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The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was

Calmodulin transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+-driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators.

Key points Ca2+ waves are known to trigger delayed after‐depolarizations that can cause malignant cardiac arrhythmias. However, modelling Ca2+ waves using physiologically realistic models has remained a major challenge. Existing models with low Ca2+ sensitivity of ryanodine receptors (RyRs) necessitate large release currents, leading to an unrealistically large Ca2+ transient amplitude incompatible with the experimental observations. Consequently, current physiologically detailed models of delayed after‐depolarizations resort to unrealistic cell architectures to produce Ca2+ waves with a normal Ca2+ transient amplitude. Here, we address these challenges by incorporating RyR cooperativity into a physiologically detailed model with a realistic cell architecture. We represent RyR cooperativity phenomenologically through a Hill coefficient within the sigmoid function of RyR open probability. Simulations in permeabilized myocytes with high Ca2+ sensitivity reveal that a sufficiently large Hill coefficient is required for Ca2+ wave propagation via the fire–diffuse–fire mechanism. In intact myocytes, propagating Ca2+ waves can occur only within an intermediate Hill coefficient range. Within this range, the spark rate is neither too low, enabling Ca2+ wave propagation, nor too high, allowing for the maintenance of a high sarcoplasmic reticulum load during diastole of the action potential. Moreover, this model successfully replicates other experimentally observed manifestations of Ca2+‐wave‐mediated triggered activity, including phase 2 and phase 3 early after‐depolarizations and high‐frequency voltage–Ca2+ oscillations. These oscillations feature an elevated take‐off potential with depolarization mediated by the L‐type Ca2+ current. The model also sheds light on the roles of luminal gating of RyRs and the mobile buffer ATP in the genesis of these arrhythmogenic phenomena. Existing mathematical models of Ca2+ waves use an excessively large Ca2+‐release current or unrealistic diffusive coupling between release units. Our physiologically realistic model, using a Hill coefficient in the ryanodine receptor (RyR) gating function to represent RyR cooperativity, addresses these limitations and generates organized Ca2+ waves at Hill coefficients ranging from ∼5 to 10, as opposed to the traditional value of 2. This range of Hill coefficients gives a spark rate neither too low, thereby enabling Ca2+ wave propagation, nor too high, allowing for the maintenance of a high sarcoplasmic reticulum load during the plateau phase of the action potential. Additionally, the model generates Ca2+‐wave‐mediated phase 2 and phase 3 early after‐depolarizations, and coupled membrane voltage with Ca2+ oscillations mediated by the L‐type Ca2+ current. This study suggests that pharmacologically targeting RyR cooperativity could be a promising strategy for treating cardiac arrhythmias linked to Ca2+‐wave‐mediated triggered activity. [ABSTRACT FROM AUTHOR]

Phospholamban (PLN) plays a crucial role in regulating sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Mutations within the PLN gene have been detected in patients with cardiomyopathy, with the heterozygous variant c.40_42delAGA (p.R14del) of PLN being the most prevalent. Investigations into the mechanisms underlying the pathology of PLN-R14del have revealed that cardiac cells from affected patients exhibit pathological aggregates containing PLN. Herein, we performed comprehensive molecular and cellular analyses to delineate the molecular aberrations associated with the formation of these aggregates. We determined that PLN aggregates contain autophagic proteins, indicating inefficient degradation via the autophagy pathway. Our findings demonstrate that the expression of PLN-R14del results in diminished autophagic flux due to impaired fusion between autophagosomes and lysosomes. Mechanistically, this defect is linked to aberrant recruitment of key membrane fusion proteins to autophagosomes, which is mediated in part by changes in Ca2+ homeostasis. Collectively, these results highlight a novel function of PLN-R14del in regulating autophagy, that may contribute to the formation of pathogenic aggregates in patients with cardiomyopathy. Prospective strategies tailored to ameliorate impaired autophagy may hold promise against PLN-R14del disease. [ABSTRACT FROM AUTHOR]

The resistance of pests to common insecticides is a global issue that threatens food production worldwide. Diamide insecticides target insect ryanodine receptors (RyRs), causing uncontrolled calcium release from the sarcoplasmic and endoplasmic reticulum. Despite their high potency and species selectivity, several resistance mutations have emerged. Using a chimeric RyR (chiRyR) approach and cryo-electron microscopy (cryo-EM), we investigate how insect RyRs engage two different diamide insecticides from separate families: flubendiamide, a phthalic acid derivative, and tetraniliprole, an anthranilic compound. Both compounds target the same site in the transmembrane region of the RyR, albeit with different poses, and promote channel opening through coupling with the pore-forming domain. To explore the resistance mechanisms, we also solve two cryo-EM structures of chiRyR carrying the two most common resistance mutations, I4790M and G4946E, both alone and in complex with the diamide insecticide chlorantraniliprole. The resistance mutations perturb the local structure, directly reducing the binding affinity and altering the binding pose. Our findings elucidate the mode of action of different diamide insecticides, reveal the molecular mechanism of resistance mutations, and provide important clues for the development of novel pesticides that can bypass the resistance mutations. Pesticide resistance poses a serious threat to global food security. In this study, the authors show the mode of action of various diamide insecticides and elucidate the molecular mechanisms underlying resistance mutations. [ABSTRACT FROM AUTHOR]

Background: Vitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited. Results: In this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern. Conclusions: Our results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands. [ABSTRACT FROM AUTHOR]

As the primary Ca2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, triggering agents including halogenated volatile anaesthetics bias RyR1 to an open state resulting in uncontrolled Ca2+ release, increased sarcomere tension, and heat production. Propofol does not trigger MH and is commonly used for patients at risk of MH. The atomic-level interactions of any anaesthetic with RyR1 are unknown. RyR1 opening was measured by [3H]ryanodine binding in heavy SR vesicles (wild type) and single-channel recordings of MH mutant R615C RyR1 in planar lipid bilayers, each exposed to propofol or the photoaffinity ligand analogue m -azipropofol (AziP m). Activator-mediated wild-type RyR1 opening as a function of propofol concentration was measured by Fura-2 Ca2+ imaging of human skeletal myotubes. AziP m binding sites, reflecting propofol binding, were identified on RyR1 using photoaffinity labelling. Propofol binding affinity to a photoadducted site was predicted using molecular dynamics (MD) simulation. Both propofol and AziP m decreased RyR1 opening in planar lipid bilayers (P <0.01) and heavy SR vesicles, and inhibited activator-induced Ca2+ release from human skeletal myotube SR. Several putative propofol binding sites on RyR1 were photoadducted by AziP m. MD simulation predicted propofol K D values of 55.8 μM and 1.4 μM in the V4828 pocket in open and closed RyR1, respectively. Propofol demonstrated direct binding and inhibition of RyR1 at clinically plausible concentrations, consistent with the hypothesis that propofol partially mitigates malignant hyperthermia by inhibition of induced Ca2+ flux through RyR1. [ABSTRACT FROM AUTHOR]

Intolerance to exercise is a symptom associated with chronic heart failure (CHF) resulting in SM waste and weakness in humans. The effect of CHF on skeletal muscle (SM) arose from experimental evidence in rat models to explain the underlying mechanism. We investigated SM mechanical and metabolic properties in sham rats and with coronary ligation-induced CHF. After twelve weeks of CHF, rats were catheterized to measure right auricular pressure, SM mechanical properties, SERCA-ATPase activity and plasma membrane Ca2+-ATPase (PMCA) hydrolytic activity in isolated sarcoplasmic reticulum (SR) and transverse tubule (TT membrane), respectively, in the sham and CHF. The right auricular pressure and plasma nitrite concentration in CHF increased two-fold with respect to the sham. Pleural effusion and ascites were detected in CHF, confirming CHF. SERCA activity was conserved in CHF. In TT membranes from CHF, the glucose transporter GLUT4 increased seven-fold, and the PMCA hydrolytic activity increased five-fold, but in isolated muscle, the mechanical properties were unaffected. The absence of a deleterious effect of coronary ligation-induced CHF in the rat model on SM could be explained by the increased activity of PMCA and increased presence of GLUT-4 on the TT membrane, which may be involved in the mechanical outcome of the EDL. [ABSTRACT FROM AUTHOR]

Background: Cytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins. Methods: Given the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle. Results: In affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs. Conclusions: Our study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines. [ABSTRACT FROM AUTHOR]

Key points Previous studies demonstrated that the mitochondrial Ca2+ uniporter MCU and the Na+‐Ca2+ exchanger NCLX exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria‐SR Ca2+ interaction. However, the physiological relevance of the mitochondria‐SR Ca2+ interaction has remained unsolved. Furthermore, although mitochondrial Ca2+ has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH‐producing dehydrogenases, the contribution of the Ca2+‐dependent regulatory mechanisms to cellular functions under physiological conditions has been controversial. In this study, we constructed a new integrated model of human ventricular myocyte with excitation‐contraction‐energetics coupling and investigated systematically the contribution of mitochondria‐SR Ca2+ interaction, especially focusing on cardiac energetics during dynamic workload transitions in exercise. Simulation analyses revealed that the spatial coupling of mitochondria and SR, particularly via mitochondrial Ca2+ uniport activity‐RyR, was the primary determinant of mitochondrial Ca2+ concentration, and that the Ca2+‐dependent regulatory mechanism facilitated mitochondrial NADH recovery during exercise and contributed to the stability of NADH in the workload transition by about 40%, while oxygen consumption rate and cytoplasmic ATP level were not influenced. We concluded that the mitochondria‐SR Ca2+ interaction, created via the uneven distribution of Ca2+ handling proteins, optimizes the contribution of the mitochondrial Ca2+‐dependent regulatory mechanism to stabilizing NADH during exercise. The mitochondrial Ca2+ uniporter protein MCU and the Na+‐Ca2+ exchanger protein NCLX are reported to exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria‐SR Ca2+ interaction in cardiomyocytes. Mitochondrial Ca2+ (Ca2+mit) has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH‐producing dehydrogenases. Here we constructed an integrated model of a human ventricular myocyte with excitation‐contraction‐energetics coupling and investigated the role of the mitochondria‐SR Ca2+ interaction in cardiac energetics during exercise. Simulation analyses revealed that the spatial coupling particularly via mitochondrial Ca2+ uniport activity‐RyR is the primary determinant of Ca2+mit concentration, and that the activation of NADH‐producing dehydrogenases by Ca2+mit contributes to NADH stability during exercise. The mitochondria‐SR Ca2+ interaction optimizes the contribution of Ca2+mit to the activation of NADH‐producing dehydrogenases. [ABSTRACT FROM AUTHOR]

Ryanodine receptors (RyRs) are large Ca2+ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited. [ABSTRACT FROM AUTHOR]

EMC1 is part of the endoplasmic reticulum (ER) membrane protein complex, whose functions include the insertion of transmembrane proteins into the ER membrane, ER–mitochondria contact, and lipid exchange. Here, we show that the Drosophila melanogaster EMC1 gene is expressed in the somatic musculature and the protein localizes to the sarcoplasmic reticulum (SR) network. Muscle-specific EMC1 RNAi led to severe motility defects and partial late pupae/early adulthood lethality, phenotypes that are rescued by co-expression with an EMC1 transgene. Motility impairment in EMC1-depleted flies was associated with aberrations in muscle morphology in embryos, larvae, and adults, including tortuous and misaligned fibers with reduced size and weakness. They were also associated with an altered SR network, cytosolic calcium overload, and mitochondrial dysfunction and dysmorphology that impaired membrane potential and oxidative phosphorylation capacity. Genes coding for ER stress sensors, mitochondrial biogenesis/dynamics, and other EMC components showed altered expression and were mostly rescued by the EMC1 transgene expression. In conclusion, EMC1 is required for the SR network's mitochondrial integrity and influences underlying programs involved in the regulation of muscle mass and shape. We believe our data can contribute to the biology of human diseases caused by EMC1 mutations. [ABSTRACT FROM AUTHOR]

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H+) act to directly and indirectly (via decreased Ca2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases. [ABSTRACT FROM AUTHOR]

Introduction: The herbal preparation STW 5 ameliorates functional dyspepsia partly by relaxing smooth muscle of the proximal stomach, thus improving gastric accommodation. We explored the unknown pathways responsible for this effect by testing targets known to modulate gastric smooth muscle relaxation. Methods: STW 5‐induced relaxation of smooth muscle strips from guinea pig gastric corpus before and after pharmacological interventions were recorded with force transducers in an organ bath. ORAI1 mRNA expression was tested in the proximal stomach. Key Results: Blockade of Ca2+‐activated K+ and Cl− channels, voltage‐gated L‐ or T‐type Ca2+ channels, TRPA1‐, TRPV1‐, adenosine or 5‐HT4 receptors, antagonizing ryanodine receptors, inhibiting cyclooxygenase or sarcoplasmic reticulum calcium ATPase did not affect STW 5‐evoked relaxation. Likewise, protein‐kinase A or G were not involved. However, the relaxation evoked by STW 5 was significantly reduced by phorbol‐12‐myristat‐13‐acetat, an activator of protein‐kinase C, by 2‐ aminoethyldiphenylborinate, an inhibitor of the IP3 receptor‐mediated Ca2+ release from the sarcoplasmic reticulum or by SKF‐96365, a nonselective store‐operated calcium entry (SOCE) blocker. Furthermore, the mixed TRPC3/SOCE inhibitor Pyr3, but not the selective TRPC3 blocker Pyr10, reduced the effect of STW 5. Finally, BTP2, a potent blocker of ORAI‐coupled SOCE, almost abolished STW 5‐evoked relaxation. Expression of ORAI1 could be demonstrated in the corpus/fundus. Conclusions & Inferences: STW 5 inhibited SOCE, most likely ORAI channels, which are modulated by IP3‐ and PKC‐dependent mechanisms. Our findings impact on the design of drugs to induce muscle relaxation and help identify phytochemicals with similar modes of actions to treat gastrointestinal disturbances. [ABSTRACT FROM AUTHOR]

Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavβ4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavβ4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavβ4. Finally, we found that overexpression of Cavβ4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavβ4 subunit expression. [ABSTRACT FROM AUTHOR]

Gprotein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of Food and Drug Administration-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of tools to separate GPCR signaling at the plasma membrane from the ones initiated at intracellular compartments. We leveraged the structural and pharmacological information available for β-adrenergic receptors (βARs) and focused on β1AR as exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell-impermeable βAR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited β1AR on the plasma membrane. In contrast, a cell-permeable βAR antagonist containing a nonsulfonated fluorophore efficiently inhibited both the plasma membrane and Golgi pools of β1ARs. Furthermore, the cell-impermeable antagonist selectively inhibited the phosphorylation of PKA downstream effectors near the plasma membrane, which regulate sarcoplasmic reticulum (SR) Ca2+ release in adult cardiomyocytes, while the β1AR Golgi pool remained active. Our tools offer promising avenues for investigating compartmentalized βAR signaling in various contexts, potentially advancing our understanding of βAR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems. [ABSTRACT FROM AUTHOR]

Ca2+ release from the sarcoplasmic reticulum (SR) plays a central role in excitation-contraction coupling (ECC) in skeletal muscles. However, the mechanism by which activation of the voltage-sensors/dihydropyridine receptors (DHPRs) in the membrane of the transverse tubular system leads to activation of the Ca2+-release channels/ryanodine receptors (RyRs) in the SR is not fully understood. Recent observations showing that a very small Ca2+ leak through RyR1s in mammalian skeletal muscle can markedly raise the background [Ca2+] in the junctional space (JS) above the Ca2+ level in the bulk of the cytosol indicate that there is a diffusional barrier between the JS and the cytosol at large. Here, I use a mathematical model to explore the hypothesis that a sudden rise in Ca2+ leak through DHPR-coupled RyR1s, caused by reduced inhibition at the RyR1 Ca2+/Mg2+ inhibitory I1-sites when the associated DHPRs are activated, is sufficient to enable synchronized responses that trigger a regenerative rise of Ca2+ release that remains under voltage control. In this way, the characteristic response to Ca2+ of RyR channels is key not only for the Ca2+ release mechanism in cardiac muscle and other tissues, but also for the DHPRdependent Ca2+ release in skeletal muscle. [ABSTRACT FROM AUTHOR]

Heart failure with preserved ejection fraction (HFpEF) is commonly found in persons living with HIV (PLWH) even when antiretroviral therapy suppresses HIV viremia. However, studying this condition has been challenging because an appropriate animal model is not available. In this article, we studied calcium transient in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in culture to simulate the cardiomyocyte relaxation defect noted in PLWH and HFpEF and assess whether various drugs have an effect. We show that treatment of hiPSC-CMs with inflammatory cytokines (such as interferon-γ or TNF-α) impairs their Ca2+ uptake into sarcoplasmic reticulum and that SGLT2 inhibitors, clinically proven as effective for HFpEF, reverse this effect. Additionally, treatment with mitochondrial antioxidants (like mito-Tempo) and certain antiretrovirals resulted in the reversal of the effects of these cytokines on calcium transient. Finally, incubation of hiPSC-CMs with serum from HIV patients with and without diastolic dysfunction did not alter their Ca2+-decay time, indicating that the exposure to the serum of these patients is not sufficient to induce the decrease in Ca2+ uptake in vitro. Together, our results indicate that hiPSC-CMs can be used as a model to study molecular mechanisms of inflammation-mediated abnormal cardiomyocyte relaxation and screen for potential new interventions. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is secondarily accompanied by Ca2+ excess in muscle fibers. Part of the Ca2+ accumulates in the mitochondria, contributing to the development of mitochondrial dysfunction and degeneration of muscles. In this work, we assessed the effect of intraperitoneal administration of rhodacyanine MKT077 (5 mg/kg/day), which is able to suppress glucose-regulated protein 75 (GRP75)-mediated Ca2+ transfer from the sarcoplasmic reticulum (SR) to mitochondria, on the Ca2+ overload of skeletal muscle mitochondria in dystrophin-deficient mdx mice and the concomitant mitochondrial dysfunction contributing to muscle pathology. MKT077 prevented Ca2+ overload of quadriceps mitochondria in mdx mice, reduced the intensity of oxidative stress, and improved mitochondrial ultrastructure, but had no effect on impaired oxidative phosphorylation. MKT077 eliminated quadriceps calcification and reduced the intensity of muscle fiber degeneration, fibrosis level, and normalized grip strength in mdx mice. However, we noted a negative effect of MKT077 on wild-type mice, expressed as a decrease in the efficiency of mitochondrial oxidative phosphorylation, SR stress development, ultrastructural disturbances in the quadriceps, and a reduction in animal endurance in the wire-hanging test. This paper discusses the impact of MKT077 modulation of mitochondrial dysfunction on the development of skeletal muscle pathology in mdx mice. [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]

Membrane IP3 receptors (IP3Rs) abound in the sarcoplasmic reticulum, nucleus and mitochondria of muscle fibers. We hypothesized that IP3R activation during muscle unloading may elicit a weak Ca2+ release signal, both cytosolic and nucleoplasmic, which promotes (perhaps in cooperation with other signaling cascades) the activation of transcription factors and thus leads to the expression or repression of genes associated with muscle phenotypes. Here, we tested this hypothesis by blocking IP3Rs with 2-APB (2-aminoethoxydiphenyl borate, 10 mg/kg in 5% DMSO i.p. daily) in a rat 7-day hindlimb suspension model of soleus muscle unloading. The blocking of IP3Rs prevented a decrease in the cross-sectional area of both slow and fast soleus muscle fibers and thus slowed down the development of atrophic processes in this postural muscle during 7-day hindlimb suspension. Such an effect of blocking IP3Rs during rat soleus muscle unloading may be due to preventing a decrease in ribosomal biogenesis and an increase in the expression of autophagy markers ULK-1 and IL-6. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the impact of developmental endothelial locus-1 (Del-1) on sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and its potential effects on spinal cord injury (SCI). A total of 48 mice were randomly assigned to the sham group, SCI group, and SCI + CE group. Each group was further divided into two subgroups. The Del treatment subgroup received tail-vein injections of Del-1 (1 μg/d) for a consecutive week, while the other group was injected with an equivalent volume of normal saline. After 1 week of experimentation, the mice were euthanized, and the spinal cord was extracted for further analysis. Initially, the impact of Del-1 on SERCA2 in the spinal cord was assessed using western blotting analysis. Subsequently, the effects of Del-1 on stress, inflammation, and apoptosis in the endoplasmic reticulum (ER) of SCI mice were analyzed through Western blotting analysis and fluorescent TUNEL. Finally, the study utilized Western blotting analysis and fluorescent TUNEL analysis to examine the consequences of Del-1 blocking SERCA2 on ER stress, inflammation, and apoptosis in mice. The results revealed that Del-1 treatment significantly increased the expression of SERCA2 in the spinal cord (P < 0.01) and mitigated SCI-induced ER stress, inflammation, and neuronal apoptosis (P < 0.01). Blocking SERCA2 expression in the spinal cord of SCI mice promoted ER stress, inflammatory response, and neuronal cell apoptosis (P < 0.01). However, Del-1 treatment did not alleviate the effects of blocking SERCA2 on ER stress, inflammatory response, and neuronal cell apoptosis (P > 0.05). In conclusion, this study proposed that Del-1 could reduce ER stress, inflammatory response, and nerve cell apoptosis in spinal cord injury by enhancing the expression of SERCA2. [ABSTRACT FROM AUTHOR]

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2′-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions. [ABSTRACT FROM AUTHOR]

CCDC78 was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a CCDC78 nonsense mutation to better understand the role of CCDC78 in muscle. Methods: We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78-mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects. Results: The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel CCDC78 transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1. Conclusions: Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4. [ABSTRACT FROM AUTHOR]

A recent study published in the Journal of General Physiology introduces a new tool for estimating the subcellular distribution of glycogen in skeletal muscle. Traditionally, glycogen content has been measured using chemical methods that provide an average measurement, but this overlooks the uneven distribution of glycogen granules within muscle fibers. The study demonstrates how artificial intelligence can be used to quantify the subcellular distribution of glycogen granules, which could provide valuable insights into the relationship between glycogen content and glucose uptake in muscle cells. The article also discusses the need for further research into the subcellular distribution and regulation of glycogen in skeletal muscle, suggesting the use of transmission electron microscopy and artificial intelligence for quantitative image analysis. [Extracted from the article]

Aim: Parvalbumin (PV) is a primary calcium buffer in mouse fast skeletal muscle fibers. Previous work showed that PV ablation has a limited impact on cytosolic Ca2+ ([Ca2+]cyto) transients and contractile response, while it enhances mitochondrial density and mitochondrial matrix‐free calcium concentration ([Ca2+]mito). Here, we aimed to quantitatively test the hypothesis that mitochondria act to compensate for PV deficiency. Methods: We determined the free Ca2+ redistribution during a 2 s 60 Hz tetanic stimulation in the sarcoplasmic reticulum, cytosol, and mitochondria. Via a reaction–diffusion Ca2+ model, we quantitatively evaluated mitochondrial uptake and storage capacity requirements to compensate for PV lack and analyzed possible extracellular export. Results: [Ca2+]mito during tetanic stimulation is greater in knock‐out (KO) (1362 ± 392 nM) than in wild‐type (WT) (855 ± 392 nM), p < 0.05. Under the assumption of a non‐linear intramitochondrial buffering, the model predicts an accumulation of 725 μmoles/Lfiber (buffering ratio 1:11 000) in KO, much higher than in WT (137 μmoles/Lfiber, ratio 1:4500). The required transport rate via mitochondrial calcium uniporter (MCU) reaches 3 mM/s, compatible with available literature. TEM images of calcium entry units and Mn2+ quenching showed a greater capacity of store‐operated calcium entry in KO compared to WT. However, levels of [Ca2+]cyto during tetanic stimulation were not modulated to variations of extracellular calcium. Conclusions: The model‐based analysis of experimentally determined calcium distribution during tetanic stimulation showed that mitochondria can act as a buffer to compensate for the lack of PV. This result contributes to a better understanding of mitochondria's role in modulating [Ca2+]cyto in skeletal muscle fibers. [ABSTRACT FROM AUTHOR]

BackgroundNitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.MethodsWe measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).ResultsBoth WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.ConclusionsWe conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Abstract Background Vitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited. Results In this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern. Conclusions Our results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands.

BackgroundCalcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis.MethodsWe developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting.ResultsSM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions.ConclusionsSparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.

In the conducted studies, the effect of vincanine and pyrazoline indole alkaloids on cardiac muscle cell sarcoplasmic reticulum (SR) Ca2+-transport systems (RyR2 and SERCA2a) was investigated under normal and hypoxic conditions. The effect of vincanine and pyrazoline on ryanodine receptor (RyR2) was evaluated in the presence of ruthenium red and it was found that the role of RyR2 in the positive inotropic effect of these alkaloids is small. Also, in the presence of cyclopiazonic acid (IC50 = 5.6 µM), an inhibitor of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), the positive inotropic effect of vincanine and pyrazoline alkaloids was reduced. In general, the positive inotropic effects and cardioprotective properties of vincanine and pyrazoline alkaloids are mediated by modulation of SERCA2a function. [ABSTRACT FROM AUTHOR]

Tubular aggregate myopathy (TAM) is a rare myopathy characterized by muscle weakness and myalgia. Muscle fibers from TAM patients show characteristic accumulation of membrane tubules that contain proteins from the sarcoplasmic reticulum (SR). Gain-of-function mutations in STIM1 and ORAI1, the key proteins participating in the Store-Operated Ca2+ Entry (SOCE) mechanism, were identified in patients with TAM. Recently, the CASQ1 gene was also found to be mutated in patients with TAM. CASQ1 is the main Ca2+ buffer of the SR and a negative regulator of SOCE. Previous characterization of CASQ1 mutants in non-muscle cells revealed that they display altered Ca2+dependent polymerization, reduced Ca2+storage capacity and alteration in SOCE inhibition. We thus aimed to assess how mutations in CASQ1 affect calcium regulation in skeletal muscles, where CASQ1 is naturally expressed. We thus expressed CASQ1 mutants in muscle fibers from Casq1 knockout mice, which provide a valuable model for studying the Ca2+ storage capacity of TAM-associated mutants. Moreover, since Casq1 knockout mice display a constitutively active SOCE, the effect of CASQ1 mutants on SOCE inhibition can be also properly examined in fibers from these mice. Analysis of intracellular Ca2+ confirmed that CASQ1 mutants have impaired ability to store Ca2+and lose their ability to inhibit skeletal muscle SOCE; this is in agreement with the evidence that alterations in Ca2+entry due to mutations in either STIM1, ORAI1 or CASQ1 represents a hallmark of TAM. [ABSTRACT FROM AUTHOR]

Background: The ryanodine receptor 3 (RYR3) is involved in skeletal muscle contraction by releasing calcium from the sarcoplasmic reticulum and subsequent T-tubule depolarization. It is also expressed in the brain, and variants in the RYR3 gene can lead to congenital myopathy type 20 (MIM: #620310). Methods: We retrospectively analyzed the clinical characteristics and prognosis of a case of West syndrome, developmental and epileptic encephalopathy (DEE) caused by a missense variant in the RYR3 gene. We also reviewed and summarized the literature on epilepsy cases caused by RYR3 gene variants. Results: A 10-month-old female child with delayed psychomotor development and recurrent spasm-like seizures was diagnosed with infantile spasm syndrome and DEE. Treatment with various antiepileptic drugs resulted in initial improvement but ultimately failed to control the seizures. Whole-exome sequencing revealed a novel heterozygous variant c.10943C  >  T/p.T3648M in the RYR3 gene, and genome-wide sequencing ruled out other potentially pathogenic variants. Three previous reports have described RYR3 variants causing DEE, two of which were attributed to de novo heterozygous variants, and one was a compound heterozygote. Conclusion: The present case of DEE caused by a RYR3 heterozygous variant is consistent with previous rare cases of epilepsy caused by RYR3 gene variants in terms of pathogenesis and clinical features, but significantly different from congenital myopathy type 20. Our findings provide important evidence for the diagnosis of RYR3-related DEE, and we hypothesize that RYR3 gain-of-function variants resulting in “leaky” Ca2+ release channels may be a molecular genetic feature leading to DEE rather than myopathy. [ABSTRACT FROM AUTHOR]

This article explores the movement of sarcomeres in the heart muscle and the role of the protein titin in this process. It challenges previous beliefs by showing that sarcomeres do not all move in synchrony during muscle contraction. The asynchrony is influenced by factors such as sarcomere length and calcium levels. The article suggests that this asynchrony may contribute to impaired heart function in conditions like dilated cardiomyopathy. Further research is needed to understand the link between sarcomere movement and heart disease. [Extracted from the article]

Mutations in ubiquitously expressed presenilin genes (PSENs) lead to early-onset familial Alzheimer's disease (FAD), but patients carrying the mutation also suffer from heart diseases. To elucidate the cardiac myocyte specific effects of PSEN ΔE9, we studied cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) from patients carrying AD-causing PSEN1 exon 9 deletion (PSEN1 ΔE9). When compared with their isogenic controls, PSEN1 ΔE9 cardiomyocytes showed increased sarcoplasmic reticulum (SR) Ca2+ leak that was resistant to blockage of ryanodine receptors (RyRs) by tetracaine or inositol-3-reseceptors (IP 3 Rs) by 2-ABP. The SR Ca2+ leak did not affect electrophysiological properties of the hiPSC-CMs, but according to experiments and in silico simulations the leak induces a diastolic buildup of [Ca2+] near the perinuclear SR and reduces the releasable Ca2+ during systole. This demonstrates that PSEN1 ΔE9 induced SR Ca2+ leak has specific effects in iPSC-CMs, reflecting their unique structural and calcium signaling features. The results shed light on the physiological and pathological mechanisms of PSEN1 in cardiac myocytes and explain the intricacies of comorbidity associated with AD-causing mutations in PSEN1. [Display omitted] • PSEN1 ΔE9 mutation causes sarcoplasmic reticulum Ca2+ leak in hiPSC-cardiomyocytes. • Increased Ca2+-leak causes a diastolic buildup of [Ca2+] near the perinuclear SR. • SR Ca2+-leak reduces the releasable Ca2+ during systole. • The leak does not affect electrical properties of the hiPSC-cardiomyocytes. • In silico simulations reveal complex autoregulation of SR Ca2+ loading in iPSC-CMs. [ABSTRACT FROM AUTHOR]

Interventions aimed at weight control often have limited effectiveness in combating obesity. This review explores how obesity‐induced dysfunction in white (WAT) and brown adipose tissue (BAT), skeletal muscle, and the brain blunt weight loss, leading to retention of stored fat. In obesity, increased adrenergic stimulation and inflammation downregulate β‐adrenoreceptors and impair catecholaminergic signalling in adipocytes. This disrupts adrenergic‐mediated lipolysis, diminishing lipid oxidation in both white and brown adipocytes, lowering thermogenesis and blunting fat loss. Emerging evidence suggests that WAT fibrosis is associated with worse weight loss outcomes; indeed, limiting collagen and laminin‐α4 deposition mitigates WAT accumulation, enhances browning, and protects against high‐fat‐diet‐induced obesity. Obesity compromises mitochondrial oxidative capacity and lipid oxidation in skeletal muscle, impairing its ability to switch between glucose and lipid metabolism in response to varying nutrient levels and exercise. This dysfunctional phenotype in muscle is exacerbated in the presence of obesity‐associated sarcopenia. Additionally, obesity suppresses sarcolipin‐induced sarcoplasmic reticulum calcium ATPase (SERCA) activation, resulting in reduced oxidative capacity, diminished energy expenditure, and increased adiposity. In the hypothalamus, obesity and overnutrition impair insulin and leptin signalling. This blunts central satiety signals, favouring a shift in energy balance toward energy conservation and body fat retention. Moreover, both obese animals and humans demonstrate impaired dopaminergic signalling and diminished responses to nutrient intake in the striatum, which tend to persist after weight loss. This may result in enduring inclinations toward overeating and a sedentary lifestyle. Collectively, the tissue adaptations described pose significant challenges to effectively achieving and sustaining weight loss in obesity. [ABSTRACT FROM AUTHOR]

Aims: Hyperactivity of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII‐induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII‐dependent RyR2 phosphorylation for CaMKII‐induced heart failure development in vivo. Methods and results: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2‐S2814A knock‐in mice that are resistant to CaMKII‐dependent RyR2 phosphorylation. Ca2+‐spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+‐leak induction in the CaMKIIδC TG, both regarding Ca2+‐spark size and frequency, demonstrating that the CaMKIIδC‐induced SR Ca2+ leak entirely depends on the CaMKII‐specific RyR2‐S2814 phosphorylation. Yet, the RyR2‐S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. Conclusions: S2814 phosphoresistance of RyR2 prevents the CaMKII‐dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms—independent of SR Ca2+ leak—are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure. [ABSTRACT FROM AUTHOR]

(1) Background: The main factors and their interrelationships contributing to cardiac repolarization alternans (CRA) remain unclear. This study aimed to elucidate the calcium (Ca2+)-related mechanisms underlying myocardial ischemia (MI)-induced CRA. (2) Materials and Methods: CRA was induced using S1 stimuli for pacing in an in silico ventricular model with MI. The standard deviations of nine Ca2+-related subcellular parameters among heartbeats from 100 respective nodes with and without alternans were chosen as features, including the maximum systole and end-diastole and corresponding differences in the Ca2+ concentration in the intracellular region([Ca2+]i) and junctional sarcoplasmic reticulum ([Ca2+]jsr), as well as the maximum opening of the L-type Ca2+ current (ICaL) voltage-dependent activation gate (d-gate), maximum closing of the inactivation gate (ff-gate), and the gated channel opening time (GCOT). Feature selection was applied to determine the importance of these features. (3) Results: The major parameters affecting CRA were the differences in [Ca2+]i at end-diastole, followed by the extent of d-gate activation and GCOT among beats. (4) Conclusions: MI-induced CRA is primarily characterized by functional changes in Ca2+ re-uptake, leading to alternans of [Ca2+]i and subsequent alternans of ICaL-dependent properties. The combination of computational simulation and machine learning shows promise in researching the underlying mechanisms of cardiac electrophysiology. [ABSTRACT FROM AUTHOR]

Store-operated Ca2+ entry (SOCE) is a ubiquitous cellular mechanism that cells use to activate extracellular Ca2+ entry when intracellular Ca2+ stores are depleted. In skeletal muscle, SOCE occurs within Ca2+ entry units (CEUs), intracellular junctions between stacks of SR membranes containing STIM1 and transverse tubules (TTs) containing ORAI1. Gain-of-function mutations in STIM1 and ORAI1 are linked to tubular aggregate (TA) myopathy, a disease characterized by the atypical accumulation of tubes of SR origin. Moreover, SOCE and TAs are increased in the muscles of aged male mice. Here, we assessed the longitudinal effects (from 4–6 months to 10–14 months of age) of constitutive, muscle-specific Orai1 knockout (cOrai1 KO) on skeletal muscle structure, function, and the assembly of TAs and CEUs. The results from these studies indicate that cOrai1 KO mice exhibit a shorter lifespan, reduced body weight, exercise intolerance, decreased muscle-specific force and rate of force production, and an increased number of structurally damaged mitochondria. In addition, electron microscopy analyses revealed (i) the absence of TAs with increasing age and (ii) an increased number of SR stacks without adjacent TTs (i.e., incomplete CEUs) in cOrai1 KO mice. The absence of TAs is consistent with TAs being formed as a result of excessive ORAI1-dependent Ca2+ entry. [ABSTRACT FROM AUTHOR]

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Spending time in a microgravity environment is known to cause significant skeletal muscle atrophy and weakness via muscle unloading, which can be partly attributed to Ca2+ dysregulation. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for bringing Ca2+ from the cytosol into its storage site, the sarcoplasmic reticulum (SR), at the expense of ATP. We have recently demonstrated that, in the soleus of space-flown mice, the Ca2+ uptake ability of the SERCA pump is severely impaired and this may be attributed to increases in reactive oxygen/nitrogen species (RONS), to which SERCA is highly susceptible. The purpose of this study was therefore to investigate whether treatment with the antioxidant, Manganese(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+ (BuOE), could attenuate muscle atrophy and SERCA dysfunction. We received soleus muscles from the rodent research 18 mission which had male mice housed on the international space station for 35 days and treated with either saline or BuOE. Spaceflight significantly reduced the soleus:body mass ratio and significantly increased SERCA's ionophore ratio, a measure of SR Ca2+ leak, and 4-HNE content (marker of RONS), none of which could be rescued by BuOE treatment. In conclusion, we find that spaceflight induces significant soleus muscle atrophy and SR Ca2+ leak that cannot be counteracted with BuOE treatment. Future work should investigate alternative therapeutics that are specifically aimed at increasing SERCA activation or reducing Ca2+ leak. [ABSTRACT FROM AUTHOR]

S100A1, a small homodimeric EF-hand Ca2+-binding protein (~21 kDa), plays an important regulatory role in Ca2+ signaling pathways involved in various biological functions including Ca2+ cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca2+ release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca2+. Ca2+-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca2+-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca2+-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca2+ release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders. [ABSTRACT FROM AUTHOR]

Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 μM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+/Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+] and [Ca2+]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+] revealed that MGO increases cytosolic [Na+] by 57% from the maximal effect produced by the Na+-K+ ATPase inhibitor ouabain (20 μM). This increase in cytosolic [Na+] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+-K+ ATPase. An increase in cytosolic [Na+] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves. [ABSTRACT FROM AUTHOR]

Calreticulin (CRT) is an intrinsically disordered multifunctional protein that plays essential roles intra-and extra-cellularly. The Michalak laboratory has proposed that CRT was initially identified in 1974 by the MacLennan laboratory as the high-affinity Ca2+-binding protein (HACBP) of the sarcoplasmic reticulin (SR). This widely accepted belief has been ingrained in the scientific literature but has never been rigorously tested. In our report, we have undertaken a comprehensive reexamination of this assumption by meticulously examining the majority of published studies that present a proteomic analysis of the SR. These analyses have utilized proteomic analysis of purified SR preparations or purified components of the SR, namely the longitudinal tubules and junctional terminal cisternae. These studies have consistently failed to detect the HACBP or CRT in skeletal muscle SR. We propose that the existence of the HACBP has failed the test of reproducibility and should be retired to the annals of antiquity. Therefore, the scientific dogma that the HACBP and CRT are identical proteins is a non sequitur. [ABSTRACT FROM AUTHOR]

Heterozygous RTN2 variants have been previously identified in a limited cohort of families affected by autosomal dominant spastic paraplegia (SPG12-OMIM:604805) with a variable age of onset. Nevertheless, the definitive validity of SPG12 remains to be confidently confirmed due to the scarcity of supporting evidence. In this study, we identified and validated seven novel or ultra-rare homozygous loss-of-function RTN2 variants in 14 individuals from seven consanguineous families with distal hereditary motor neuropathy (dHMN) using exome, genome and Sanger sequencing coupled with deep-phenotyping. All affected individuals (seven males and seven females, aged 9–50 years) exhibited weakness in the distal upper and lower limbs, lower limb spasticity and hyperreflexia, with onset in the first decade of life. Nerve conduction studies revealed axonal motor neuropathy with neurogenic changes in the electromyography. Despite a slowly progressive disease course, all patients remained ambulatory over a mean disease duration of 19.71 ± 13.70 years. Characterization of Caenorhabditis elegans RTN2 homologous loss-of-function variants demonstrated morphological and behavioural differences compared with the parental strain. Treatment of the mutant with an endoplasmic/sarcoplasmic reticulum Ca2+ reuptake inhibitor (2,5-di-tert-butylhydroquinone) rescued key phenotypic differences, suggesting a potential therapeutic benefit for RTN2-disorder. Despite RTN2 being an endoplasmic reticulum (ER)-resident membrane shaping protein, our analysis of patient fibroblast cells did not find significant alterations in ER structure or the response to ER stress. Our findings delineate a distinct form of autosomal recessive dHMN with pyramidal features associated with RTN2 deficiency. This phenotype shares similarities with SIGMAR1-related dHMN and Silver-like syndromes, providing valuable insights into the clinical spectrum and potential therapeutic strategies for RTN2-related dHMN. [ABSTRACT FROM AUTHOR]

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca2+ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca2+ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC50 of -0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca2+ transient and sarcoplasmic reticulum Ca2+ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca2+ transients by DPI. The L-type Ca2+ current (ICa) was decreased concentration-dependently by DPI (IC50 of -40.3 µM) with -13.1%-inhibition at 3 µM. The frequency of Ca2+ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca2+ and Na+. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca2+ channel and RyR, thereby attenuating Ca2+-induced Ca2+ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression. [ABSTRACT FROM AUTHOR]

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and β-adrenergic receptor (βAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during βAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity. NEW & NOTEWORTHY: S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status. [ABSTRACT FROM AUTHOR]

Background: Brugada syndrome (BrS) is an inherited primary channelopathy syndrome associated with the risk of ventricular fibrillation (VF) and sudden cardiac death in a structurally normal heart. Aim of the Study: The aim of this study was to clinically and genetically evaluate a large family with severe autosomal dominant Brugada syndrome. Methods: Clinical and genetic studies were performed. Genetic analysis was conducted with NGS technologies (WES) using the Illumina instrument. According to the standard procedure, variants found by WES were confirmed in all available families by Sanger sequencing. The effect of the variants was studied by using in silico prediction of pathogenicity. Results: The proband was a 52-year-old man who was admitted to the emergency department for syncope at rest. WES of the index case identified a heterozygous VUS CASQ2, c.532T>C, p.(Tyr178His). We studied the segregation of the variation in all pedigree members. All the patients were heterozygous for the variation CASQ2 p.(Tyr178His), whereas the remaining healthy individuals in the family were homozygous for the normal allele. Structural analysis of CASQ2 p.(Tyr178His) was performed and revealed an important effect of the missense variation on monomer stability. The CASQ2 Tyr180 residue is located inside the sarcoplasmic reticulum (SR) junctional face membrane interaction domain and is predicted to disrupt filamentation. Conclusions: Our data suggest that the p.Tyr178His substitution is associated with BrS in the family investigated, affecting the stability of the protein, disrupting filamentation at the interdimer interface, and affecting the subsequent formation of tetramers and polymers that contain calcium-binding sites. [ABSTRACT FROM AUTHOR]

We studied the effect of enteral administration of the glucocorticoid deflazacort (DFC, 1.2 mg/kg per day, 28 days) on the state of skeletal muscles and tissue ultrastructure, as well as the composition of the colon microbiota in dystrophin-deficient mdx mice. DFC has been shown to reduce the intensity of degeneration/regeneration cycles in muscle fibers of mdx mice. This effect of DFC was accompanied by normalization of the size of sarcomeres of skeletal muscles of mdx mice, improvement of the ultrastructure of the subsarcolemmal population of mitochondria, and an increase in the number of organelles, as well as normalization of the number of contact interactions between the sarcoplasmic reticulum and mitochondria. In addition, DFC had a corrective effect on the colon microbiota of mdx mice, which manifested in an increase in the number of the Bifidobacterium genus microorganisms and a decrease in the level of E. coli with reduced enzymatic activity. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction (AMI) is a serious condition that occurs when part of the heart is subjected to ischemia episodes, following partial or complete occlusion of the epicardial coronary arteries. The resulting damage to heart muscle cells have a significant impact on patient's health and quality of life. About that, recent research focused on the role of the sarcoplasmic reticulum (SR) and mitochondria in the physiopathology of AMI. Moreover, SR and mitochondria get in touch each other through multiple membrane contact sites giving rise to the subcellular region called mitochondria-associated membranes (MAMs). MAMs are essential for, but not limited to, bioenergetics and cell fate. Disruption of the architecture of these regions occurs during AMI although it is still unclear the cause-consequence connection and a complete overview of the pathological changes; for sure this concurs to further damage to heart muscle. The calcium ion (Ca2+) plays a pivotal role in the pathophysiology of AMI and its dynamic signaling between the SR and mitochondria holds significant importance. In this review, we tried to summarize and update the knowledge about the roles of these organelles in AMI from a Ca2+ signaling point of view. Accordingly, we also reported some possible cardioprotective targets which are directly or indirectly related at limiting the dysfunctions caused by the deregulation of the Ca2+ signaling. [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]