Sarcoplasmic Reticulum Ca2 Research Articles

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.

Ultrastructural and biochemical changes of sarcoplasmic reticulum in spotted mackerel (Scomber australasicus Cuvier, 1832) muscle during cold storage at 5 °C

SummarySpotted mackerel were stored at 5 °C for 0–5 days. To reveal changes in freshness and provide the fundamental knowledge of sarcoplasmic reticulum (SR), structural and biochemical changes were investigated by optical microscope, transmission electron microscopy and biochemical detection, including pH, myosin Ca2+‐ATPase activity, adenosine 5′‐triphosphate (ATP) content, SR recovery field and SR Ca2+‐ATPase (SERCA) activity. The result exhibited that SR swelled on day 1 and ruptured after 2 days. Muscle structure showed significant changes from day 0 to day 5. SERCA activity and SR recovery field were 0.46 μm Pi/min/mg and 1.05 mg/g at day 0 and 0.106 μm Pi/min/mg and 0.109 mg/g at day 2, respectively, highly corresponding to the decline of pH, myosin Ca2+‐ATPase activity and ATP content with a significant difference from day 0 to days 1 and 2. The biochemical properties of SERCA exhibited the maximum activity at pH 6.8–7.0 (0.408 μmol Pi/min/mg). Treated at pH 5.5 for 80 min or incubated at 35 °C for 40 min inactivated SERCA of 80%. Therefore, keeping at a freezing temperature of 5 °C and maintaining SR functionality was essential to delay freshness decrease.

Cardiac Protein Kinase D1 ablation alters the myocytes β-adrenergic response

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.

Targeting p21-activated kinase 1 for development of a novel anti-arrhythmic drug class.

Evidence accumulated over the past decade suggests that p21-activated kinase 1 (PAK1) is a critical cardiac-protective signalling molecule. The present article provides an updated review of recent findings regarding the role of PAK1 in maintaining normal cardiac electrophysiological function through its regulation of membrane and Ca2+ clocks. We first overviewed the PAK1 activation mechanism. We then discussed recent updated results showing the action mechanisms of PAK1 signalling on Cav1.2/Cav1.3 (ICaL)-mediated Ca2+ entry, ryanodine receptor type 2-mediated sarcoplasmic reticulum (SR) Ca2+ release, transcriptional regulation of SR Ca2+-ATPase 2a, Na+/Ca2+ exchangers, and Ca2+/calmodulin-dependent protein kinase II. Finally, we proposed a new and exciting route for developing a PAK1-based therapeutic strategy for cardiac arrhythmias. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

The effects of doxorubicin on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

Anthracyclines such as doxorubicin (DOX) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, previous studies show anthracyclines perturb aspects of excitation-contraction coupling (ECC), though to our knowledge this has yet to be demonstrated in a large animal model. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced reactive oxygen species (ROS) production / oxidative stress remains ambiguous. This is important given the key role of oxidative stress in other cardiotoxic processes. Therefore, we aimed to (1) characterise the effects of DOX on Ca handling and contractility in sheep ventricular myocytes and (2) quantify the dependence of any effect on oxidative stress.Sheep myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986. Cells were loaded with fura-2 (0.1 μM) then field stimulated at 0.5 Hz. Fura-2 was excited sequentially at 340 nm and 380 nm (200 Hz) and changes of cytoplasmic Ca inferred from the ratio of emitted light. Sarcomere length was measured simultaneously using a Myocam-S CCD camera and SarcLen acquisition module (Ion Optix, USA). Changes to sarcoplasmic reticulum (SR) Ca content were estimated by rapid application of caffeine (10 mM). Two-way ANOVA was used for statistical analysis.1 nM DOX reduced the amplitude of systolic Ca and shortening by 39 ± 3 % (n=49, p<0.001) and 42 ± 4 % (n=45, p<0.001) respectively. The amplitude of the caffeine-evoked Ca transient decreased by 33 ± 5 % (n=24, p<0.001). The rate of decay of the systolic Ca transient ( k sys ) represents the combined activity of SERCA and NCX and was reduced by 17 ± 3 % (n=46, p<0.001). The rate of decay of the caffeine-evoked Ca transient ( k caff ) represents NCX alone and was increased by 64 ± 26 % (n=23, p=0.002).Subtraction of k caff from k sys indicates SERCA activity ( k SERCA ), which was decreased by 34 ± 8 % (n=20, p<0.001). When the experiment was repeated in the presence of the antioxidant N-acetylcysteine (NAC), the effect of DOX on systolic Ca, systolic shortening and the caffeine-evoked Ca transient was attenuated by 52 % (n=29, p<0.001), 58 % (n=29, p<0.001) and 45 % (n=15, p=0.003) respectively. Furthermore, NAC prevented the effect of DOX on k sys , k caff and k SERCA .These data suggest DOX increases NCX activity and reduces SERCA activity which decreases SR Ca content thence systolic Ca and contractility. The effect of DOX on NCX and SERCA appears to be entirely ROS / oxidative stress dependent. However, other non-oxidative mechanisms likely contribute to net DOX-mediated negative cellular inotropy. Our next experiments will investigate this and the sources of anthracycline-induced oxidative stress. This work was funded by Kidscan childrens cancer research, UK. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants.

We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Ca2+ handling remodelling in a porcine model of right ventricular dysfunction

Increased afterload is the primary pathophysiologic mechanism for right ventricular failure (RVF), which is the most critical determinant of survival in patients with pulmonary hypertension (PH). The cellular and molecular processes involved in the RV remodeling, including Ca2+ handling as an instrumental actor in the heart, remain incompletely understood. We gain insights into the cellular Ca2+ signaling in a large animal model of RV failure, the chronic thromboembolic pulmonary hypertension (CTEPH) piglet model, to develop specific therapy targeting RVF. CTEPH piglet model consisted of a primary left pulmonary artery ligation followed by weekly embolization in the right lower lobe leading to PH and progressive RV hypertrophy and failure. After six weeks, the piglet model replicated all the features of human CTEPH: increased pulmonary vascular resistance, mean pulmonary arterial pressure (PAP), and RV wall thickness associated with RV dysfunction as assessed by the reduced tricuspid annular plane systolic excursion (TAPSE). RV histological analyses revealed hypertrophied cardiomyocytes and aberrant fibrosis. At the cell level, RV myocytes from CTEPH piglets presented reduced L-type Ca2+ currents and CACNA1C mRNA, lower and slower [Ca2+]i transients, decreased sarcoplasmic reticulum Ca2+ content, and cell shortening to myocytes from sham piglets. This was related to reduce Sarco/Endoplasmic Reticulum Ca2+-ATPase isoform 2a (SERCA2a) protein expression. We found that store-operated Ca2+ entry is increased in hypertrophied RV myocytes, which is correlated with a de novo expression of the SR Ca2+ sensor STIM1L (long stromal interacting molecule 1). Finally, we found that Ca2+ sparks are larger and longer in RV myocytes from CTEPH piglets, whereas their frequencies and amplitude were unchanged compared to myocytes from sham. This was associated with profound disorganization of T tubules and ryanodine receptors (RyR) clustering without changes in RyR phosphorylation status. Our data highlight a cellular Ca2+ cycling remodeling that participates to the pathogenesis of RVF in CTEPH piglets.

PO-01-178 MAPPING OF PATHOGENIC VARIANT HOTSPOTS ON THE STRUCTURE OF RYANODINE RECEPTOR TYPE 2 DRIVES A RATIONAL DRUG DESIGN APPROACH FOR CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

Heritable mutations in RYR2 cause sudden-death predisposing arrhythmia syndromes, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), through increased sarcoplasmic reticulum (SR) diastolic Ca2+ leak. RYR2-encoded ryanodine receptor 2 is an intracellular transmembrane Ca2+ release channel in the SR of cardiomyocytes. Even on optimal medical therapy, breakthrough life-threatening arrhythmias in patients with CPVT are common. We sought to correlate identified pathogenic hotspots with key functional domains on the 3D structure of RyR2 and possible mobile binding sites to determine small molecule targets for reducing SR Ca2+ leak. We performed “signal” to “noise” (S:N) analysis in RYR2 to establish pathogenic hotspots. The minor allele frequency (MAF) of literature-derived disease-associated genetic variants at a given amino acid locus was normalized to the MAF of rare population-based variants in Genome Aggregation Database (gnomAD). Using PyMOL, a heat map overlay was generated using S:N levels and possible molecular binding pockets were identified using software PyMOL and Maestro. The highest cumulative S:N intensity of pathogenic hotspots of each domain localized on the channel domain (28.2%), helical domain 1 (25.6%), central domain (17.7%), and N-terminal domain (16.5%) of RyR2. The SPRY1 (0.498%), P1 (0.147%), handle domain (2.32%), and central U motif (8.88%) had lower S:N. Cumulative S:N intensities were nonexistent for SPRY2, SPRY3, P2, and helical domain 2. Putative small molecule binding pockets were identified in all domains while the largest number localized to the channel domain. Once binding pockets were combined with the heat map structure of RyR2, activator binding pockets of low S:N threshold in the open state of RyR2 were shown to be located heavily on the P2 domain, where CaMKII and PKA binds. The highest S:N intensity was in the channel domain with high S:N levels in other proximal domains localizing near the SR transmembrane in the lumen. Mobile binding pockets between the open and closed state of RyR2 with low S:N intensity may be alternative areas for drug targeting, especially on domains that contain binding pockets for activators of RYR2.

Timing mechanisms to control heart rhythm and initiate arrhythmias: roles for intracellular organelles, signalling pathways and subsarcolemmal Ca2.

Rhythms of electrical activity in all regions of the heart can be influenced by a variety of intracellular membrane bound organelles. This is true both for normal pacemaker activity and for abnormal rhythms including those caused by early and delayed afterdepolarizations under pathological conditions. The influence of the sarcoplasmic reticulum (SR) on cardiac electrical activity is widely recognized, but other intracellular organelles including lysosomes and mitochondria also contribute. Intracellular organelles can provide a timing mechanism (such as an SR clock driven by cyclic uptake and release of Ca2+, with an important influence of intraluminal Ca2+), and/or can act as a Ca2+ store involved in signalling mechanisms. Ca2+ plays many diverse roles including carrying electric current, driving electrogenic sodium-calcium exchange (NCX) particularly when Ca2+ is extruded across the surface membrane causing depolarization, and activation of enzymes which target organelles and surface membrane proteins. Heart function is also influenced by Ca2+ mobilizing agents (cADP-ribose, nicotinic acid adenine dinucleotide phosphate and inositol trisphosphate) acting on intracellular organelles. Lysosomal Ca2+ release exerts its effects via calcium/calmodulin-dependent protein kinase II to promote SR Ca2+ uptake, and contributes to arrhythmias resulting from excessive beta-adrenoceptor stimulation. A separate arrhythmogenic mechanism involves lysosomes, mitochondria and SR. Interacting intracellular organelles, therefore, have profound effects on heart rhythms and NCX plays a central role. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Phospholamban R14del disease: The past, the present and the future.

Arrhythmogenic cardiomyopathy affects significant number of patients worldwide and is characterized by life-threatening ventricular arrhythmias and sudden cardiac death. Mutations in multiple genes with diverse functions have been reported to date including phospholamban (PLN), a key regulator of sarcoplasmic reticulum (SR) Ca2+ homeostasis and cardiac contractility. The PLN-R14del variant in specific is recognized as the cause in an increasing number of patients worldwide, and extensive investigations have enabled rapid advances towards the delineation of PLN-R14del disease pathogenesis and discovery of an effective treatment. We provide a critical overview of current knowledge on PLN-R14del disease pathophysiology, including clinical, animal model, cellular and biochemical studies, as well as diverse therapeutic approaches that are being pursued. The milestones achieved in <20 years, since the discovery of the PLN R14del mutation (2006), serve as a paradigm of international scientific collaboration and patient involvement towards finding a cure.

Flecainide induces a sustained countercurrent dependent effect on RyR2 in permeabilized WT ventricular myocytes but not in intact cells.

Background and purpose: While flecainide is now an accepted treatment for arrhythmias associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), its mechanism of action remains controversial. In studies on myocytes from CPVT mice, inhibition of proarrhythmic Ca2+ waves was initially attributed to a novel action on the type-2 ryanodine receptor (RyR2). However, subsequent work on wild type (WT) myocytes questioned the conclusion that flecainide has a direct action on RyR2. In the present study, the effects of flecainide were compared in intact and permeabilized WT myocytes. Experimental approach: Intracellular Ca2+ was measured using confocal microscopy in intact or saponin permeabilized adult rat ventricular myocytes (ARVM). In some experiments on permeabilized cells, flecainide was studied following partial inhibition of the sarcoplasmic reticulum (SR) counter-current. Key results: Flecainide induced sustained changes Ca2+ sparks and waves in permeabilized ARVM, which were comparable to those reported in intact or permeabilized myocytes from CPVT mice. However, a relatively high level of flecainide (25μM) was required to induce these effects. Inhibition of the SR counter-current potentiated the effects of flecainide on SR Ca2+ waves. In intact field stimulated ARVM, prolonged exposure to 15μM flecainide decreased wave frequency but RyR2 dependent effects on Ca2+ sparks were absent; higher drug concentrations blocked field stimulation, consistent with inhibition of Nav1.5. Conclusions and implications: In intact ARVM, the absence of effects on Ca2+ sparks suggests that the intracellular flecainide concentration was insufficient to influence RyR2. Wave inhibition in intact ARVM may reflect secondary effects of Nav1.5 inhibition. Potentiation of flecainide's action by counter-current inhibition can be explained if transient polarization of the SR membrane during SR Ca2+ release facilitates its action on RyR2.