOCs under constant calcium concentration conditions, fluctuations in oscillation amplitude indistinguishable from HSOs were observed. Additionally, like HSO, a positive Lyapunov exponent was measured. S4C is likely a crucial sarcomeric property supporting the rapid and flexible ventricular diastole with each heartbeat of the heart. [ABSTRACT FROM AUTHOR]

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation–contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches. [ABSTRACT FROM AUTHOR]

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction. [ABSTRACT FROM AUTHOR]

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

This presentation reviews images of electron micrographs from various skeletal muscles identifying a consistent association of diydropyridine receptors (DHPR) tetrads with alternate ryanodine receptors. Imaging of the junctional gap in triads from various sources provide direct evidence for the association of four diydropyridine receptors (DHPRs), clustered into tetrads, with alternate ryanodine receptors (RyRs). It is not clear whether firing of all four components of a tetrad is necessary to fully activate the opening of the RyR channel.

Regulation of intracellular calcium is a critical component of cardiac electrophysiology and excitation-contraction coupling. The calcium spark, the fundamental element of the intracellular calcium transient, is initiated in specialized nanodomains which co-locate the ryanodine receptors and L-type calcium channels. However, calcium homeostasis is ultimately regulated at the cellular scale, by the interaction of spatially separated but diffusively coupled nanodomains with other sub-cellular and surface-membrane calcium transport channels with strong non-linear interactions; and cardiac electrophysiology and arrhythmia mechanisms are ultimately tissue-scale phenomena, regulated by the interaction of a heterogeneous population of coupled myocytes. Recent advances in imaging modalities and image-analysis are enabling the super-resolution reconstruction of the structures responsible for regulating calcium homeostasis, including the internal structure of nanodomains themselves. Extrapolating functional and imaging data from the nanodomain to the whole-heart is non-trivial, yet essential for translational insight into disease mechanisms. Computational modeling has important roles to play in relating structural and functional data at the sub-cellular scale and translating data across the scales. This review covers recent methodological advances that enable image-based modeling of the single nanodomain and whole cardiomyocyte, as well as the development of multi-scale simulation approaches to integrate data from nanometer to whole-heart. Firstly, methods to overcome the computational challenges of simulating spatial calcium dynamics in the nanodomain are discussed, including image-based modeling at this scale. Then, recent whole-cell models, capable of capturing a range of different structures (such as the T-system and mitochondria) and cellular heterogeneity/variability are discussed at two different levels of discretization. Novel methods to integrate the models and data across the scales and simulate stochastic dynamics in tissue-scale models are then discussed, enabling elucidation of the mechanisms by which nanodomain remodeling underlies arrhythmia and contractile dysfunction. Perspectives on model differences and future directions are provided throughout.

Pulmonary artery hypertension (PAH) is characterised by increased pulmonary vascular resistance (PVR) resulting in elevated pressure in the pulmonary artery supplying the pulmonary circulation. Disease of the right ventricle (RV) often manifests as a result of PAH placing excessive pressure on the right side of the heart. Although a relatively rare disease in humans, the impact of sustained PAH is severe, with poor outcomes even in treated individuals. As PAH develops, the blood flow is restricted through the pulmonary arteries and the right ventricle hypertrophies due to the increased strain of pumping blood through the pulmonary circulation. With time, RV hypertrophy progresses to right heart failure, impacting the supply of blood to the left ventricle and systemic circulation. Although right heart failure can currently be treated, it cannot be cured. There is therefore a need for more research into the physiological changes that cause the heart to fail under pressure overload. This review aims to evaluate the monocrotaline (MCT) rat model of PAH as a means of studying the cellular mechanisms associated with the development of RV hypertrophy and right heart failure.

Experimental evidence, both in vitro and in vivo, has indicated cardioprotective effects of extracellular vesicles (EVs) derived from various cell types, including induced pluripotent stem cell-derived cardiomyocytes. The biological effects of EV secretion, particularly in the context of ischemia and cardiac electrophysiology, remain to be fully explored. Therefore, the goal of this study was to unveil the effects of exosome (EXO)-mediated cell–cell signaling during hypoxia by employing a simulated preconditioning approach on human-induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs). Electrophysiological activity of hIPSC-CMs was measured using a multielectrode array (MEA) system. A total of 16 h of hypoxic stress drastically increased the beat period. Moreover, hIPSC-CMs preconditioned with EXOs displayed significantly longer beat periods compared with non-treated cells after 16 h of hypoxia (+15.7%, p < 0.05). Furthermore, preconditioning with hypoxic EXOs resulted in faster excitation–contraction (EC) coupling compared with non-treated hIPSC-CMs after 16 h of hypoxia (−25.3%, p < 0.05). Additionally, microRNA (miR) sequencing and gene target prediction analysis of the non-treated and pre-conditioned hIPSC-CMs identified 10 differentially regulated miRs and 44 gene targets. These results shed light on the intricate involvement of miRs, emphasizing gene targets associated with cell survival, contraction, apoptosis, reactive oxygen species (ROS) regulation, and ion channel modulation. Overall, this study demonstrates that EXOs secreted by hIPSC-CM during hypoxia beneficially alter electrophysiological properties in recipient cells exposed to hypoxic stress, which could play a crucial role in the development of targeted interventions to improve outcomes in ischemic heart conditions.

Background Sodium-calcium (Ca2+) exchanger isoform 1 (NCX1) is the dominant Ca2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca2+ homeostasis during excitation-contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen-inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation-contraction coupling and intracellular Ca2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury. Methods and Results We used the α-myosin heavy chain promoter (Myh6)-MerCreMer promoter to create a tamoxifen-inducible cardiac-specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca2+ increased despite adaptive reductions in Ca2+ current and action potential duration and compensatory increases in excitation-contraction coupling gain, sarcoplasmic reticulum Ca2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca2+ normalized and SR Ca2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular-related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion. Conclusions Tamoxifen-inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca2+ influx, increasing alternative Ca2+ efflux pathways, and increasing excitation-contraction coupling gain to maintain contractility at the cost of mild Ca2+-activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.

Heating cardiomyocytes to 38–42°C induces hyperthermal sarcomeric oscillations (HSOs), which combine chaotic instability and homeostatic stability. These properties are likely important for achieving periodic and rapid ventricular expansion during the diastole phase of the heartbeat. Compared with spontaneous oscillatory contractions in cardiomyocytes, which are sarcomeric oscillations induced in the presence of a constant calcium concentration, we found that calcium concentration fluctuations cause chaotic instability during HSOs. We believe that the experimental fact that sarcomeres, autonomously oscillating, exhibit such instability due to the action of calcium concentration changes is important for understanding the physiological function of sarcomeres. Therefore, we have named this chaotic sarcomere instability that appears under conditions involving changes in calcium concentration as Sarcomere Chaos with Changes in Calcium Concentration (S4C). Interestingly, sarcomere instability that could be considered S4C has also been observed in the relaxation dynamics of EC coupling. Unlike ADP-SPOCs and Cell-SPOCs under constant calcium concentration conditions, fluctuations in oscillation amplitude indistinguishable from HSOs were observed. Additionally, like HSO, a positive Lyapunov exponent was measured. S4C is likely a crucial sarcomeric property supporting the rapid and flexible ventricular diastole with each heartbeat of the heart.

Fluorofenidone (AKF-PD) is a novel pyridone derivative that inhibits fibrosis and inflammation in many tissues. Accordingly, it has been effective in disease models, such as liver failure, nephropathy, and pulmonary fibrosis. However, its potential role in cardiac physiology and pathology has yet to be elucidated. Thus, this paper investigated a possible functional impact of AKF-PD on adult rat cardiac myocytes. Cells were kept in culture for 1–2 days under either control conditions or the presence of AKF-PD (500 μM). They were next examined concerning cell contractility, intracellular Ca2+ homeostasis, and activity of voltage-gated Ca2+ channels. Remarkably, AKF-PD enhanced the percentage of cell shortening and rates of both contraction and relaxation by nearly 100%. A stimulus in Ca2+-induced Ca2+ release (CICR) most likely accounts for these effects because AKF-PD also increased the magnitude of electrically evoked Ca2+ transients. Of note, the compound did not alter the peak value of caffeine-elicited Ca2+ transients, indicating stimulation of CICR at constant sarcoplasmic reticulum Ca2+ load. Since CICR is triggered by the entry of Ca2+ through Ca V 1.2 (I Ca), a possible effect on these Ca2+ channels was also investigated. AKF-PD increased the magnitude of both I Ca and maximal macroscopic Ca2+ conductance (G max) by about 50%. However, no differences were found in either voltage dependence of inactivation or the amount of maximal immobilization-resistant charge movement (Q max). Thus, the effect on I Ca could be explained by a higher channel's open probability (P o) rather than a greater abundance of channel proteins. Additional data indicate that AKF-PD reduces the rate of Ca2+ extrusion in the presence of caffeine, suggesting inhibition of the Na/Ca exchanger. Overall, these results indicate that AKF-PD upregulates the P o of Ca V 1.2 and then sequentially enhances I Ca , CICR, and contractility. Therefore, the novel compound is also a candidate to be tested in cardiac disease models. • Fluorofenidone (AKF-PF) is a novel anti-fibrotic and anti-inflammatory compound. • Here, its possible influence on the physiology of cardiac myocytes was assessed. • AKF-PF exerted a chronic upregulation of voltage-gated Ca2+ channels (Ca V 1.2). • The latter effect, in turn, enhances intracellular Ca2+ release and contractility. • These findings greatly improve the potential of AKF-PF as a cardiovascular agent. [ABSTRACT FROM AUTHOR]

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation–contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t‐tubules was the same, but male cardiomyocytes had more longitudinal t‐tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field‐stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. Key points: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum.Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight‐or‐flight response.In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology.The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes.Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice. [ABSTRACT FROM AUTHOR]

Our previous studies revealed the protection of stachydrine hydrochloride (STA) against cardiopathological remodeling. One of the underlying mechanisms involves the calcium/calmodulin-dependent protein kinase Ⅱ (CaMKII). However, the way STA influences CaMKII needs to be further investigated. The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-coupled reactive oxygen species (ROS) overproduction putatively induces the oxidative activation of CaMKII, resulting in the occurrence of pathological cardiac remodeling and dysfunction in experimental models of mice. Thus, in this study, we assessed the role of the NOX2-ROS signal axis in STA cardioprotection. The transverse aortic constriction (TAC)-induced heart failure model of mice, the phenylephrine-induced hypertrophic model of neonatal rat primary cardiomyocytes, and the H2O2-induced oxidative stress models of adult mouse primary cardiomyocytes and H9c2 cells were employed. The echocardiography and histological staining were applied to assess the cardiac effect of STA (6 mg/kg/d or 12 mg/kg/d), which was given by gavage. NOX2, ROS, and excitation-contraction (EC) coupling were detected by Western blotting, immunofluorescence, and calcium transient-contraction synchronous recordings. ROS and ROS-dependent cardiac fibrosis were alleviated in STA-treated TAC mice, demonstrating improved left ventricular ejection fraction and hypertrophy. In the heart failure model of mice and the hypertrophic model of cardiomyocytes, STA depressed NOX2 protein expression and activation, as shown by inhibited translocation of its phosphorylation, p67phox and p47phox, from the cytoplasm to the cell membrane. Furthermore, in cardiomyocytes under oxidative stress, STA suppressed NOX2-related cytosolic Ca2+ overload, enhanced cell contractility, and decreased Ca2+-dependent regulatory protein expression, including CaMKⅡ and Ryanodine receptor calcium release channels. Cardioprotection of STA against pressure overload-induced pathological cardiac remodeling correlates with the NOX2-coupled ROS signaling cascade. [ABSTRACT FROM AUTHOR]

Cardiac excitation-contraction coupling (ECC) depends on Ca2+ release from intracellular stores via ryanodine receptors (RyRs) triggered by L-type Ca2+ channels (LCCs). Uncertain numbers of RyRs and LCCs form 'couplons' whose activation produces Ca2+ sparks, which summate to form a cell-wide Ca2+ transient that switches on contraction. Voltage (V m) changes during the action potential (AP) and stochasticity in channel gating should create variability in Ca2+ spark timing, but Ca2+ transient wavefronts have remarkable uniformity. To examine how this is achieved, we measured the V m -dependence of evoked Ca2+ spark probability (P spark) and latency over a wide voltage range in rat ventricular cells. With depolarising steps, Ca2+ spark latency showed a U-shaped V m -dependence, while repolarising steps from 50 mV produced Ca2+ spark latencies that increased monotonically with V m. A computer model based on reported channel gating and geometry reproduced our experimental data and revealed a likely RyR:LCC stoichiometry of ∼ 5:1 for the Ca2+ spark initiating complex (IC). Using the experimental AP waveform, the model revealed a high coupling fidelity (P cpl ∼ 0.5) between each LCC opening and IC activation. The presence of ∼ 4 ICs per couplon reduced Ca2+ spark latency and increased P spark to match experimental data. Variability in AP release timing is less than that seen with voltage steps because the AP overshoot and later repolarization decrease P spark due to effects on LCC flux and LCC deactivation respectively. This work provides a framework for explaining the V m - and time-dependence of P spark , and indicates how ion channel dispersion in disease can contribute to dyssynchrony in Ca 2 + release. [Display omitted] • Evoked Ca2+ spark latency was measured in rat cardiomyocytes using voltage clamp. • The latency was highly voltage dependent and different for action potentials and steps. • Computer simulations of experiments were used to analyse the origin of Ca2+ latency. • 4 initiating complexes of 3–8 RyRs per LCC could explain Ca2+ transient latency. • During an action potential, 50% of LCC openings could trigger a Ca2+ spark. [ABSTRACT FROM AUTHOR]

Synergist ablation (SA) is an experimental procedure for the induction of hypertrophy. However, SA causes a decrease in specific force (i.e., force per cross-sectional area), likely due to excessive muscle use. Here, we investigated the mechanisms behind the SA-induced intrinsic contractile dysfunction, especially focusing on the excitation-contraction (EC) coupling. Male Wistar rats had unilateral surgical ablation of gastrocnemius and soleus muscles to induce compensatory hypertrophy in the plantaris muscles. Two weeks after SA, plantaris muscle was dissected from each animal and used for later analyses. SA significantly increased the mean fiber cross-sectional area (+18%). On the other hand, the ratio of depolarization-induced force to the maximum Ca2+-activated specific force, an indicator of sarcoplasmic reticulum (SR) Ca2+ release, was markedly reduced in mechanically skinned fibers from the SA group (-51%). These functional defects were accompanied by an extensive fragmentation of the SR Ca2+ release channel, the ryanodine receptor 1 (RyR1), and a decrease in the amount of other triad proteins (i.e., DHPR, STAC3, and junctophilin1). SA treatment also caused activation of calpain-1 and increased the amount of NADPH oxidase 2, endoplasmic reticulum (ER) stress proteins (i.e., Grp78, Grp94, PDI, and Ero1), and lipid peroxidation [i.e., 4-hydroxynonenal (4-HNE)] in SA-treated muscles. Our findings show that SA causes skeletal muscle weakness due to impaired EC coupling. This is likely to be induced by Ca2+-dependent degradation of triad proteins, which may result from Ca2+ leak from fragmented RyR1 triggered by increased oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals. Methods: To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach. Results: Manual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000–2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. Conclusions: KairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.

The postnatal mammalian heart undergoes remarkable developmental changes, which are stimulated by the transition from the intrauterine to extrauterine environment. With birth, increased oxygen levels promote metabolic, structural and biophysical maturation of cardiomyocytes, resulting in mature muscle with increased efficiency, contractility and electrical conduction. In this Topical Review article, we highlight key studies that inform our current understanding of human cardiomyocyte maturation. Collectively, these studies suggest that human atrial and ventricular myocytes evolve quickly within the first year but might not reach a fully mature adult phenotype until nearly the first decade of life. However, it is important to note that fetal, neonatal and paediatric cardiac physiology studies are hindered by a number of limitations, including the scarcity of human tissue, small sample size and a heavy reliance on diseased tissue samples, often without age‐matched healthy controls. Future developmental studies are warranted to expand our understanding of normal cardiac physiology/pathophysiology and inform age‐appropriate treatment strategies for cardiac disease. [ABSTRACT FROM AUTHOR]

Most patients with malignant hyperthermia susceptibility diagnosed by the in vitro caffeine–halothane contracture test (CHCT) develop excessive force in response to halothane but not caffeine (halothane-hypersensitive). Hallmarks of halothane-hypersensitive patients include high incidence of musculoskeletal symptoms at rest and abnormal calcium events in muscle. By measuring sensitivity to halothane of myotubes and extending clinical observations and cell-level studies to a large group of patients, we reach new insights into the pathological mechanism of malignant hyperthermia susceptibility. Patients with malignant hyperthermia susceptibility were classified into subgroups HH and HS (positive to halothane only and positive to both caffeine and halothane). The effects on [Ca2+] cyto of halothane concentrations between 0.5 and 3 % were measured in myotubes and compared with CHCT responses of muscle. A clinical index that summarises patient symptoms was determined for 67 patients, together with a calcium index summarising resting [Ca2+] cyto and spontaneous and electrically evoked Ca2+ events in their primary myotubes. Halothane-hypersensitive myotubes showed a higher response to halothane 0.5% than the caffeine-halothane hypersensitive myotubes (P <0.001), but a lower response to higher concentrations, comparable with that used in the CHCT (P =0.055). The HH group had a higher calcium index (P <0.001), but their clinical index was not significantly elevated vs the HS. Principal component analysis identified electrically evoked Ca2+ spikes and resting [Ca2+] cyto as the strongest variables for separation of subgroups. Enhanced sensitivity to depolarisation and to halothane appear to be the primary, mutually reinforcing and phenotype-defining defects of halothane-hypersensitive patients with malignant hyperthermia susceptibility. [ABSTRACT FROM AUTHOR]