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Our laboratory previously showed lipid hydroperoxides and oxylipin levels are elevated in response to loss of skeletal muscle innervation and are associated with muscle pathologies. To elucidate the pathological impact of lipid hydroperoxides, we overexpressed glutathione peroxidase 4 (GPx4), an enzyme that targets reduction of lipid hydroperoxides in membranes, in adult CuZn superoxide dismutase knockout (Sod1KO) mice that show accelerated muscle atrophy associated with loss of innervation. The gastrocnemius muscle from Sod1KO mice shows reduced mitochondrial respiration and elevated oxidative stress (F2‐isoprostanes and hydroperoxides) compared to wild‐type (WT) mice. Overexpression of GPx4 improved mitochondrial respiration and reduced hydroperoxide generation in Sod1KO mice, but did not attenuate the muscle loss that occurs in Sod1KO mice. In contrast, contractile force generation is reduced in EDL muscle in Sod1KO mice relative to WT mice, and overexpression of GPx4 restored force generation to WT levels in Sod1KO mice. GPx4 overexpression also prevented loss of muscle contractility at the single fibre level in fast‐twitch fibres from Sod1KO mice. Muscle fibres from Sod1KO mice were less sensitive to both depolarization and calcium at the single fibre level and exhibited a reduced activation by S‐glutathionylation. GPx4 overexpression in Sod1KO mice rescued the deficits in both membrane excitability and calcium sensitivity of fast‐twitch muscle fibres. Overexpression of GPx4 also restored the sarco/endoplasmic reticulum Ca2+‐ATPase activity in Sod1KO gastrocnemius muscles. These data suggest that GPx4 plays an important role in preserving excitation–contraction coupling function and Ca2+ homeostasis, and in maintaining muscle and mitochondrial function in oxidative stress‐induced sarcopenia. Key points: Knockout of CuZn superoxide dismutase (Sod1KO) induces elevated oxidative stress with accelerated muscle atrophy and weakness.Glutathione peroxidase 4 (GPx4) plays a fundamental role in the reduction of lipid hydroperoxides in membranes, and overexpression of GPx4 improves mitochondrial respiration and reduces hydroperoxide generation in Sod1KO mice.Muscle contractile function deficits in Sod1KO mice are alleviated by the overexpression of GPx4.GPx4 overexpression in Sod1KO mice rescues the impaired muscle membrane excitability of fast‐twitch muscle fibres and improves their calcium sensitivity.Sarco/endoplasmic reticulum Ca2+‐ATPase activity in Sod1KO muscles is decreased, and it is restored by the overexpression of GPx4.Our results confirm that GPx4 plays an important role in preserving excitation–contraction coupling function and Ca2+ homeostasis, and maintaining muscle and mitochondrial function in oxidative stress‐induced sarcopenia. [ABSTRACT FROM AUTHOR]

Heart failure is the final stage of various cardiovascular diseases and seriously threatens human health. Increasing mediators have been found to be involved in the pathogenesis of heart failure, including the RNA binding protein RBFox2. It participates in multiple aspects of the regulation of cardiac function and plays a critical role in the process of heart failure. However, how RBFox2 itself is regulated remains unclear. Here, we dissected transcriptomic signatures, including mRNAs and miRNAs, in a mouse model of heart failure after TAC surgery. A global analysis showed that an asymmetric alternation in gene expression and a large-scale upregulation of miRNAs occurred in heart failure. An association analysis revealed that the latter not only contributed to the degradation of numerous mRNA transcripts, but also suppressed the translation of key proteins such as RBFox2. With the aid of Ago2 CLIP-seq data, luciferase assays verified that RBFox2 was targeted by multiple miRNAs, including Let-7, miR-16, and miR-200b, which were significantly upregulated in heart failure. The overexpression of these miRNAs suppressed the RBFox2 protein and its downstream effects in cardiomyocytes, which was evidenced by the suppressed alternative splicing of the Enah gene and impaired E–C coupling via the repression of the Jph2 protein. The inhibition of Let-7, the most abundant miRNA family targeting RBFox2, could restore the RBFox2 protein as well as its downstream effects in dysfunctional cardiomyocytes induced by ISO treatment. In all, these findings revealed the molecular mechanism leading to RBFox2 depression in heart failure, and provided an approach to rescue RBFox2 through miRNA inhibition for the treatment of heart failure. [ABSTRACT FROM AUTHOR]

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/−) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/− myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus. [Display omitted] • RyR2 is a critical component of cardiac excitation-contraction coupling. • Heterozygous knock-out of RyR2 in rabbits produces ~50% loss of channel expression. • RyR2 deficiency leads to mild subcellular changes in Ca2+ handling. • Cardiac structure and function are normal in heterozygous RyR2-KO rabbits. • Excitation-contraction coupling has a safety factor that mitigates RyR2 deficiency. [ABSTRACT FROM AUTHOR]

Heart failure is the final stage of various cardiovascular diseases and seriously threatens human health. Increasing mediators have been found to be involved in the pathogenesis of heart failure, including the RNA binding protein RBFox2. It participates in multiple aspects of the regulation of cardiac function and plays a critical role in the process of heart failure. However, how RBFox2 itself is regulated remains unclear. Here, we dissected transcriptomic signatures, including mRNAs and miRNAs, in a mouse model of heart failure after TAC surgery. A global analysis showed that an asymmetric alternation in gene expression and a large-scale upregulation of miRNAs occurred in heart failure. An association analysis revealed that the latter not only contributed to the degradation of numerous mRNA transcripts, but also suppressed the translation of key proteins such as RBFox2. With the aid of Ago2 CLIP-seq data, luciferase assays verified that RBFox2 was targeted by multiple miRNAs, including Let-7, miR-16, and miR-200b, which were significantly upregulated in heart failure. The overexpression of these miRNAs suppressed the RBFox2 protein and its downstream effects in cardiomyocytes, which was evidenced by the suppressed alternative splicing of the Enah gene and impaired E–C coupling via the repression of the Jph2 protein. The inhibition of Let-7, the most abundant miRNA family targeting RBFox2, could restore the RBFox2 protein as well as its downstream effects in dysfunctional cardiomyocytes induced by ISO treatment. In all, these findings revealed the molecular mechanism leading to RBFox2 depression in heart failure, and provided an approach to rescue RBFox2 through miRNA inhibition for the treatment of heart failure.

Defects in cardiac contractility and heart failure (HF) are common following doxorubicin (DOX) administration. Different miRs play a role in HF, and their targeting was suggested as a promising therapy. We aimed to target miR-24, a suppressor upstream of junctophilin-2 (JP-2), which is required to affix the sarcoplasmic reticulum to T-tubules, and hence the release of Ca2+ in excitation–contraction coupling using pachymic acid (PA) and/or losartan (LN). HF was induced with DOX (3.5 mg/kg, i.p., six doses, twice weekly) in 24 rats. PA and LN (10 mg/kg, daily) were administered orally for four weeks starting the next day of the last DOX dose. Echocardiography, left ventricle (LV) biochemical and histological assessment and electron microscopy were conducted. DOX increased serum BNP, HW/TL, HW/BW, mitochondrial number/size and LV expression of miR-24 but decreased EF, cardiomyocyte fiber diameter, LV content of JP-2 and ryanodine receptors-2 (RyR2). Treatment with either PA or LN reversed these changes. Combined PA + LN attained better results than monotherapies. In conclusion, HF progression following DOX administration can be prevented or even delayed by targeting miR-24 and its downstream JP-2. Our results, therefore, suggest the possibility of using PA alone or as an adjuvant therapy with LN to attain better management of HF patients, especially those who developed tolerance toward LN.

Abstract Background Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle. Methods A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed “mitoflash.” Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation. Results We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity. Conclusions The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation–contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na+ and K+ contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca2+ channels. We show that cell contraction can be mediated directly by the inward Ca2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation. [ABSTRACT FROM AUTHOR]

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

Background: Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle. Methods: A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed "mitoflash." Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation. Results: We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity. Conclusions: The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle. [ABSTRACT FROM AUTHOR]

Transverse tubules (T-tubules) are orderly invaginations of the sarcolemma in mammalian cardiomyocytes. The integrity of T-tubule architecture is critical for cardiac excitation-contraction coupling function. T-tubule remodeling is recognized as a key player in cardiac dysfunction. Early studies on T-tubule structure were based on electron microscopy, which uncovered important information about the T-tubule architecture. The advent of fluorescent membrane probes allowed the application of confocal microscopy to investigations of T-tubule structure. Studies have now been extended beyond single cardiomyocytes to examine the T-tubule network in intact hearts through in situ confocal imaging of Langendorff-perfused hearts. This technique has allowed visualization of T-tubule organization in their natural habitat, avoiding the damage induced by isolation of cardiomyocytes. Additionally, it is possible to obtain T-tubule images in different subepicardial regions in a single intact heart. We review how this state-of-the-art imaging technique has provided important mechanistic insights into maturation of T-tubules in developing hearts and defined the role of T-tubule remodeling in development and progression of heart failure.

Pathological enhancement of late Na+ current (INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INa can be a source of intracellular Na+ ([Na+]i) overload. Late INa was enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na+]i was determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22-24°C, resting [Na+]i was 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa 8.7-fold. [Na+]i measured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM (P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INa with GS-967 (1 µM) prevented Na+i accumulation. A strong positive correlation was observed between the late INa and the pacing-induced increase of [Na+]i (R² = 0.88) and between the rise in [Na+]i and the increases in cytosolic Ca2+ (R² = 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na+]i in quiescent myocytes suggesting that late INa was solely responsible for triggering the ATX-II effect on [Na+]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na+]i overload associated with the increase in late INa caused by ATX-II. Enhancement of late INa can cause intracellular Na+ overload in ventricular myocytes. [ABSTRACT FROM AUTHOR]

The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status, and immobilization. Furthermore, these defined pools may serve specific functions in the cell. Specifically, reduced levels of these pools of glycogen are associated with reduced SR Ca2+ release, muscle relaxation rate, and membrane excitability. Collectively, the available literature strongly demonstrates that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state of the muscle fiber to energy utilization. [ABSTRACT FROM AUTHOR]

A simulator of the cardiac cell (E-C coupling process) is developed in this work, which solves the electro-chemical model of the cell proposed by Roche et al. (2004). It also contains other mathematical models as well, namely, those by Tang & Othmer (1994) and Fox et al. (2001), for comparison purposes. The simulator allows the modification of the model parameters in correspondence with the different cellular elements, to emulate cells of different species or cells under different conditions. The paper also contains an application of the simulator for testing the sensitivity of the E-C process to drugs and inhibitors, showing its intended use as a research tool in experiment design and cardiac pathology treatment. [ABSTRACT FROM AUTHOR]

Aims Contraction of the heart is regulated by electrically evoked Ca2+ transients (CaTs). H+ ions, the end products of metabolism, modulate CaTs through direct interactions with Ca2+-handling proteins and via Na+-mediated coupling between acid-extruding proteins (e.g. Na+/H+ exchange, NHE1) and Na+/Ca2+ exchange. Restricted H+ diffusivity in cytoplasm predisposes pH-sensitive Ca2+ signalling to becoming non-uniform, but the involvement of readily diffusible intracellular Na+ ions may provide a means for combatting this. Methods and results CaTs were imaged in fluo3-loaded rat ventricular myocytes paced at 2 Hz. Cytoplasmic [Na+] ([Na+]i) was imaged using SBFI. Intracellular acidification by acetate exposure raised diastolic and systolic [Ca2+] (also observed with acid-loading by ammonium prepulse or CO2 exposure). The systolic [Ca2+] response correlated with a rise in [Na+]i and sarcoplasmic reticulum Ca2+ load, and was blocked by the NHE1 inhibitor cariporide (CO2/HCO3−-free media). Exposure of one half of a myocyte to acetate using dual microperfusion (CO2/HCO3−-free media) raised diastolic [Ca2+] locally in the acidified region. Systolic [Ca2+] and CaT amplitude increased more uniformly along the length of the cell, but only when NHE1 was functional. Cytoplasmic Na+ diffusivity (DNa) was measured in quiescent cells, with strophanthidin present to inhibit the Na+/K+ pump. With regional acetate exposure to activate a local NHE-driven Na+-influx, DNa was found to be sufficiently fast (680 µm2/s) for transmitting the pH–systolic Ca2+ interaction over long distances. Conclusions Na+ ions are rapidly diffusible messengers that expand the spatial scale of cytoplasmic pH–CaT interactions, helping to co-ordinate global Ca2+ signalling during conditions of intracellular pH non-uniformity. [ABSTRACT FROM AUTHOR]

Aim Spontaneous activity of embryonic cardiomyocytes originates from sarcoplasmic reticulum ( SR) Ca2+ release during early cardiogenesis. However, the regulation of heart rate during embryonic development is still not clear. The aim of this study was to determine how endothelin-1 ( ET-1) affects the heart rate of embryonic mice, as well as the pathway through which it exerts its effects. Methods The effects of ET-1 and ET-1 receptor inhibition on cardiac contraction were studied using confocal Ca2+ imaging of isolated mouse embryonic ventricular cardiomyocytes and ultrasonographic examination of embryonic cardiac contractions in utero. In addition, the amount of ET-1 peptide and ET receptor a ( ETa) and b ( ETb) m RNA levels were measured during different stages of development of the cardiac muscle. Results High ET-1 concentration and expression of both ETa and ETb receptors was observed in early cardiac tissue. ET-1 was found to increase the frequency of spontaneous Ca2+ oscillations in E10.5 embryonic cardiomyocytes in vitro. Non-specific inhibition of ET receptors with tezosentan caused arrhythmia and bradycardia in isolated embryonic cardiomyocytes and in whole embryonic hearts both in vitro (E10.5) and in utero (E12.5). ET-1-mediated stimulation of early heart rate was found to occur via ETb receptors and subsequent inositol trisphosphate receptor activation and increased SR Ca2+ leak. Conclusion Endothelin-1 is required to maintain a sufficient heart rate, as well as to prevent arrhythmia during early development of the mouse heart. This is achieved through ETb receptor, which stimulates Ca2+ leak through IP3 receptors. [ABSTRACT FROM AUTHOR]

Aims Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation–contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in cardiomyocytes. Methods and results Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; αMHC-shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by >70% in αMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes implicated in TT development remained mostly unchanged. At the same time, intracellular Ca2+ handling was disrupted in ventricular myocytes from αMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection fraction, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8. Conclusion Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network. [ABSTRACT FROM AUTHOR]

In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research., Competing Interests: The authors declare no competing interests., (© 2022 Imperial College London.)

Abstract: The development of local control theories in cardiac excitation–contraction coupling solved a major problem in the calcium-induced calcium release (CICR) hypothesis. Local control explained how regeneration, inherent in the CICR mechanism, might be limited spatially to enable graded Ca release (and force production). The key lies in the stochastic recruitment of individual calcium release units (couplons or CRUs) where adjacent CRUs are partially uncoupled by the distance between them. In the CRU, individual groups of sarcoplasmic reticulum calcium release channels (RyRs) are very close to the surface membrane where calcium influx, controlled by membrane depolarization, leads to high local Ca levels that enable a high speed response from RyRs that have a very low probability to opening at resting Ca levels. However, calcium diffusion from an activated CRU results in adjacent CRUs being exposed to much lower levels of Ca and probability of activation. This effectively uncouples the CRUs and limits overall regenerative gain to enable stability without compromising sensitivity. Nevertheless, it is still unclear how the CRU terminates its release of calcium on the physiological timescale, and possible mechanisms (and problems) are briefly reviewed. We suggest that modulation in RyR gating may serve to control average SR Ca levels to regulate other metabolic functions of the sarco(endo)plasmic reticulum beyond regulating contractility. This article is part of a special issue entitled "Local Signaling in Myocytes." [Copyright &y& Elsevier]

Abstract: Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5–10nM) increases channel open probability (Po) twofold, by lengthening open dwell time. The degree of channel activation is concentration-dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((−)-epigallocatechin) and EC ((−)-epicatechin) showed a rank order of activity toward RyR1 (EGCG>ECG≫EGC >>> EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by ≤100μM cytoplasmic Ca2+ without altering inhibitory potency by >100μM Ca2+. EGCG as high as 10μM in the extracellular medium potentiated Ca2+ transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca2+ release or slowing Ca2+ transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities. [Copyright &y& Elsevier]

Abstract: Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD22,23 mice) or PKC sites at Ser 42 and 44 (cTnIAD22,23DD42,44) displayed differential force–frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD22,23 transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca2+] i , while cTnIAD22,23DD42,44 TG mice had a flat FFR with normal peak systolic [Ca2+] i , thus suggestive of diminished responsiveness to [Ca2+] i at higher frequencies. Force–[Ca2+] i hysteresis analysis revealed that cTnIDD22,23 mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca2+] i , whereas cTnIAD22,23DD42,44 transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca2+ at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force–[Ca2+] i hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to β-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development. [Copyright &y& Elsevier]

The article presents a study that examines the relation of diurnal variations in excitation-contraction (E-C) coupling in rat ventricles. The study isolates left ventricular mycocytes of rat during resting or active periods and observes responses of basal contraction and [calcium (Ca)2+]i in isoproterenol. Result shows higher Ca2+ and contraction strength in rest period myocytes, while showing greater arrhythmic activity with the active period isolated myocytes.

The purpose of the present study was to determine whether exposure to exogenous physiological concentrations of caffeine influence contractility, Ca2+ handling, and fatigue development in isolated single Xenopus laevis skeletal muscle fibers. After isolation, two identical contractile periods (separated by 60-mm rest) were conducted in each single myofiber (n = 8) at 20°C. During the first contractile period, four fibers were perfused with a noncaffeinated Ringer solution, while the other four fibers were perfused with a caffeinated (70 μM) Ringer solution. The order was reverse for the second contractile period. The single myofibers were stimulated during each contractile period at increasing frequencies (0.16, 0.20, 0.25, 0.33, 0.50, and 1.0 tetanic contractions/s), with each stimulation frequency lasting 2 mm until fatigue ensued, defined in this study as a fall in tension development to 66% of maximum. Tension development and free cytosolic [Ca2+]fura-2 fluorescence spectroscopy) were simultaneously measured. There was no significant difference in the peak force generation, time to fatigue, cytosolic Ca2+ levels, or relaxation times between the noncaffeinated and caffeinated trials. These results demonstrate that physiological levels of caffeine have no significant effect on Xenopus single myofiber contractility, Ca2+ handling, and fatigue development, and suggest that any ergogenic effects of physiological levels of caffeine on muscle performance during contractions of moderate to high intensity are likely related to factors extraneous to the muscle fiber. [ABSTRACT FROM AUTHOR]

Abstract: We previously proposed a systematic motor model for muscle with two parallel Maxwell elements and a force generator P. The motor model showed the non-linear behavior of a muscle, such as the force–velocity relation and the force depression and enhancement, by using weight functions. Our newly proposed muscle model is based on the molecular mechanism of myosin cross-bridges. We assume that each parallel Maxwell element represents the mechanical properties of weak and strong binding of the myosin head to actin. Furthermore, we introduce a controller to simulate the excitation–contraction coupling of the muscle. The new muscle model satisfies all the properties obtained in our previous model and reduces the wasted energy of the viscous component to less than 5% of the total energy. The controller enables us to simulate contractions of slow and fast twitch muscles, which are driven by an artificial action potential or a processing electromyography signal despite their same mechanical components. The maximum velocities are calculated to be 3.4L 0 m/s for the fast twitch muscle model and 2.5L 0 m/s for the slow twitch muscle model, where L 0 is the initial length of the muscle model. [Copyright &y& Elsevier]

Aims: The Na+/K+-ATPase (NKA) α2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca2+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA α2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. [ABSTRACT FROM PUBLISHER]

Abstract: Membrane depolarization triggers Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca2+ channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca2+ entry through VGCCs triggers RyR-mediated Ca2+ release via a Ca2+-induced Ca2+ release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca2+ release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP3Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca2+]i. The initial abrupt sub-PM [Ca2+]i upstroke was all but abolished by block of VGCCs (by 5μM nicardipine), depletion of intracellular Ca2+ stores (with 10μM cyclopiazonic acid) or inhibition of IP3Rs (by 2μM xestospongin C or 30μM 2-APB), but was not affected by block of RyRs (by 50–100μM tetracaine or 100μM ryanodine). Inhibition of either IP3Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation–contraction coupling in this phasic visceral smooth muscle occurs by Ca2+ entry through VGCCs which evokes an initial IP3R-mediated Ca2+ release activated via a CICR mechanism. [Copyright &y& Elsevier]

Nitric oxide (NO) is a highly reactive, free radical signalling molecule that is constitutively released in cardiomyocytes by both the endothelial and neuronal isoforms of nitric oxide synthase (eNOS and nNOS, respectively). There are increasing data indicating that NO modulates various proteins involved in excitation–contraction coupling (ECC), and here we discuss the evidence that NO may modulate the function of the ryanodine receptor Ca2+ release channel (RyR2) on the cardiac sarcoplasmic reticulum (SR). Both constitutive isoforms of NOS have been shown to co-immunoprecipitate with RyR2, suggesting that the channel may be a target protein for NO. eNOS gene deletion has been shown to abolish the increase in spontaneous Ca2+ spark frequency in cardiomyocytes exposed to sustained stretch, whereas the effect of nNOS-derived NO on RyR2 function remains to be investigated. Single channel studies have been performed with RyR2 reconstituted in planar lipid bilayers and exposed to various NO donors and, under these conditions, NO appears to have a dose-dependent, stimulatory effect on channel open probability (Popen). We discuss whether NO has a direct effect on RyR2 via covalent S-nitrosylation of reactive thiol residues within the protein, or whether there are downstream effects via cyclic nucleotides, phosphodiesterases, and protein kinases. Finally, we consider whether the proposed migration of nNOS from the SR to the sarcolemma in the failing heart may have consequences for the nitrosative vs. oxidative balance at the level of the RyR2, and whether this may contribute to an increased diastolic Ca2+ leak, depleted SR Ca2+ store, and reduced contractility in heart failure. [ABSTRACT FROM PUBLISHER]

Development of cardiac arrhythmias in several degenerative cardiac disorders such as heart failure is precipitated by abnormalities in intracellular calcium regulation. Recently, the identification of mutations in proteins responsible for the control of intracellular calcium has been associated with an inherited arrhythmogenic syndrome called catecholaminergic polymorphic ventricular tachycardia (CPVT). Here, we review the current knowledge about the molecular pathophysiology of CPVT and we discuss some potentially innovative strategies for controlling calcium-handling abnormalities in CPVT that may provide novel therapeutic options for affected patients. [ABSTRACT FROM PUBLISHER]

Aims: Peroxynitrite production increases during the pathogenesis of numerous cardiac disorders (e.g. heart failure). However, limited studies have investigated the mechanism through which peroxynitrite exerts anti-adrenergic effects. Thus, the purpose of this study is to investigate the contribution of phospholamban (PLB), a critical excitation–contraction coupling protein, to the peroxynitrite-induced dysfunction. [ABSTRACT FROM PUBLISHER]

Abstract: A comprehensive understanding of excitation–contraction (E–C) coupling in skeletal and cardiac muscle requires that all the major components of the Ca2+ release machinery be resolved. We utilized a unique immuno-proteomic approach to generate a monoclonal antibody library that targets proteins localized to the skeletal muscle triad junction, which provides a structural context to allow efficient E–C coupling. Screening of this library has identified several mitsugumins (MG); proteins that can be localized to the triad junction in mammalian skeletal muscle. Many of these proteins, including MG29 and junctophilin, are important components in maintaining the structural integrity of the triad junction. Other triad proteins, such as calumin, play a more direct role in regulation of muscle Ca2+ homeostasis. We have recently identified a family of trimeric intracellular cation-selective (TRIC) channels that allow for K+ movement into the endoplasmic or sarcoplasmic reticulum to counter a portion of the transient negative charge produced by Ca2+ release into the cytosol. Further study of TRIC channel function and other novel mitsugumins will increase our understanding of E–C coupling and Ca2+ homoeostasis in muscle physiology and pathophysiology. [Copyright &y& Elsevier]

Abstract: End-stage heart failure is believed to involve depressed cardiomyocyte contractility and Ca2+ transients. However, the time course of these alterations is poorly understood. We examined alterations in myocyte excitation–contraction coupling in a mouse model of early congestive heart failure (CHF) following myocardial infarction. One week after myocardial infarction was induced by ligation of the left coronary artery, CHF mice were selected based on established criteria (increased left atrial diameter, increased lung weight). Sham-operated animals (SHAM) served as controls. Echocardiographic measurements showed decreased global function in early CHF relative to SHAM, but increased local function in viable regions of the myocardium which deteriorated with time. Cardiomyocytes isolated from the non-infarcted septum also exhibited larger contractions in early CHF than SHAM (CHF=219.6±15.3% of SHAM values, P <0.05; 1 Hz field stimulation), and relaxation was more rapid (time to 50% relaxation=82.9±5.5% of SHAM values, P <0.05). Ca2+ transients (fluo-4 AM) were larger and decayed more rapidly in CHF than SHAM during both field stimulation (1 Hz) and voltage-clamp steps. Sarcoplasmic reticulum (SR) Ca2+ content was increased. Western blots showed that while SR Ca2+ ATPase (SERCA) expression was unaltered in CHF, phospholamban (PLB) was downregulated (60±11% of SHAM values, P <0.05). Thus, an increased SERCA/PLB ratio in CHF may promote SR Ca2+ re-uptake. Additionally, peak L-type Ca2+ current and Na+/Ca2+ exchanger expression were increased in CHF, suggesting increased sarcolemmal Ca2+ flux. Thus, in early CHF, alterations in Ca2+ homeostasis improve cardiomyocyte contractility which may compensate for loss of function in the infarction area. [Copyright &y& Elsevier]

Abstract: Objective: The presence of both α1- and α2-isoforms of the Na+/K+-ATPase (NKA) in cardiomyocytes indicates different functions. We hypothesized that preferential localization of the α2-isoform to the t-tubules, locally controlling the Na+/Ca2+-exchanger (NCX), underlies a specific role in Ca2+ handling. Methods: We studied NKA isoform distribution in isolated cardiomyocytes from Wistar rats using immunocytochemistry. NKA pump and NCX currents (Ipump and INCX) were measured in control and detubulated cardiomyocytes. Intracellular Na+ concentration [Na+] i was assessed with the fluorescent dye SBFI. Results: The α2-isoform abundance was higher in the t-tubules than in the surface sarcolemma. We established that 0.3 μM ouabain specifically blocked the α2-isoform in isolated rat cardiomyocytes. This low concentration blocked 10.7±0.6% of Ipump in control, but only 6.0±0.5% in detubulated cardiomyocytes. Moreover, measured and calculated α1-specific and α2-specific Ipump in control (547±29 pA and 66 pA, respectively) and in detubulated cells (495±30 pA and 31 pA, respectively) showed that 53% of the α2-isoform, but only 9.5% of the α1-isoform, were localized to the t-tubules. Despite the small abundance of the α2-isoform (∼11% of total NKA), selective inhibition of this isoform induced a 40% increase in contractility in field stimulated cardiomyocytes, but no increase in global [Na+] i . However, inhibition of the α2-isoform increased INCX indicating local subsarcolemmal accumulation of Na+ near NCX. Conclusions: The α2-isoform of the NKA is functionally coupled to the NCX and can regulate Ca2+ handling without changing global [Na+] i . [Copyright &y& Elsevier]

Abstract: Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment. [Copyright &y& Elsevier]

Abstract: Background: The cardiac sodium–calcium exchanger (NCX1) is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. Because heart failure is associated with increased expression of NCX1, heterozygous (HET) and homozygous (HOM) transgenic mice overexpressing NCX1 were developed and evaluated. Methods and Results: The NCX1 transgenic mice display 2.3-fold (HET) and 3.1-fold (HOM) increases in exchanger activity from wild-type (WT) mice. Functional information was obtained by echocardiography and catheterizations before and after hemodynamic stress from pregnancy, treadmill exercise or transaortic constriction (TAC). HET and HOM mice exhibited hypertrophy and blunted responses with β-adrenergic stimulation. Postpartum mice from all groups were hypertrophied, but only the HOM mice exhibited premature death from heart failure. HOM mice became exercise intolerant after 6 weeks of daily treadmill running. After 21 days TAC, HET, and HOM mice exhibited significant contractile dysfunction and 15% to 40% mortality with clinical evidence of heart failure. Conclusions: Hemodynamic stress results in a compensated hypertrophy in WT mice, but NCX1 transgenic mice exhibit decreased contractile function and heart failure in proportion to their level of NCX1 expression. Thus exchanger overexpression in mice leads to abnormal calcium handling and a decompensatory transition to heart failure with stress. [Copyright &y& Elsevier]

Myometrial contractility is a complex and dynamic physiological process that changes substantially during pregnancy and culminates in childbirth. Uterine contractions are initiated by transient rises in cytoplasmic Ca2+ concentration ([Ca2+]i), which in turn are triggered and controlled by myometrial action potentials. The sequence of events between the action potential generation and the contraction initiation is referred to as excitation–contraction coupling. Hormones and other physiologically active substances affect myometrial contractility by modulating different steps in the excitation–contraction coupling process. It is therefore imperative that we understand that process to understand the regulation of myometrial contractility. The complex action potentials generated by human myometrium result from the activity of many ion channels, transporters, and pumps. Two types of myometrial action potential waveform have been described in the literature: a plateau type and a spike type. Parameters of the myometrial [Ca2+]i transients and contractions differ depending on the type of action potential that triggers them. Some aspects of the excitation–contraction coupling are unique to human myometrium and cannot be found in animal models; some others are common between many species. This article reviews the current state and discusses future directions of physiological research on human myometrial excitation–contraction coupling. [ABSTRACT FROM AUTHOR]

Abstract: Calcium (Ca2+) is the central second messenger in the translation of electrical signals into mechanical activity of the heart. This highly coordinated process, termed excitation–contraction coupling or ECC, is based on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). In recent years it has become increasingly clear that several Ca2+-dependent proteins contribute to the fine tuning of ECC. One of these is the Ca2+/calmodulin-dependent protein kinase (CaMK) of which CaMKII is the predominant cardiac isoform. During ECC CaMKII phosphorylates several Ca2+ handling proteins with multiple functional consequences. CaMKII may also be co-localized to distinct target proteins. CaMKII expression as well as activity are reported to be increased in heart failure and CaMKII overexpression can exert distinct and novel effects on ECC in the heart and in isolated myocytes of animals. In the present review we summarize important aspects of the role of CaMKII in ECC with an emphasis on recent novel findings. [Copyright &y& Elsevier]

Abstract: Background: AVE0118 (2′-{[2-(4-Methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide) blocks atrial ultrarapid delayed rectifier currents (I Kur) and prolongs the atrial action potential (AP) plateau without affecting ventricular repolarisation. In patients with atrial contractile dysfunction due to atrial tachyarrhythmias, this response might increase atrial contractility without risk of ventricular proarrhythmia. This study was designed to evaluate the inotropic mechanisms of AVE0118. Methods and results: In isometrically contracting atrial trabeculae, AVE0118 increased contractile force by 55.4% in sinus rhythm patients (n =9) and by 107.4% in patients with atrial fibrillation (n =8). In freshly isolated canine atrial myocytes studied under perforated patch current clamp (37 °C), AVE0118 increased myocyte fractional shortening from 3.8±0.6 to 9.6±0.8% and prolonged action potential duration at 30% repolarisation from 9±2 to 102±11 ms. Clamping cells to an AP waveform recorded during exposure to AVE0118 produced the same inotropic response as the drug itself. In action potential clamp, peak Ca2+ inward current (I CaL) current declined from 5.5±1.3 pA/pF during control to 4.1±0.7 pA/pF when an AP recorded in the presence of AVE0118 was used as command waveform. However, I CaL was more sustained with AVE0118 and the time integral did not change (135±37 vs. 173±30 pA/pF⁎ms, p =ns). Importantly, blockade of reverse mode Na+/Ca2+-exchanger activity with 5 μM KBR7943 or using a Na+-free pipette solution abolished the positive inotropic effect of the AP recorded in the presence of AVE0118. In ventricular myocytes AVE0118 did not elicit a positive inotropic response. Conclusions: Block of I Kur by AVE0118 enhances atrial contractility both in patients with sinus rhythm and atrial fibrillation. The positive inotropic effect is atrial-specific and due to the changes of the action potential configuration which enhances Ca2+ entry via reverse mode Na+/Ca2+ exchange. [Copyright &y& Elsevier]

Abstract: Background: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release from the sarcoplasmic reticulum (SR) in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. While the adenosine A2A receptor (A2AR) is known to regulate cyclic AMP levels, expression and function of this receptor in human cardiac myocytes has not been investigated. Methods: PCR, western blotting and immunofluorescence were used to identify the A2AR, and functional effects of A2AR stimulation were measured with confocal calcium imaging and patch-clamp technique. Results: The A2AR is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the ryanodine receptor. A2AR stimulation caused a protein kinase A dependent increase in spontaneous SR calcium release in isolated human atrial myocytes. The A2AR agonist CGS21680 increased the frequency of calcium sparks from 0.12±0.03 to 0.31±0.08 sparks·μm min−1 (p <0.05) and calcium waves from 0.65±0.31 to 5.11±1.84 waves·min−1 (p <0.03). Moreover, spontaneous Na–Ca exchange currents (I NCX) increased from 1.19±0.17 to 2.50±0.42 min−1 (p <0.001). In contrast, CGS21680 did not alter caffeine inducible calcium release (6.98±0.52 vs. 6.82±0.57 amol pF−1, p =0.6) or the spontaneous I NCX amplitude (0.32±0.05 vs. 0.29±0.04 pA pF−1, p =0.2). Current–voltage relationship and amplitude of the L-type calcium current (1.62±0.18 vs. 1.80±0.18 pA pF−1) were not altered, but calcium release dependent inactivation was faster with CGS21680 (13.4±0.7 vs. 15.8±1.0 ms, p <0.001). Conclusions: Adenosine A2A receptors are expressed in the human atrial myocardium and modulate the frequency of spontaneous calcium release from the SR. [Copyright &y& Elsevier]

The ovariectomized (OVX) rat model was used to investigate the effects of estrogen treatment on soleus muscle functionality in situ following muscle injury induced by downhill running. Fifty immature, 24- to 26-d-old, OVX rats were randomly assigned to 5 separate experimental groups: sedentary controls (OVX-Sed), placebo-treated and studied immediately after exercise (OVX-Pb0), placebo-treated and studied 72 h after exercise (OVX-Pb72), estradiol-treated and studied immediately after exercise (OVX-Ed0), and estradiol-treated and studied 72 h after exercise (OVX-Ed72). At the age of 9 weeks, under ketamine and xylazine anesthesia i.p., the rats were subcutaneously implanted with either placebo or 17β-estradiol-impregnated pellets (0.05 mg/pellet, 3 week release). Treatment with 17β-estradiol increased the estradiol plasma levels in OVX animals to those normally seen during the proestrous cycle of normal animals. Three weeks after the implantation the rats were subjected to a 90 min intermittent downhill running protocol. Our results indicate that the exercise protocol used in the study induced injury in the soleus muscle, as it was detected by the significant reduction in unfused (stimulation at 10, 20, and 40 Hz) and maximal (Po) tetanic force, as well as the decreased ability of the soleus muscle to maintain tension (stimulation at 40 Hz for 3 min) in OVX-Pb0 and OVX-Pb72 placebo-treated animals subjected to downhill running (injured muscles) as compared with OVX-Sed control rats (uninjured muscle). Estradiol replacement in OVX rats partially protected the soleus muscle from the injury normally induced by downhill running. As compared with the OVX-Pb0 and OVX-Pb72 placebo-treated groups, the soleus muscles of OVX-Ed0 and OVX-Ed72 estradiol-treated rats were capable of producing significantly greater unfused tetanic force and had an increased ability to maintain tension after fatigue. However, estrogen at the dose administered did not prevent the decrease in maximal tetanic force. We postulate that the protective effect of estrogens on muscle strength may be related to the ability of estrogen hormones to attenuate the E–C coupling failure and (or) the disorganization of the contractile apparatus associated with eccentric exercise through a mechanism or mechanisms yet to be fully understood. [ABSTRACT FROM AUTHOR]

Abstract: Intracellular concentrations of redox-active molecules can significantly increase in the heart as a result of activation of specific signal transduction pathways or the development of certain pathophysiological conditions. Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca2+ levels ([Ca2+]i) and [Ca2+]i regulation, redox modification of Ca2+ channels and transporters has a profound effect on cardiac function. The sarcoplasmic reticulum (SR) Ca2+ release channel, or ryanodine receptor (RyR), is one of the well-characterized redox-sensitive ion channels in the heart. The redox modulation of RyR activity is mediated by the redox modification of sulfhydryl groups of cysteine residues. Other key components of cardiac excitation–contraction (e–c) coupling such as the SR Ca2+ ATPase and L-type Ca2+ channel are subject to redox modulation. Redox-mediated alteration of the activity of ion channels and pumps is directly involved in cardiac pathologies such as ischemia–reperfusion injury. Significant bursts of reactive oxygen species (ROS) generation occur during reperfusion of the ischemic heart, and changes in the activity of the major components of [Ca2+]i regulation, such as RyR, Na+–Ca2+ exchange and Ca2+ ATPases, are likely to play an important role in ischemia-related Ca2+ overload. This article summarizes recent findings on redox regulation of cardiac Ca2+ transport systems and discusses contributions of this redox regulation to normal and pathological cardiac function. [Copyright &y& Elsevier]

Abstract: We utilized Wistar rats with monocrotaline (MCT)-induced right ventricular hypertrophy (RVH) in order to evaluate the T-type Ca2+ channel current (I CaT) for myocardial contraction. RT-PCR provides that mRNA for T-type Ca2+ channel α1-subunits in hypertrophied myocytes was significantly higher than those in control rats (α1G; 264±36%, α1H; 191±34%; P <0.05). By whole-cell patch-clamp study, I CaT was recorded only in hypertrophied myocytes but not in control myocytes. The application of 50nmol/L nifedipine reduced the twitch tension of the right ventricles equally in the control and RVH rats. On the other hand, 0.5μmol/L mibefradil, a T-type Ca2+ channel blocker, strongly inhibited the twitch tension of the RVH muscle (control 6.4±0.8% vs. RVH 20.0±2.3% at 5Hz; P <0.01). In conclusion, our results indicate the functional expression of T-type Ca2+ channels in the hypertrophied heart and their contribution to the remodeling of excitation–contraction coupling in the cardiac myocyte. [Copyright &y& Elsevier]

The tubular (t-) system is the main interface between the myoplasm and the extracellular environment and is responsible for the rapid inward spread of excitation from the sarcolemma to the inner parts of the skeletal muscle fibre as well as for signal transfer to the sarcoplasmic reticulum to release Ca2+ that, in turn, activates the contractile apparatus. In this review, I explore the insights provided by the mechanically skinned muscle fibre preparation to the better understanding of the importance of the t-system excitability in determining the force response under physiologically relevant conditions. In the mechanically skinned muscle fibre, the t-system seals off after is physically separated from the sarcolemma and its excitability can be investigated by electrical stimulation under controlled conditions. Parameters that can be assessed include the threshold for action potential generation, specific electrical resistance and time constant of the tubular wall, quantity of charge transferred during an action potential, refractory period, length constant and velocity of excitation propagation. Results obtained with mechanically skinned fibres from fast-twitch muscles show that decreased t-system excitability does not necessarily translate into reduced force output, but for any particular set of physiologically relevant conditions there is a level below which a further decrease in t-system excitability markedly decreases the force output. There are several built-in mechanisms linked to the metabolic/energetic state of the muscle fibre which prevent complete action potential failure in the t-system, thus allowing the muscle to respond to nerve stimulation, even if the response becomes markedly attenuated. [ABSTRACT FROM AUTHOR]

Sphingosylphosphorylcholine (SPC) is a vasoconstricting lysosphingolipid, and the RhoA/Rho-kinase pathway plays an important role in SPC-induced contraction. Since RhoA/Rho-kinase-mediated signaling is involved in the generation and/or maintenance of hypertension, we compared the effect of SPC on the contractility of endothelium-denuded small mesenteric arteries in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Fura-2 Ca2+ signals, contractile responses, and phosphorylation of 20-kDa myosin light chains (MLC20) were measured. Ten μM SPC induced a gradual and sustained vasoconstriction, which was greater in arteries of the SHR (82.5±4.3%, n=9) than in those of the WKY (26.7±4.5%, n=10). In Ca2+-free media, SPC gradually increased vascular tone in the SHR, but caused little vasoconstriction in the WKY. In the SHR and WKY, SPC evoked a greater vasoconstriction than did high K+depolarization at a given Ca2+ ratio, and the Ca2+ ratio–tension curve induced by SPC was significantly shifted to the left compared with that induced by high K+ depolarization. However, the magnitude of shift to the left was greater in the SHR than in the WKY. The Rho-kinase inhibitor Y-27632 significantly inhibited SPC-induced contractions, but neither the protein kinase C inhibitor calphostin-C nor PD98059, which inhibits activation of some mitogen-activated protein kinases, had any effect on the SHR or the WKY. SPC significantly increased the phosphorylation of MLC20 in both the SHR and the WKY, and Y-27632 inhibited the SPC-induced increase in MLC20 phosphorylation in the SHR. Our results suggest that SPC induces greater vascular tone in the SHR than in the WKY. Furthermore, our results indicate that activation of the Rho-kinase pathway plays an important role in the SPC-induced Ca2+ sensitization in the SHR. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Urocortin II (UcnII), a peptide of the corticotropin-releasing factor (CRF) family, exerts profound actions on the cardiovascular system. Direct effects of UcnII on adult cardiomyocytes have not been evaluated before. Our aim was to characterize functional effects of UcnII on cardiomyocytes and to elucidate the underlying signaling pathway(s) and cellular mechanisms. Methods: Rabbit ventricular cardiomyocytes were stimulated at 0.5 Hz (22–25 °C). Unloaded cell shortening (FS, edge detection), [Ca2+]i transients (Fluo-4), and L-type Ca2+ currents (I Ca, whole-cell patch clamping) were measured. Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid application of caffeine (20 mmol/L). Results: UcnII increased cell shortening and accelerated relaxation in a time- and concentration-dependent manner (EC50: 10.7 nmol/L). The inotropic effect of UcnII was maximal at 100 nmol/L (35%±11% increase in FS, n =8, P <0.05). The inotropic and lusitropic actions of UcnII were largely eliminated by inhibition of CRF2 receptors (10 nmol/L antisauvagine-30, n =5) or protein kinase A (PKA, 500 nmol/L H-89, n =5). UcnII increased [Ca2+]i transient amplitude (by 63%±35%, n =7, P <0.05) and decreased the time constant for decay (from 800±63 to 218±27 ms, n =7, P <0.001). UcnII also increased SR Ca2+ load (by 19%±7%, n =7, P <0.05) and fractional Ca2+ release (from 57%±7% to 98%±2%, n =7, P <0.01). I Ca was augmented by 32.7%±10.0% (n =9, P <0.05) and the I Ca–V relationship was shifted by −15 mV during UcnII treatment. Conclusion: UcnII exerts positive inotropic and lusitropic effects in cardiomyocytes via CRF2 receptor-mediated stimulation of PKA which augments I Ca and SR Ca2+ load to increase SR Ca2+ release and [Ca2+]i transients. [Copyright &y& Elsevier]

Abstract: Objective: Src has been proposed to activate L-type calcium channel activity by binding to the α1c subunit. In the II–III linker region of this subunit there is a novel consensus sequence for Src binding. We have examined whether this site is a functional Src interaction site and investigated the effect displacing Src from this region has on calcium channel activity. Methods: In vitro binding assays were performed to map α1 subunit interaction sites. Cardiac myocytes were isolated enzymatically from rat ventricles. Whole cell patch-clamp technique was used to record Ca2+ channel currents in cells that had been loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with the α1 subunit. Results: Peptides corresponding to the II–III linker region and C-terminal tail of the α1c subunit, but not scrambled peptide controls, were found to inhibit Src SH3 domain binding to the channel and significantly reduced the channel current amplitude. The II–III linker region peptide shifted the inactivation curve to the left whereas the C-terminal tail region peptide shifted the activation curve to the right when compared to scramble peptide controls. PP1-pre-treatment of myocytes also reduced the current amplitude, decreased the V 1/2 for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to co-associate with Src and the channel, but PP1 pre-treatment reduced this co-association. Conclusions: Src binds to both the II–III linker and C-terminal tail regions of the α1c subunit to differentially modulate channel activity. PYK2 is also able to co-complex with Src when bound to this region of the channel but only when Src is catalytically active. Together the two kinases may synergistically regulate channel activity. [Copyright &y& Elsevier]

Abstract: Objective: The role of phosphorylation of the ryanodine receptor at serine-2808 (RyRS2808) in congestive heart failure (CHF) is controversial, and effects of RyRS2808 phosphorylation on contraction are unclear. It has been reported that diastolic sarcomere length (SL) fluctuations accompany propagating contractile waves due to propagating SR Ca2+ release in trabeculae from rats with CHF. Here, we studied the influence of RyR destabilization by FK506 and isoproterenol on twitch force (F tw) and SL fluctuations in right ventricular (RV) trabeculae. We measured phosphorylation of RyRS2808 in rats with myocardial infarction (MI) with or without β-blockade and in rats during isoproterenol stimulation in order to assess the role of RyRS2808 phosphorylation in SL fluctuations in failing hearts. Methods: Five groups of male Lewis Brown–Norway rats were studied 3 months after MI: i) Sham; ii) MI with CHF (cMI); iii) MI without CHF; iv) metoprolol-treated MI, with and without CHF. The root mean square (RMSSL) of SL fluctuations in RV trabeculae was calculated. Results: RMSSL increased strongly both following a short train of stimuli at 2.5 Hz and following catecholamine activation in trabeculae from MI with CHF, resulting in a decrease in F tw in proportion to RMSSL. RyRS2808 phosphorylation was increased significantly in the left ventricle (LV; ∼58%, P <0.05) but not in the RV (n.s.) in MI rats with CHF. FK506 tripled high frequency stimulation-induced RMSSL in nonfailing trabecula but did not further enhance RMSSL in failing trabecula. Isoproterenol increased RMSSL in nonfailing trabeculae only modestly despite a substantial increase in RyRS2808 phosphorylation in the RV (∼60%, P <0.05). Isoproterenol induced SL fluctuation without an increase in RV-RyRS2808 phosphorylation in failing trabeculae. Chronic β-blockade decreased high frequency and catecholamine stimulation-induced RMSSL while RyRS2808 phosphorylation in the RV was indistinguishable from that in cMI. Conclusions: Acute RyRS2808 phosphorylation by itself does not cause spontaneous contractile waves owing to RyR2 destabilization. Spontaneous contractile waves in CHF are not caused by RyRS2808 phosphorylation alone, suggesting that factors other than RyRS2808 phosphorylation affect RyR function. [Copyright &y& Elsevier]

Abstract: Objective: Rabbit ventricular myocardium is characterized by a biphasic response to stretch with an initial, rapid increase in force followed by a delayed, slow increase in force (slow force response, SFR). The initial phase is attributed to increased myofilament Ca2+ sensitivity, but the mechanisms of the delayed phase are only incompletely understood. We tested whether stretch-dependent stimulation of Na+/H+ exchange (NHE1) and consecutive changes in pHi and/or [Na+]i may underlie the SFR. Methods: Isometric contractions of rabbit ventricular muscles were recorded in bicarbonate-containing Tyrode''s (Tyrode) or bicarbonate-free HEPES-buffered solution (HEPES). Muscles were loaded with the Ca2+ indicator aequorin, the pH indicator BCECF, or the Na+ indicator SBFI and rapidly stretched from 88% (L 88) to 98% (L 98) of optimal length. The resulting immediate and slow increases in twitch force (1st phase and SFR) as well as changes in [Ca2+]i, [Na+]i, or pHi were quantified before and after inhibition of NHE1 by HOE 642 (3 μM) or reverse-mode Na+/Ca2+ exchange (NCX) by KB-R 7943 (5 μM). Results: In both Tyrode (n =21) and HEPES (n =22), developed force increased to ∼160% during the 1st phase followed by a further increase to ∼205% during the SFR. The SFR was accompanied by a 21% increase of the aequorin light transient (n =4; normalized to the 1st phase) and a ∼3 mM increase in [Na+]i (n =4–7). The SFR was also associated with an increase in pHi. However, this increase was delayed and was significant only after the SFR had reached its maximum. The delayed pHi increase was larger in HEPES than in Tyrode. HOE 642 and/or KB-R 7943 reduced the SFR by ∼30–40%. In addition, HOE 642 diminished the stretch-mediated elevation of [Na+]i by 72% and the delayed alkalinization. Conclusions: The data are consistent with the hypothesis that SFR results from increases in [Ca2+]i secondary to altered flux via NCX in part resulting from increases in [Na+]i mediated by NHE1. [Copyright &y& Elsevier]