Sarcoplasmic Reticulum Ca2 Research Articles

Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells.

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.

Increased Risk for Atrial Alternans in Rabbit Heart Failure: The Role of Ca2+/Calmodulin-Dependent Kinase II and Inositol-1,4,5-trisphosphate Signaling.

Heart failure (HF) increases the probability of cardiac arrhythmias, including atrial fibrillation (AF), but the mechanisms linking HF to AF are poorly understood. We investigated disturbances in Ca2+ signaling and electrophysiology in rabbit atrial myocytes from normal and failing hearts and identified mechanisms that contribute to the higher risk of atrial arrhythmias in HF. Ca2+ transient (CaT) alternans-beat-to-beat alternations in CaT amplitude-served as indicator of increased arrhythmogenicity. We demonstrate that HF atrial myocytes were more prone to alternans despite no change in action potentials duration and only moderate decrease of L-type Ca2+ current. Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition suppressed CaT alternans. Activation of IP3 signaling by endothelin-1 (ET-1) and angiotensin II (Ang II) resulted in acute, but transient reduction of CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ load, and lowered the alternans risk. However, prolonged exposure to ET-1 and Ang II enhanced SR Ca2+ release and increased the degree of alternans. Inhibition of IP3 receptors prevented the transient ET-1 and Ang II effects and by itself increased the degree of CaT alternans. Our data suggest that activation of CaMKII and IP3 signaling contribute to atrial arrhythmogenesis in HF.

Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.

Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation “two-pore channel” (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2−/−) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2−/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Summer fades, deer change: Photoperiodic control of cellular seasonal acclimatization of skeletal muscle

We found major seasonal changes of polyunsaturated fatty acids (PUFAs) in muscular phospholipids (PL) in a large non-hibernating mammal, the red deer (Cervus elaphus). Dietary supply of essential linoleic acid (LA) and α-linolenic acid (ALA) had no, or only weak influence, respectively. We further found correlations of PL PUFA concentrations with the activity of key metabolic enzymes, independent of higher winter expression. Activity of the sarcoplasmic reticulum (SR) Ca++-ATPase increased with SR PL concentrations of n-6 PUFA, and of cytochrome c oxidase and citrate synthase, indicators of ATP-production, with concentrations of eicosapentaenoic acid in mitochondrial PL. All detected cyclic molecular changes were controlled by photoperiod and are likely of general relevance for mammals living in seasonal environments, including humans. During winter, these changes at the molecular level presumably compensate for Arrhenius effects in the colder peripheral body parts and thus enable a thrifty life at lower body temperature.

In vivo heat production dynamics during a contraction-relaxation cycle in rat single skeletal muscle fibers

Skeletal muscle generates heat via contraction-dependent (shivering) and independent (nonshivering) mechanisms. While this thermogenic capacity of skeletal muscle undoubtedly contributes to the body temperature homeostasis of animals and impacts various cellular functions, the intracellular temperature and its dynamics in skeletal muscle in vivo remain elusive. We aimed to determine the intracellular temperature and its changes within skeletal muscle in vivo during contraction and following relaxation. In addition, we tested the hypothesis that sarcoplasmic reticulum Ca2+ ATPase (SERCA) generates heat and increases the myocyte temperature during a transitory Ca2+-induced contraction-relaxation cycle. The intact spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and loaded with the fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 μM) by microinjection over 1 s. The fluorescence ratio (i.e., 580 nm/515 nm) was measured in vivo during 1) temperature increases induced by means of an external heater, and 2) Ca2+ injection (3.9 nL, 2.0 mM). The fluorescence ratio increased as a linear function of muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). Ca2+ injection (3.9 nL, 2.0 mM) significantly increased myocyte intracellular temperature: An effect that was suppressed by SERCA inhibition with cyclopiazonic acid (CPA, Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). While muscle shortening occurred immediately after the Ca2+ injection, the increased muscle temperature was maintained during the relaxation phase. In this investigation, we demonstrated a novel model for measuring the intracellular temperature of skeletal muscle in vivo and further that heat generation occurs concomitant principally with SERCA functioning and muscle relaxation.

Dapansutrile Ameliorates Atrial Inflammation and Vulnerability to Atrial Fibrillation in HFpEF Rats

Numerous studies have demonstrated that NLRP3 inflammasomes are key players in the progression of atrial fibrillation (AF) in heart failure with preserved ejection fraction (HFpEF). This study aimed to analyse the effect of pharmacological inhibition of NLRP3 inflammasomes using dapansutrile (DAPA), an oral NLRP3-specific inhibitor. Dahl salt-sensitive rats were fed a high-salt diet (HSD, 8% NaCl) to induce HFpEF. Either DAPA (200 mg/kg/day) or saline was administered daily via gavage for 4 weeks. Electrophysiological studies were performed to assess the AF inducibility. Confocal fluorescence microscopy and western blot analysis were used to study calcium handling. The DAPA-treated HFpEF rats were less prone to AF induction by programmed electrical stimulation. Atrial fibrosis and inflammation were attenuated in DAPA-treated HFpEF hearts. Dapansutrile treatment showed an increase in the Ca2+ transient sarcoplasmic reticulum-Ca2+ load, and protein expression of SERCA2; NCX1 and phosphorylation of PLB at Thr17 were decreased following DAPA treatment. The increased frequency of spontaneous Ca2+ spark in the HFpEF rats was related to the hyperphosphorylation of RyR2 at Ser2814, which was blunted in DAPA treatment. Dapansutrile treatment also decreased the phosphorylation of CaMKII expression in the HFpEF rats. Mechanistically, DAPA exerts an anti-arrhythmic effect, mainly by inhibiting activation of the NLRP3 inflammasome. These data provide evidence that the beneficial cardiac effects of DAPA are associated with reduced atrial inflammation and improved CaMKII-dependent Ca2+-handling abnormalities via blunting activation of the NLRP3 inflammasome, and DAPA may be beneficial in a rat model of HFpEF-induced AF.

Impaired intracellular Ca2+ signaling contributes to age-related cerebral small vessel disease in Col4a1 mutant mice.

Humans and mice with mutations in COL4A1 and COL4A2 manifest hallmarks of cerebral small vessel disease (cSVD). Mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1+/G1344D) exhibit age-dependent intracerebral hemorrhages (ICHs) and brain lesions. Here, we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Furthermore, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ (BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca2+ signaling. Col4a1 mutations impair protein folding, which can cause SR stress. Treating Col4a1+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca2+ signaling, maintained BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICHs. We conclude that alterations in SR Ca2+ handling that impair ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone and contribute to age-related cSVD in Col4a1+/G1344D mice.

Ethanol-induced atrial fibrillation results from late INa and can be prevented by ranolazine

Abstract Background Ethanol consumption is the most important self-triggered cause of atrial fibrillation (AF). As abstinence often fails, alternative therapies are needed. CaMKII-activation upon ethanol with consecutive Ca2+-leak from the sarcoplasmic reticulum (SR) appears to be an important pathomechanism for ethanol-induced AF, but is not a druggable target. CaMKII has previously been shown to induce late sodium current (late INa) which could be proarrhythmogenic. Purpose We hypothesized that late INa might be involved in the development of AF upon acute ethanol exposition and that thus, the antianginal drug ranolazine (an inhibitor of late INa) could be repurposed to prevent ethanol-induced AF. Methods Acute effects of ethanol were investigated in vivo and in vitro as compared to vehicle. The occurrence of AF in vivo was assessed by programmed electrical stimulation in mice. To test the role of late INa, we used ranolazine in vivo and in vitro, as well as tetrodotoxin in vitro. We measured late INa and cytosolic sodium concentration, recorded stimulated action potentials and spontaneous delayed afterdepolarizations (DAD), and assessed SR Ca2+-leak by recording Ca2+-sparks. Mechanistically, we inhibited CaMKII using AIP and used NaV1.5 S571A mice to specifically investigate the role of CaMKII-mediated late INa. Human biopsies were acquired during surgery and were used for cell isolation or chunk incubation. Results Ethanol acutely induced late INa, sodium overload, SR Ca2+-leak and delayed afterdepolarizations in isolated atrial cardiomyocytes, as well as atrial fibrillation in vivo (10 of 10 mice). All of these cellular ethanol effects were prevented by pharmacologic inhibition of late INa. Importantly, ranolazine prevented the development of AF upon ethanol exposure in vivo. Mechanistically, CaMKII-activation and, accordingly, CaMKII-dependent NaV1.5 phosphorylation (S571) were increased by ethanol exposure. Indeed, the phosphoresistant NaV1.5 S571A mutation prevented late INa, SR Ca2+-leak and in vivo AF upon ethanol, demonstrating a critical role of CaMKII-mediated NaV1.5 phosphorylation for acute ethanol effects. Conclusion We show that ethanol acutely induces late INa upstream of arrhythmogenic SR Ca2+-leak and delayed afterdepolarizations, dependent on NaV1.5 S571 phosphorylation, leading to atrial fibrillation in vivo. Importantly, these effects as well as AF in vivo can be prevented using the late INa inhibitor ranolazine. We suggest that this antianginal drug could be repurposed to prevent ethanol-induced AF.Late INa (Patch-Clamp)In vivo AF (programmed stimulation)

Ketogenic Diet Regulates Cardiac Remodeling and Calcium Homeostasis in Diabetic Rat Cardiomyopathy.

A ketogenic diet (KD) might alleviate patients with diabetic cardiomyopathy. However, the underlying mechanism remains unclear. Myocardial function and arrhythmogenesis are closely linked to calcium (Ca2+) homeostasis. We investigated the effects of a KD on Ca2+ homeostasis and electrophysiology in diabetic cardiomyopathy. Male Wistar rats were created to have diabetes mellitus (DM) using streptozotocin (65 mg/kg, intraperitoneally), and subsequently treated for 6 weeks with either a normal diet (ND) or a KD. Our electrophysiological and Western blot analyses assessed myocardial Ca2+ homeostasis in ventricular preparations in vivo. Unlike those on the KD, DM rats treated with an ND exhibited a prolonged QTc interval and action potential duration. Compared to the control and DM rats on the KD, DM rats treated with an ND also showed lower intracellular Ca2+ transients, sarcoplasmic reticular Ca2+ content, sodium (Na+)-Ca2+ exchanger currents (reverse mode), L-type Ca2+ contents, sarcoplasmic reticulum ATPase contents, Cav1.2 contents. Furthermore, these rats exhibited elevated ratios of phosphorylated to total proteins across multiple Ca2+ handling proteins, including ryanodine receptor 2 (RyR2) at serine 2808, phospholamban (PLB)-Ser16, and calmodulin-dependent protein kinase II (CaMKII). Additionally, DM rats treated with an ND demonstrated a higher frequency and incidence of Ca2+ leak, cytosolic reactive oxygen species, Na+/hydrogen-exchanger currents, and late Na+ currents than the control and DM rats on the KD. KD treatment may attenuate the effects of DM-dysregulated Na+ and Ca2+ homeostasis, contributing to its cardioprotection in DM.

CaMKIIdelta-mediated SR Ca leak contributes to long-term Doxorubicin-induced cardiotoxicity

Abstract Background Cardiotoxicity represents a major complication of cancer therapy with anthracyclines, whose main known compound is doxorubicine (DOX). Although mechanisms of DOX-induced toxic cardiomyopathy (DICM) are not entirely understood, CaMKIIδ-activity is known to be increased upon acute DOX exposure, contributing to impaired Calcium (Ca) handling. However, currently no studies have addressed the question, whether CaMKIIδ is involved in DOX-induced long-term cardiotoxicity. Purpose We aimed to investigate cardiac function and intracellular Ca handling using an in-vivo DOX treated mouse model, examining whether CaMKIIδ is involved in DOX-induced long-term cardiotoxicity. Methods Cardiac function and excitation-contraction coupling of CaMKIIdWT and CaMKIId-/- (global CaMKIId knockout) mice were tested in an in-vivo DOX treated mouse model (cumulative dose 12 mg/kg, intraperitoneal injection). Cardiac function was determined by echocardiography after 12 weeks. Ca handling of freshly isolated cardiomyocytes was assessed by epifluorescence microscopy (Fura2-AM) and confocal microscopy (Fluo4-AM). Histological analyses regarding cardiac fibrosis and cell dimensions were examined. Results Long-term DOX exposure reduced left ventricular ejection fraction (EF) by ∼13% and markedly increased cardiac fibrosis in CaMKIIδWT mice. Moreover, CaMKIIdWT hearts displayed decreased cell volume indicating atrophy following DOX treatment. Coinciding with the reduction in EF, CaMKIIdWT mice showed depressed Ca transient amplitudes as well as slowed Ca transient decay indicating depressed Ca uptake into the sarcoplasmic reticulum (SR). In addition, spontaneous diastolic Ca leakage from the SR was dramatically increased. In contrast, CaMKIId-/- mice showed preserved EF following 12 weeks of DOX treatment as compared to CaMKIIdWT in line with maintained Ca transient amplitudes. As an underlying cause, SR Ca leakage was not increased in CaMKIId-/- myocytes despite DOX-treatment. Pharmacological CaMKII inhibition using AIP (1µM) prevented SR Ca leak and restored Ca transient amplitudes in DOX-treated CaMKIIdWT myocytes. Conclusion Our study shows that CaMKIId contributes to long-term DOX-induced cardiotoxicity. We found that chronic DOX treatment induced depressed cardiac function and myocardial fibrosis, associated with impaired calcium handling as a consequence of CaMKIId-mediated SR Ca leak, which was absent in our CaMKIId-/- mouse model. In line, pharmacological inhibition of CaMKII rescued impaired Ca handling in DOX-treated CaMKIIdWT myocytes.

Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle

Abstract Background More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+. Purpose The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice. Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1. Methods (1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented. (2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test). Results (1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D). (2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012). Conclusion(s) We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.

DDX5 maintains cardiac function by regulating CamkII delta alternative splicing

Abstract Background Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease. Dead-box protein 5 (DDX5) is a master regulator of many RNA processes, though its functions in heart physiology and disease remain unclear. Purpose To elucidate the function of DDX5 in HF and the mechanisms underlying its role in regulating alternative splicing. Methods and Results We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. Ddx5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. By integrating alternative splicing analysis and RNA immunoprecipitation sequencing data, we identified that DDX5 regulated the aberrant splicing of CamkIIδ, thus preventing the production of CaMKIIδA and maintaining Ca2+ homeostasis. In line with this, we found an increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5 depleted cardiomyocytes. Conclusions Our findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes.

OpiCa1-PEG-PLGA nanomicelles antagonize acute heart failure induced by the cocktail of epinephrine and caffeine

BackgroundReducing Ca2+ content in the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) by calcin is a potential intervention strategy for the SR Ca2+ overload triggered by β-adrenergic stress in acute heart diseases. MethodsOpiCal-PEG-PLGA nanomicelles were prepared by thin film dispersion, of which the antagonistic effects were observed using an acute heart failure model induced by epinephrine and caffeine in mice. In addition, cardiac targeting, self-stability as well as biotoxicity were determined. ResultsThe synthesized OpiCa1-PEG-PLGA nanomicelles were elliptical with a particle size of 72.26 nm, a PDI value of 0.3, and a molecular weight of 10.39 kDa. The nanomicelles showed a significant antagonistic effect with 100 % survival rate to the death induced by epinephrine and caffeine, which was supported by echocardiography with significantly recovered heart rate, ejection fraction and left ventricular fractional shortening rate. The FITC labeled nanomicelles had a strong membrance penetrating capacity within 2 h and cardiac targeting within 12 h that was further confirmed by immunohistochemistry with a self-prepared OpiCa1 polyclonal antibody. Meanwhile, the nanomicelles can keep better stability and dispersibility in vitro at 4 °C rather than 20 °C or 37 °C, while maintain a low but stable plasma OpiCa1 concentration in vivo within 72 h. Finally, no obvious biotoxicities were observed by CCK-8, flow cytometry, H&E staining and blood biochemical examinations. ConclusionOur study also provide a novel nanodelivery pathway for targeting RyRs and antagonizing the SR Ca2+ disordered heart diseases by actively releasing SR Ca2+ through RyRs with calcin.

Abstract 15846: Impaired Contraction and High Susceptibility to Arrhythmias in a Mouse Model of Desmoplakin-Mediated Arrhythmogenic Cardiomyopathy

Introduction: Desmoplakin (DSP) is a key protein for cardiac myocyte cell-to-cell adhesion that connects the desmosomes present in intercalated discs to the cytoplasmic network of desmin intermediate filaments. Pathogenic variants of the human DSP gene often lead to left ventricular (LV)-dominant and biventricular forms of arrhythmogenic cardiomyopathy characterized by a dilated LV, arrhythmias, and an elevated risk of sudden cardiac death. Nonetheless, the pathophysiological pathways triggered by DSP variants remain unknown. Hypothesis: We hypothesized that DSP pathogenic variants modify cardiomyocyte excitation-contraction coupling leading to life-threatening ventricular arrhythmias. Methods: We created a novel knock-in mouse model based on the human DSP p.S2859LfsX5 variant that leads to a murine protein truncated from its last 9 amino-acids. We compared one-year-old sedentary heterozygous (Dsp +/- ) animals to WT littermates and defined histologic, cellular, and molecular outcomes of the variant. Results: Western blotting revealed 50% Dsp reduction (N= 5 hearts/group; P &lt; 0.05) in mutant right ventricle (RV) indicating mutant protein instability. Histologic assessments of Dsp +/- hearts revealed LV chamber enlargement and RV wall thinning in the absence of fibrosis and lipid infiltration. Dsp +/- myocytes isolated from LV and RV free walls and field stimulated at 3 Hz exhibited 30-40% reduction in sarcomere shortening (n= 17-25 myocytes/ventricle/3-4 hearts/group; P &lt; 0.05), while their Ca 2+ handling properties remained normal. Pacing of Dsp +/- isolated myocytes (LV and RV) in the presence of (100 nmol/L) isoproterenol provoked a 20-30% increase in Ca 2+ release due to sarcoplasmic reticulum Ca 2+ overload (n= 15-20 myocytes/ventricle/3-4 hearts/group; P &lt; 0.05). Finally, surface ECGs showed that activation of the sympathetic pathway (isoproterenol + caffeine) in 6-month-old Dsp +/- mice increased mutant heart susceptibility to ventricular arrhythmias. Conclusions: The pathogenic variant p.S2859LfsX5 promotes biventricular dysfunction due to the loss in myocyte contraction capacity and catecholaminergic-mediated ventricular arrhythmias from cardiomyocyte Ca 2+ overload.

Lack of Evidence for the Role of the p.(Ser96Ala) Polymorphism in Histidine-Rich Calcium Binding Protein as a Secondary Hit in Cardiomyopathies.

Inherited forms of arrhythmogenic and dilated cardiomyopathy (ACM and DCM) are characterized by variable disease expression and age-related penetrance. Calcium (Ca2+) is crucially important for proper cardiac function, and dysregulation of Ca2+ homeostasis seems to underly cardiomyopathy etiology. A polymorphism, c.286T>G p.(Ser96Ala), in the gene encoding the histidine-rich Ca2+ binding (HRC) protein, relevant for sarcoplasmic reticulum Ca2+ cycling, has previously been associated with a marked increased risk of life-threatening arrhythmias among idiopathic DCM patients. Following this finding, we investigated whether p.(Ser96Ala) affects major cardiac disease manifestations in carriers of the phospholamban (PLN) c.40_42delAGA; p.(Arg14del) pathogenic variant (cohort 1); patients diagnosed with, or predisposed to, ACM (cohort 2); and DCM patients (cohort 3). We found that the allele frequency of the p.(Ser96Ala) polymorphism was similar across the general European-American population (control cohort, 40.3-42.2%) and the different cardiomyopathy cohorts (cohorts 1-3, 40.9-43.9%). Furthermore, the p.(Ser96Ala) polymorphism was not associated with life-threatening arrhythmias or heart failure-related events across various patient cohorts. We therefore conclude that there is a lack of evidence supporting the important role of the HRC p.(Ser96Ala) polymorphism as a modifier in cardiomyopathy, refuting previous findings. Further research is required to identify bona fide genomic predictors for the stratification of cardiomyopathy patients and their risk for life-threatening outcomes.

Natriuretic Peptide Receptor B Protects Against Atrial Fibrillation by Controlling Atrial cAMP Via Phosphodiesterase 2.

β-AR (β-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca2+ homeostasis via cAMP-dependent mechanisms; however, enhanced β-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca2+ homeostasis, and atrial arrhythmogenesis is incompletely understood. Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B+/-) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca2+ imaging, and in mouse and human atrial tissues using molecular biology. Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B+/- mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B+/- mice leading to larger increases in atrial cAMP in the presence of the β-AR agonist isoproterenol. NPR-B+/- mice displayed larger increases in action potential duration and L-type Ca2+ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca2+ release events and delayed afterdepolarizations in NPR-B+/- atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B+/- atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca2+ current through PDE2 in mouse and human atrial myocytes. NPR-B protects against AF by preventing enhanced atrial responses to β-adrenergic receptor agonists.

Regulation of cardiac calcium signaling by newly identified calcium pump modulators

In cardiomyocytes, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a central component of intracellular Ca2+ regulation. Several heart diseases, including heart failure, are associated with reduced myocardial contraction due to SERCA2a downregulation. Therefore, the need for developing new drugs that could improve SERCA2a function is high. We have recently identified SERCA2a modulators (Compounds 6 and 8) from our screening campaigns and confirmed activation of biochemical SERCA2a ATPase activity and Ca2+ uptake activity. In this study, confocal microscopy and in-cell Ca2+ imaging were used to characterize the effects of these SERCA2a activators on Ca2+ regulation in mouse ventricular myocytes and endoplasmic reticulum (ER) Ca2+ uptake in a HEK293 cell expressing human SERCA2a. Analysis of cytosolic Ca2+ dynamics in cardiomyocytes revealed that both Compounds (6 and 8) increase the action potential-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load. While Compound 6 induced a negligible effect on Ca2+ transients invoked by the L-type Ca2+ channel (LTCC) current, Compound 8 increased Ca2+ transients during LTCC activation, suggesting an off-target protein interaction of Compound 8. Analysis of ER Ca2+ transport by human SERCA2a in HEK cells showed that only Compound 6 increased both ER Ca2+ uptake and ER Ca2+ load significantly, whereas Compound 8 had no effect on SERCA2a Ca2+ transport. This study revealed that Compound 6 exhibits promising characteristics that can improve intracellular Ca2+ dynamics by selectively enhancing SERCA2a Ca2+ uptake.

Calcium Signaling Consequences of RyR2-S4938F Mutation Expressed in Human iPSC-Derived Cardiomyocytes.

Type-2 ryanodine receptor (RyR2) is the major Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to -50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.

Epigenetics in Heart Failure: Role of DNA Methylation in Potential Pathways Leading to Heart Failure with Preserved Ejection Fraction.

This review will focus on epigenetic modifications utilizing the DNA methylation mechanism, which is potentially involved in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). The putative pathways of HFpEF will be discussed, specifically myocardial fibrosis, myocardial inflammation, sarcoplasmic reticulum Ca2+-ATPase, oxidative-nitrosative stress, mitochondrial and metabolic defects, as well as obesity. The relationship of HFpEF to aging and atrial fibrillation will be examined from the perspective of DNA methylation.