Sarcoplasmic Reticulum Ca2 Research Articles

Personalized medicine in the dish to prevent calcium leak associated with short-coupled polymorphic ventricular tachycardia in patient-derived cardiomyocytes

BackgroundPolymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro.MethodsPreviously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs.ResultsThe voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control.ConclusionsBy testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.

Elementary intracellular Ca signals approximated as a transition of release channel system from a metastable state.

Cardiac muscle contraction is initiated by an elementary Ca signal (called Ca spark) which is achieved by collective action of Ca release channels in a cluster. The mechanism of this synchronization remains uncertain. We approached Ca spark activation as an emergent phenomenon of an interactive system of release channels. We constructed a weakly lumped Markov chain that applies an Ising model formalism to such release channel clusters and probable open channel configurations and demonstrated that spark activation is described as a system transition from a metastable to an absorbing state, analogous to the pressure required to overcome surface tension in bubble formation. This yielded quantitative estimates of the spark generation probability as a function of various system parameters. We performed numerical simulations to find spark probabilities as a function of sarcoplasmic reticulum Ca concentration, obtaining similar values for spark activation threshold as our analytic model, as well as those reported in experimental studies. Our parametric sensitivity analyses also showed that the spark activation threshold decreased as Ca sensitivity of RyR activation and RyR cluster size increased.

Biochemical and proteomic insights into sarcoplasmic reticulum Ca2+-ATPase complexes in skeletal muscles

ABSTRACT Introduction Skeletal muscles contain large numbers of high-molecular-mass protein complexes in elaborate membrane systems. Integral membrane proteins are involved in diverse cellular functions including the regulation of ion handling, membrane homeostasis, energy metabolism and force transmission. Areas covered The proteomic profiling of membrane proteins and large protein assemblies in skeletal muscles are outlined in this article. This includes a critical overview of the main biochemical separation techniques and the mass spectrometric approaches taken to study membrane proteins. As an illustrative example of an analytically challenging large protein complex, the proteomic detection and characterization of the Ca2+-ATPase of the sarcoplasmic reticulum is discussed. The biological role of this large protein complex during normal muscle functioning, in the context of fiber type diversity and in relation to mechanisms of physiological adaptations and pathophysiological abnormalities is evaluated from a proteomics perspective. Expert opinion Mass spectrometry-based muscle proteomics has decisively advanced the field of basic and applied myology. Although it is technically challenging to study membrane proteins, innovations in protein separation methodology in combination with sensitive mass spectrometry and improved systems bioinformatics has allowed the detailed proteomic detection and characterization of skeletal muscle membrane protein complexes, such as Ca2+-pump proteins of the sarcoplasmic reticulum.

Comparative Efficacy of Levosimendan, Ramipril, and Sacubitril/ Valsartan in Isoproterenol-induced Experimental Heart Failure: A Hemodynamic and Molecular Approach.

Cardiac ischemia-related myocardial damage has been considered a major reason for heart failure. We aimed to investigate the role of levosimendan (LEVO) in comparison to ramipril and sacubitril/valsartan (Sac/Val) in preventing damage associated with isoproterenol (ISO) induced myocardial infarction. Myocardial infarction was induced by injecting subcutaneous isoproterenol (5 mg/kg once for 7 consecutive days) to establish an experimental heart failure model. Simultaneously, LEVO (1 mg/kg/day), ramipril (3mg/kg/day) and Sac/Val (68 mg/kg/day) suspension were administered orally for four weeks. We observed a significant correlation between ISO-induced ischemia with cardiac remodeling and alterations in myocardial architecture. LEVO, ramipril, and Sac/Val significantly prevented lipid peroxidation and damaged antioxidant enzymes like superoxide dismutase, catalase, glutathione and thioredoxin reductase. We also observed their ameliorative effects in myocardium's cardiac hypertrophy, evidenced by reduced heart weight to body weight ratio and transforming growth factor β related collagen deposition. LEVO, ramipril, and Sac/Val also maintained cardiac biomarkers like lactate dehydrogenase, creatine kinase-MB, brain natriuretic peptide and cardiac Troponin-I, indicating reduced myocardial damage that was further demonstrated by histopathological examination. Decreased sarcoplasmic endoplasmic reticulum Ca2+ATPase2a and sodium-calcium exchanger-1 protein depletion after LEVO, ramipril, and Sac/Val administration indicated improved Ca2+ homeostasis during myocardial contractility. Our findings suggest that LEVO has comparable effects to ramipril, and Sac/Val in preventing myocardial damage via balancing oxidant-antioxidant system, decreased collagen deposition, reduced myocardial stress as well as improved Ca2+ homeostasis during myocardial contractility.

5-Methoxytryptophan alleviates atrial structural remodeling in ibrutinib-associated atrial fibrillation

BackgroundIbrutinib is an effective and well-tolerated treatment for B-cell lymphomas but is associated with an increased risk of atrial fibrillation (AF) by altering the structure of the atrium. 5-Methoxytryptophan (5-MTP) inhibits inflammatory and fibrotic processes. This study aimed to determine the effects and mechanisms of 5-MTP on the underlying mechanisms of AF caused by ibrutinib. MethodsThe effect of 5-MTP on ibrutinib-related AF was investigated in male Sprague Dawley rats using echocardiographic, electrophysiological, immunofluorescent, Masson staining, and molecular analyses. RusultsThe ibrutinib+5-MTP group showed (1) a lower incidence and shorter duration of AF and accelerated atrial conduction; (2) a decreased left atrial mass and left atrial diameter; (3) decreased myocardial fibrosis in the left atrium; (4) lower atrial inflammation; (5) increased sarcoplasmic reticulum Ca2+-ATPase 2a protein expression, decreased phosphorylation of phospholamban at Thr17, and decreased sodium/calcium exchanger 1 protein expression and phosphorylation of ryanodine receptor 2 at S2814; and (6) decreased phosphorylation of CaMKII expression. 5-MTP treatment markedly activated the PI3K-Akt signaling. Inhibiting PI3K-Akt signaling significantly reversed the protective effect of 5-MTP on ibrutinib-related AF. ConclusionsThese findings suggest that 5-MTP administration decreases the vulnerability of ibrutinib-related AF mainly caused by ameliorated maladaptive left atrial remodeling and dysregulation of calcium handling proteins. Mechanistically, 5-MTP treatment markedly enhanced the activation of cardiac PI3K-Akt signaling.

Repeated witness social stress causes cardiomyocyte contractile impairment and intracellular Ca2+ derangement in female rats

The impact of psychosocial stressors on cardiovascular health in women is of growing interest in both the popular and scientific literature. Rodent models are useful for providing direct experimental evidence of the adverse cardiovascular consequences of psychosocial stressors, yet studies in females are scarce. Here, we investigated the effects of repeated exposure to witness social defeat stress (WS) on cardiomyocyte contractile function and intracellular Ca2+ homeostasis in young adult wild-type Groningen female rats. Female rats bore witness to an aggressive social defeat episode between two males for nine consecutive days or were exposed to a control procedure. Stress-related behaviors were assessed during the first and last WS/control exposure. Twenty-four hours after the last exposure, plasma corticosterone levels were measured, and cardiomyocytes were isolated for analyses of contractile properties and Ca2+ transients, and expression levels of proteins involved in intracellular Ca2+dynamics. The results show an impairment of the intrinsic cardiac mechanical properties and prolonged intracellular Ca2+decay in WS female rats showing social stress-related behavioral (larger amounts of burying behavior) and neuroendocrine (elevated plasma corticosterone levels) phenotypes. Further, the results implicate alterations in the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex in the contractile defects described in cardiomyocytes of WS female rats. In conclusion, this study highlights the utility of the WS model as an ethologically relevant social stressor for investigating pathophysiological processes that occur in the heart of female subjects and may increase vulnerability to social stress-related cardiovascular risk.

Glucagon-like Peptide-1 Receptor Activation Reduces Pulmonary Vein Arrhythmogenesis and Regulates Calcium Homeostasis.

Glucagon-like peptide-1 (GLP-1) receptor agonists are associated with reduced atrial fibrillation risk, but the mechanisms underlying this association remain unclear. The GLP-1 receptor agonist directly impacts cardiac Ca2+ homeostasis, which is crucial in pulmonary vein (PV, the initiator of atrial fibrillation) arrhythmogenesis. This study investigated the effects of the GLP-1 receptor agonist on PV electrophysiology and Ca2+ homeostasis and elucidated the potential underlying mechanisms. Conventional microelectrodes and whole-cell patch clamp techniques were employed in rabbit PV tissues and single PV cardiomyocytes before and after GLP-1 (7-36) amide, a GLP-1 receptor agonist. Evaluations were conducted both with and without pretreatment with H89 (10 μM, an inhibitor of protein kinase A, PKA), KN93 (1 μM, an inhibitor of Ca2+/calmodulin-dependent protein kinase II, CaMKII), and KB-R7943 (10 μM, an inhibitor of Na+/Ca2+ exchanger, NCX). Results showed that GLP-1 (7-36) amide (at concentrations of 1, 10, and 100 nM) reduced PV spontaneous activity in a concentration-dependent manner without affecting sinoatrial node electrical activity. In single-cell experiments, GLP-1 (7-36) amide (at 10 nM) reduced L-type Ca2+ current, NCX current, and late Na+ current in PV cardiomyocytes without altering Na+ current. Additionally, GLP-1 (7-36) amide (at 10 nM) increased sarcoplasmic reticulum Ca2+ content in PV cardiomyocytes. Furthermore, the antiarrhythmic effects of GLP-1 (7-36) amide on PV automaticity were diminished when pretreated with H89, KN93, or KB-R7943. This suggests that the GLP-1 receptor agonist may exert its antiarrhythmic potential by regulating PKA, CaMKII, and NCX activity, as well as modulating intracellular Ca2+ homeostasis, thereby reducing PV arrhythmogenesis.

Sex and Gene Influence Arrhythmia Susceptibility in Murine Models of Calmodulinopathy.

Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.

Abstract P1034: Functional Modelling Of Arrhythmias With Human Cardiac Organoids

Engineered heart tissues (EHTs) have become a well-established model of human cardiac muscle. Metabolic maturity, improved sarcoplasmic reticulum (SR) Ca2+ handling, and the incorporation of critical non-myocyte cell populations are advantages that EHTs provide over immature 2D cardiomyocyte models. Our human cardiac organoid-EHT hybrid (hCO) platform was developed to be compatible with standardized 96-well tissue culture formats to enable high-throughput functional screening, while retaining the physiological advantages of larger EHTs. Here, we explored the applicability of hCOs as a model of various arrhythmic mechanisms. QT-prolongation could be mimicked with LTCC agonist BayK8644 or hERG blocker dofetilide, which increased the relaxation time of the hCOs and induced arrhythmia at high concentrations. Ectopy was induced by perturbing SR Ca2+ cycling with ryanodine or caffeine. HCN4 blocker ivabradine slowed rate, and at high concentrations produced a complex pause-burst phenotype. Increasing the cardiomyocyte-to-fibroblast ratio with growth factors, or the addition of extra fibroblasts prolonged contraction duration and induced ectopy. Inflammatory cytokines also increased relaxation time and provoked ectopy. From this data we propose that hCOs are a sensitive and reliable platform for studying arrhythmias and we are currently exploring the impact of CPVT-linked RYR2 and CASQ2 mutations on hCO arrhythmogenesis.

Abstract P2164: Expression Of Engineered Bacterial Sodium Channel Improves Cardiomyocyte Contractility

Introduction: Our recent studies in non-human primates have demonstrated adeno-associated virus (AAV) delivery of prokaryotic sodium channel (BacNa v ) can improve the contractile function of infarcted hearts. We hypothesized that BacNa v expression increases cardiomyocyte (CM) contractile function by indirectly augmenting Ca 2+ transient amplitude via increase in sarcoplasmic reticulum (SR) Ca 2+ stores. Methods: We developed an in vitro model of ischemia-reperfusion (I/R) injury in engineered heart tissues (cardiopatches) made from human iPSC-derived CMs (hiPSC-CMs). Prior to formation, tissues were transduced with lentivirus conferring a doxycycline (Dox)-inducible expression of BacNa v . Upon I/R injury, the tissues were treated with either vehicle or Dox for 48h followed by force testing and Ca 2+ imaging. The effect of BacNa v expression on Ca 2+ handling was additionally studied using neonatal rat ventricular myocytes (NRVMs). Results: The tissue injury from I/R was evident from increases in cleaved-caspase 3 area (0.25% vs 1.53%), ROS generation (1.2 fold), LDH release (4.1 fold), and percent dead cells (0.97% vs 4.67%). Immediately following the I/R injury, tissues showed decrease in contractile force (2.78mN vs 0.74mN) and conduction velocity (20.7cm/s vs 7.9cm/s). At 72hr post-injury, BacNa v -expressing tissues exhibited significantly higher contractile force (1.67mN vs 1.32mN) and Ca 2+ transient (1.8 fold) amplitudes compared to the vehicle control. Furthermore, in uninjured NRVM monolayers, BacNa v expression resulted in higher Ca 2+ transient amplitude (1.31 fold), as well as increased SR calcium stores (1.3 fold) and slower sodium/calcium exchanger (NCX) extrusion rate (1.03s -1 vs 0.82s -1 ) in the presence of caffeine. Blocking NCX with 10M ORM-10962 eliminated the difference in Ca 2+ transient amplitude between BacNa v -expressing and control CMs. Conclusion: Our results suggest BacNa v expression augments CM contractility and Ca 2+ transient amplitude, at least in part through the suppression of Ca 2+ efflux mode of the NCX. In addition to its antiarrhythmic effects, BacNa v gene delivery may represent an effective therapeutic strategy to improve cardiac contractility in congestive heart failure.

Abstract P3134: Impaired Calcium Homeostasis Underlies Lamin A/C Mutation R331Q Associated Atrial Fibrillation

Background: Atrial fibrillation (AF) is one of the first cardiac manifestations in patients harboring mutations in nucleoskeletal protein lamin A/C (LMNA). However, the underlying arrhythmogenic mechanisms are unclear. Objective: We hypothesize that impaired intracellular Ca 2+ -homeostasis contribute to LMNA mutation-associated atrial arrhythmogenesis. Methods: LMNA mutation R331Q was introduced in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 and cells were differentiated into atrial iPSC-derived cardiomyocytes (iPSC-CMs). L-type Ca 2+ current and intracellular Ca 2+ concentration were measured using patch-clamp technique and epifluorescence microscopy (Fluo-3), respectively. Results: R331Q iPSC-CMs demonstrated increased L-type Ca 2+ current density followed by increased amplitude of systolic Ca 2+ transient (CaT) compared to isogenic control (Fig. A). Sarcoplasmic reticulum (SR) Ca 2+ content was evaluated from caffeine-induced CaT amplitude and integrated Na + -Ca 2+ exchange current, and found to be greater in R331Q iPSC-CMs (Fig. B). Confocal line scan analysis revealed an increased Ca 2+ spark frequency in R331Q iPSC-CMs (Fig. C). In addition, by applying a stepwise increase in stimulation frequency (1-5 Hz), R331Q iPSC-CMs developed Ca 2+ alternans at lower threshold frequency (Fig. D). Conclusions: LMNA mutation R331Q is associated with disturbed intracellular Ca 2+ cycling, promoting occurrence of diastolic Ca 2+ sparks and arrhythmogenic Ca 2+ alternans. Collectively, our findings suggest that impaired Ca 2+ homeostasis may contribute to atrial arrhythmogenesis in patients harboring LMNA mutations.

Intrinsic contractile dysfunction due to impaired sarcoplasmic reticulum Ca2+ release in compensatory hypertrophied muscle fibers following synergist ablation.

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

Targeting calcium regulators as therapy for heart failure: focus on the sarcoplasmic reticulum Ca-ATPase pump.

Impaired myocardial Ca2+ cycling is a critical contributor to the development of heart failure (HF), causing changes in the contractile function and structure remodeling of the heart. Within cardiomyocytes, the regulation of sarcoplasmic reticulum (SR) Ca2+ storage and release is largely dependent on Ca2+ handling proteins, such as the SR Ca2+ ATPase (SERCA2a) pump. During the relaxation phase of the cardiac cycle (diastole), SERCA2a plays a critical role in transporting cytosolic Ca2+ back to the SR, which helps to restore both cytosolic Ca2+ levels to their resting state and SR Ca2+ content for the next contraction. However, decreased SERCA2a expression and/or pump activity are key features in HF. As a result, there is a growing interest in developing therapeutic approaches to target SERCA2a. This review provides an overview of the regulatory mechanisms of the SERCA2a pump and explores potential strategies for SERCA2a-targeted therapy, which are being investigated in both preclinical and clinical studies.

Distinct Effects of Mitochondrial Na+/Ca2+ Exchanger Inhibition and Ca2+ Uniporter Activation on Ca2+ Sparks and Arrhythmogenesis in Diabetic Rats.

Background Mitochondrial dysfunction contributes to the cardiac remodeling triggered by type 2 diabetes (T2D). Mitochondrial Ca2+ concentration ([Ca2+]m) modulates the oxidative state and cytosolic Ca2+ regulation. Thus, we investigated how T2D affects mitochondrial Ca2+ fluxes, the downstream consequences on myocyte function, and the effects of normalizing mitochondrial Ca2+ transport. Methods and Results We compared myocytes/hearts from transgenic rats with late-onset T2D (rats that develop late-onset T2D due to heterozygous expression of human amylin in the pancreatic β-cells [HIP] model) and their nondiabetic wild-type (WT) littermates. [Ca2+]m was significantly lower in myocytes from diabetic HIP rats compared with WT cells. Ca2+ extrusion through the mitochondrial Na+/Ca2+ exchanger (mitoNCX) was elevated in HIP versus WT myocytes, particularly at moderate and high [Ca2+]m, while mitochondrial Ca2+ uptake was diminished. Mitochondrial Na+ concentration was comparable in WT and HIP rat myocytes and remained remarkably stable while manipulating mitoNCX activity. Lower [Ca2+]m was associated with oxidative stress, increased sarcoplasmic reticulum Ca2+ leak in the form of Ca2+ sparks, and mitochondrial dysfunction in T2D hearts. MitoNCX inhibition with CGP-37157 reduced oxidative stress, Ca2+ spark frequency, and stress-induced arrhythmias in HIP rat hearts while having no significant effect in WT rats. In contrast, activation of the mitochondrial Ca2+ uniporter with SB-202190 enhanced spontaneous sarcoplasmic reticulum Ca2+ release and had no significant effect on arrhythmias in both WT and HIP rat hearts. Conclusions [Ca2+]m is reduced in myocytes from rats with T2D due to a combination of exacerbated mitochondrial Ca2+ extrusion through mitoNCX and impaired mitochondrial Ca2+ uptake. Partial mitoNCX inhibition limits sarcoplasmic reticulum Ca2+ leak and arrhythmias in T2D hearts, whereas mitochondrial Ca2+ uniporter activation does not.

Effect of nitrosyl iron complexes and their thio ligands on the activity of phosphodiesterase and sarcoplasmic reticulum Ca2+-ATPase

Effect of nitrosyl iron complexes and their thio ligands on the activity of phosphodiesterase and sarcoplasmic reticulum Ca2+-ATPase

Dapagliflozin attenuates cardiac remodeling and dysfunction in rats with β-adrenergic receptor overactivation through restoring calcium handling and suppressing cardiomyocyte apoptosis.

Background: Long-term β-adrenergic receptor (β-AR) activation can impair myocardial structure and function. Dapagliflozin (DAPA) has been reported to improve clinical prognosis in heart failure patients, whereas the exact mechanism remains unclear. Here, we investigated the effects of DAPA against β-AR overactivation toxicity and explored the underlying mechanism.Methods and Results: Rats were randomized to receive saline + placebo, isoproterenol (ISO, 5mg/kg/day, intraperitoneally) + placebo, or ISO + DAPA (1mg/kg/day, intragastrically) for 2-week. DAPA treatment improved cardiac function, alleviated myocardial fibrosis, prevented cardiomyocytes (CMs) apoptosis, and decreased the expression of ER stress-mediated apoptosis markers in ISO-treated hearts. In isolated CMs, 2-week ISO stimulation resulted in deteriorated kinetics of cellular contraction and relaxation, increased diastolic intracellular Ca2+ level and decay time constant of Ca2+ transient (CaT) but decreased CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ level. However, DAPA treatment prevented abnormal Ca2+ handling and contractile dysfunction in CMs from ISO-treated hearts. Consistently, DAPA treatment upregulated the expression of SR Ca2+-ATPase protein and ryanodine receptor 2 (RyR2) but reduced the expression of phosphorylated-RyR2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphorylated-CaMKII in ventricles from ISO-treated rats.Conclusion: DAPA prevented myocardial remodeling and cardiac dysfunction in rats with β-AR overactivation via restoring calcium handling and suppressing ER stress-related CMs apoptosis.

Increased Ca2+ Transient Underlies RyR2-Related Left Ventricular Noncompaction.

A loss-of-function cardiac ryanodine receptor (RyR2) mutation, I4855M+/-, has recently been linked to a new cardiac disorder termed RyR2 Ca2+ release deficiency syndrome (CRDS) as well as left ventricular noncompaction (LVNC). The mechanism by which RyR2 loss-of-function causes CRDS has been extensively studied, but the mechanism underlying RyR2 loss-of-function-associated LVNC is unknown. Here, we determined the impact of a CRDS-LVNC-associated RyR2-I4855M+/- loss-of-function mutation on cardiac structure and function. We generated a mouse model expressing the CRDS-LVNC-associated RyR2-I4855M+/- mutation. Histological analysis, echocardiography, ECG recording, and intact heart Ca2+ imaging were performed to characterize the structural and functional consequences of the RyR2-I4855M+/- mutation. As in humans, RyR2-I4855M+/- mice displayed LVNC characterized by cardiac hypertrabeculation and noncompaction. RyR2-I4855M+/- mice were highly susceptible to electrical stimulation-induced ventricular arrhythmias but protected from stress-induced ventricular arrhythmias. Unexpectedly, the RyR2-I4855M+/- mutation increased the peak Ca2+ transient but did not alter the L-type Ca2+ current, suggesting an increase in Ca2+-induced Ca2+ release gain. The RyR2-I4855M+/- mutation abolished sarcoplasmic reticulum store overload-induced Ca2+ release or Ca2+ leak, elevated sarcoplasmic reticulum Ca2+ load, prolonged Ca2+ transient decay, and elevated end-diastolic Ca2+ level upon rapid pacing. Immunoblotting revealed increased level of phosphorylated CaMKII (Ca2+-calmodulin dependent protein kinases II) but unchanged levels of CaMKII, calcineurin, and other Ca2+ handling proteins in the RyR2-I4855M+/- mutant compared with wild type. The RyR2-I4855M+/- mutant mice represent the first RyR2-associated LVNC animal model that recapitulates the CRDS-LVNC overlapping phenotype in humans. The RyR2-I4855M+/- mutation increases the peak Ca2+ transient by increasing the Ca2+-induced Ca2+ release gain and the end-diastolic Ca2+ level by prolonging Ca2+ transient decay. Our data suggest that the increased peak-systolic and end-diastolic Ca2+ levels may underlie RyR2-associated LVNC.

FARL-11 (STRIP1/2) is Required for Sarcomere and Sarcoplasmic Reticulum Organization in C. elegans.

Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B''' regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and C. elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Since the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans. CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo, and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca+2 release channel UNC-68.

Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/- rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/- myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/- rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/- myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/- cardiomyocytes.

Glutathione Depression Alters Cellular Mechanisms of Skeletal Muscle Fatigue in Early Stage of Recovery and Prolongs Force Depression in Late Stage of Recovery.

The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body weight/day) treatment for 5 d, which decreased GSH content to ~10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N =17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR.