RyR2 Phosphorylation Research Articles

Calcium handling abnormalities increase arrhythmia susceptibility in DMSXL myotonic dystrophy type 1 mice

BackgroundMyotonic dystrophy type 1 (DM1) is a multiorgan disorder with significant cardiac involvement. ECG abnormalities, including arrhythmias, occur in 80 % of DM1 patients and are the second-most common cause of death after respiratory complications; however, the mechanisms underlying the arrhythmogenesis remain unclear. The objective of this study was to investigate the basis of the electrophysiological abnormalities in DM1 using the DMSXL mouse model. MethodsECG parameters were evaluated at baseline and post flecainide challenge. Calcium transient and action potential parameters were evaluated in Langendorff-perfused hearts using fluorescence optical mapping. Calcium transient/sparks were evaluated in ventricular myocytes via confocal microscopy. Protein and mRNA levels for calcium handling proteins were evaluated using western blot and RT-qPCR, respectively. ResultsDMSXL mice showed arrhythmic events on ECG including premature ventricular contractions and sinus block. DMSXL mice showed increased calcium transient time to peak without any change to voltage parameters. Calcium alternans and both sustained and non-sustained ventricular tachyarrhythmias were readily inducible in DMSXL mice. The confocal experiments also showed calcium transient alternans and increased frequency of calcium sparks in DMSXL cardiomyocytes. These calcium abnormalities were correlated with increased RyR2 phosphorylation without changes to the other calcium handling proteins. ConclusionsThe DMSXL mouse model of DM1 exhibited enhanced arrhythmogenicity associated with abnormal intracellular calcium handling due to hyperphosphorylation of RyR2, pointing to RyR2 as a potential new therapeutic target in DM1 treatment.

Abstract Mo060: SPEG mediates increased atrial fibrillation incidence in chronic kidney disease

Introduction: Chronic kidney disease (CKD) is a major public health problem. CKD is a risk factor of cardiovascular morbidity and mortality due to the increased incidence of atrial fibrillation (AF). AF onset is known to be the consequence of cardiomyocyte intracellular calcium (Ca 2+ ) mishandling where increased ryanodine receptor type 2 (RyR2) activity causes triggered activity. RyR2 activity is regulated by phosphorylation of serine 2367 (S2367) by the ‘striated muscle preferentially expressed protein’ (SPEG). Reduced RyR2 phosphorylation at S2367 increases the risk of AF. We hypothesized that changes in atrial SPEG underly the increased risk of AF in CKD patients. Methods: The experimental 5/6 nephrectomy model was used to mimic CKD disease in mice. AF inducibility was determined using intracardiac programmed electric stimulation. Echocardiography was used to analyze cardiac structure. RYR2 phosphorylation and SPEG levels were measured by western blotting. Intracellular Ca2+ dynamics were assessed by confocal microscopy. Results: CKD mice showed increased serum phosphate (14.1±1.4 vs 9.3±0.4mg/dL; p<0.01), creatinine (0.4±0.1 vs 0.8±0.3 mg/dL; p<0.001) and urea (30.5±1.1 vs 20.9±0.4 mg/dL; p<0.001). AF incidence was 4-fold higher in CKD respect sham mice (60 vs 14.2%; p<0.05). CKD mice showed longer P waves (14.3±0.4 vs 12.5±0.3; p<0.01) and greater increase of left atrial size than Sham mice (35.0±7.5 vs 13.9±4.6; p<0.05). Ejection fraction was reduced in CKD mice after 6 weeks of CKD progression (57.9±0.6 vs 61.7±0.8%; p<0.001) while no changes were observed in Sham mice. SPEG levels were decreased in atria from CKD mice compared to sham (0.64±0.11 vs 1.19±0.13; p<0.05). RyR2 phosphorylation at S2367 was reduced in CKD mice atria (0.66±0.16 vs 1.25±0.26 in sham; p=0.098). Atrial cardiomyocytes from CKD mice exhibited altered Ca 2+ handling, with decreased SERCA activity (p<0.05), increased spark-mediated diastolic Ca 2+ leak (p<0.01) and increased NCX activity (p<0.05). Finally, CKD-induced AF onset was prevented in a mice with a mutation that mimics RYR2 phosphorylation at SPEG site (S2367D mice) Conclusion: This study shows for the first time that SPEG levels are decreased in atria from CKD mice what induces increased RyR2 activity leading to increased AF incidence. By conserving RYR2 phosphorylation at Ser2367, CKD-induced AF was prevented. Thus, we propose SPEG as a possible therapeutic target to reduce the increased incidence of AF in CKD patients.

Abstract Tu064: Abnormal Calcium Regulation Leads to Pathological Cardiac Hypertrophy During Pregnancy in the GSNOR-Deficient Mouse Model of Preeclampsia

Introduction: Preeclampsia (PE), a leading cause of maternal mortality, is linked to cardiac hypertrophy and impaired relaxation, predisposing the mother to an increased risk of cardiovascular (CV) injury during pregnancy. Mice lacking S-nitrosoglutathione reductase (GSNOR –/– mice) exhibit all the clinical features of PE. Calcium regulation plays an important role in cardiac relaxation and hypertrophy, but whether alterations in calcium handling contribute to the impairment in these PE mice is unknown. Hypothesis: Cardiac hypertrophy and impaired relaxation in pregnant GSNOR –/– mice may be due in part to altered Ca 2+ handling mechanisms. Methods: Pregnant control (WT [C57B6]) and GSNOR –/– mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) were isolated (N=3-4 mice/group; n=8-24 CMs/heart) and assessed for intracellular Ca 2+ and sarcomere shortening using IonOptix. Ca 2+ decay and sarcomere relaxation rates were recorded. Ventricular tissue homogenates were assessed for Ca 2+ handling and sarcomere-associated protein expression and post-translational modifications by Western Blot. Results: At late pregnancy, the CMs of GSNOR –/– mothers were significantly wider and systolic and diastolic Ca 2+ levels were elevated as compared to WT. At 17.5 dpc, expression of Ca 2+ handling proteins, RyR2, L-TCC and NCX1 were increased, and phosphorylation of RyR2 and L-TCC were increased in the GSNOR –/– mothers as compared to WT. The Ca 2+ decay rate improved in CMs from pregnant versus NP WT CMs but not in GSNOR –/– CMs. PLB phosphorylation at Thr-17 was augmented in pregnant WT hearts, while phosphorylation at Ser-16 was reduced in pregnant GSNOR –/– . Sarcomere relaxation parameters and sarcomere resting length were improved in CMs from pregnant WT mice but were impaired in pregnant versus NP GSNOR –/– mice in association with increased expression of MyBPC in pregnant GSNOR –/– . Conclusion: The PE-related cardiac hypertrophy and impaired relaxation at late pregnancy in GSNOR –/– mice were associated with elevated cytosolic Ca 2+ levels, altered expression and post-translational modifications of Ca 2+ handling and sarcomeric proteins, which may predispose these mice to higher CV injury during pregnancy.

Abstract Tu079: The Calcium Handling Machinery and Electrophysiology is Remodeled in Friedreich’s Ataxia

Background: Friedreich's ataxia (FA) is a progressive neurodegenerative disorder. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. Objective: The objective of our study is to determine whether abnormal calcium handling is a molecular mechanism of cardiac dysfunction in FA. Methods: We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium-handling proteins was assessed using proteomics and westernblot. We used echocardiography and telemetric ECG to assess cardiac function in the mice. We also performed in-vivo programmed electrical stimulation (PES) to assess ventricular arrhythmogenesis in the mouse heart. Results: Proteomics and western blot showed that SERCA2, Ryr2, PLN, CASQ2, and CaMKII were downregulated and that the phosphorylation of Ryr2 and PLN (Ser16 and Thr17) were also significantly decreased in left ventricular tissue from humans with FA and from KO mice. The mitochondrial calcium handling proteins VDAC and MCU were upregulated in left ventricular tissue from humans with FA and from KO mice. On telemetric ECG, the RR tended to prolong in the KO animals compared to wild types (p=0.053), and heart rate variability analysis indicated heightened cardiac parasympathetic input. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. In PES, ventricular arrhythmogenesis was greater in KO mice compared to wild types. Conclusions: The reduced expression of calcium handling proteins may contribute to cardiac contractile and electrophysiological abnormalities in FA. Keywords: Friedreich's ataxia, SERCA2, Ryr2, PLN, CASQ2, and CaMKII.

Modulation of the heart rhythm by RyR2 phosphorylation

Modulation of the heart rhythm by RyR2 phosphorylation

Cardioprotective effect of the anti-ageing protein Klotho on ischemic heart disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII, Ministerio de Universidades, Education and Research Council of Madrid, Biomedicine Network Comunidad de Madrid Background Cardiovascular diseases are the leading cause of death and disability worldwide. Among them, ischemic heart disease, due to myocardial infarction (MI) remains the first cause of cardiovascular death. The anti-ageing protein Klotho is mainly expressed in membrane at renal level. However, Klotho also has a soluble form with different pleiotropic functions that are still undetermined, including a possible cardioprotective role that is currently the target of several studies. Purpose Firstly, we evaluated circulating Klotho levels related to cardiac damage in a cohort of 146 patients after ST-elevation myocardial infarction (STEMI) and in an experimental post-myocardial infarction (PMI) model. Secondly, we investigated soluble Klotho supplementation as a potential novel therapeutic strategy for the prevention of cardiac dysfunction, deleterious remodeling, cardiomyocyte calcium (Ca2+) mishandling and arrhythmias in PMI mice. Methods Study with human samples: we analysed Klotho and NT-proBNP plasma levels in STEMIpatients with preserved renal function. Experimental study: C57BL6 mice subjected to PMI by ligation of the left anterior descending coronary artery were treated with vehicle solution or recombinant Klotho (10 mg/Kg/day) for 15 days after MI. Cardiac function was studied through non-invasive methods including electrocardiogram (ECG) and cardiac magnetic resonance imaging (CMRI). Intracellular Ca2+ handling was analysed using confocal microscopy in isolated ventricular cardiomyocytes. Cardiac remodeling was studied through histological and biomolecular analysis of several markers of cardiac damage and target proteins involved in Ca2+ handling. Results Plasma Klotho levels decrease rapidly after MI in both STEMI patients and PMI mice. Additionally, MI patients in the lowest Klotho tertile showed significantly elevated NT-proBNP levels. Klotho treatment decreased the infarct area in PMI mice accompanied by a decrease in both cardiac hypertrophy and fibrosis. Moreover, reduction in ejection fraction and MI-related ECG alterations were reduced in PMI mice treated with Klotho, including increased QRS, JT, QTc intervals and occurrence of premature ventricular contractions. Klotho also prevented systolic Ca2+ release and cell shortening disturbances in cardiomyocytes from PMI mice without Klotho. Increased diastolic Ca2+ leak was prevented in mice treated with Klotho via CaMKII-dependent pathway by avoiding its phosphorylation and the RyR2 phosphorylation at CaMKII site. Conclusions Klotho supplementation protect against MI by avoiding deleterious cardiac remodeling and ameliorating one of the main complications of MI such as arrhythmias. Thus, recombinant Klotho prevents the intracardiomyocyte Ca2+ mishandling after MI. Our translational findings support the importance of Klotho as a novel biomarker of ventricular damage just after MI and as a novel therapeutic strategy for prevention cardiac events associated to ischemic heart disease.

Leaky RyR2 channels as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): American Heart Association National Institutes of Health Introduction Atrial arrhythmias, like atrial fibrillation, occur in 20–40% of patients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Atrial fibrillation in ARVC patients is often associated with an increased risk of sustained ventricular arrhythmias and inappropriate ICD shocks. However, the pathophysiology behind the development of atrial arrhythmias in ARVC is far from clear. A genome-wide association study, based on UK-biobank data, revealed a link between atrial fibrillation and variants in the desmosomal gene plakophilin-2 (PKP2). Previous studies show that PKP2 is necessary to maintain the transcription of genes that control intracellular calcium (Ca2+) cycling and that loss of PKP2 expression in adult mouse ventricular tissue leads to disruption of intracellular Ca2+ homeostasis. In this study, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial myocytes. Methods Experiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). Cre-negative, flox-positive, TAM-injected littermates were used as controls. We used RNA sequencing, quantitative imaging modalities, as well as biochemical and electrophysiological methods to study the functional and structural properties of atrial PKP2cKO tissue. Studies were carried out 21 days post-tamoxifen injection, a time point at which an arrhythmogenic cardiomyopathy of right ventricular predominance is manifest. Results In contrast to the previously-reported observations in PKP2cKO ventricular myocytes, atrial PKP2cKO myocytes presented no significant differences in Ca2+ transient dynamics, SR load and action potential duration. However, PKP2cKO atrial myocytes showed an increased frequency and amplitude of Ca2+ sparks, in combination with increased diastolic Ca2+ levels as well as an increase in cellular ROS levels. RNAseq data showed an under-representation of mRNA for Integrinα7, a stabilizer of Ryr2 that reduces its phosphorylation. Separate studies showed that PKP2cKO atrial myocytes present impaired relaxation and enhanced sarcomere shortening, a finding consistent with a downregulation in the expression of Myosin Binding Protein C. Isoproterenol (ISO) exposure further increased the frequency of Ca2+ sparks and the levels of diastolic Ca2+, and these effects were reversed by acute (30 minute) in vivo treatment with Ryanodex (medical grade Dantrolene), a known RyR blocker. Conclusion Loss of PKP2 expression affects cellular Ca2+ handling and electrophysiology differently in the atrial versus ventricular myocardium. We speculate that an increased probability of opening of Ryr2 channels, caused by ROS-induced RyR2 oxidation and/or Integrinα7-induced RyR2 phosphorylation, serve as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium, that these effects can be amplified by a catecholaminergic input and that RyR blockers can dampen these pro-arrhythmic effects.

Phosphorylation of RyR2 simultaneously expands the dyad and rearranges the tetramers.

We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately, making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. Here, we used the β-agonist isoproterenol and mice homozygous for one of the following clinically relevant mutations: S2030A, S2808A, S2814A, or S2814D. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that the S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers, suggesting a direct link between the phosphorylation state of the tetramer and its microarchitecture. S2808A and S2814A mutant mice, as well as wild types, had significant expansions of their dyads in response to isoproterenol, while S2030A mutants did not. In agreement with functional data from these mutants, S2030 and S2808 were necessary for a complete β-adrenergic response, unlike S2814 mutants. Additionally, all mutants had unique effects on the organization of their tetramer arrays. Lastly, the correlation of structural with functional changes suggests that tetramer-tetramer contacts play an important functional role. We thus conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a β-adrenergic receptor agonist.

Effects of the angiotensin-converting enzyme inhibitor captopril on occlusal-disharmony-induced cardiac dysfunction in mice

Occlusal disharmony is known to affect not only the oral cavity environment, but also the autonomic nervous system in the heart. Since the renin-angiotensin system (RAS) inhibitor captopril (Cap) is one of the first-line drugs for preventing cardiac remodeling in patients with heart failure, we hypothesized that Cap might prevent cardiac dysfunction induced by occlusal disharmony. Here, to test this idea, we used our bite-opening (BO) mouse model, which was developed by cementing a suitable appliance onto the mandibular incisor. Mice were divided into four groups: (1) Control, (2) BO, (3) Cap, and (4) BO + Cap. After 2 weeks, we evaluated cardiac function by echocardiography and confirmed that cardiac function was significantly decreased in the BO group compared to the control, while Cap ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and oxidative stress-induced myocardial damage in the BO group were significantly increased versus the control, and these increases were suppressed by Cap. Cardiac dysfunction induced by BO was associated with dual phosphorylation on PKCδ (Tyr-311/Thr-505), leading to activation of CaMKII with increased phosphorylation of RyR2 and phospholamban. Our results suggest that the RAS might play an important role in the development of cardiac diseases induced by occlusal anomalies.

Empagliflozin attenuates doxorubicin-impaired cardiac contractility by suppressing reactive oxygen species in isolated myocytes.

In animal studies, sodium-glucose co-transporter-2 inhibitors-such as empagliflozin-have been shown to improve heart failure and impaired cardiac contractility induced by anthracyclines-including doxorubicin-although the therapeutic mechanism remains unclear. Moreover, abnormalities in Ca2+ handling within ventricular myocytes are the predominant feature of heart failure. Accordingly, this study aimed to investigate whether empagliflozin can alleviate Ca2+ handling disorders induced by acute doxorubicin exposure and elucidate the underlying mechanisms. To this end, ventricular myocytes were isolated from C57BL/6 mice. Contraction function, Ca2+ handling, and mitochondrial reactive oxygen species (ROS) generation were then evaluated using IonOptix or confocal microscopy. Ca2+ handling proteins were detected by western blotting. Results show that incubation with 1μmol/L of doxorubicin for 120-min impaired cardiac contractility in isolated myocytes, which was significantly alleviated by pretreatment with 1μmol/L of empagliflozin. Doxorubicin also markedly induced Ca2+ handling disorders, including decreased Ca2+ transients, prolonged Ca2+ transient decay time, enhanced frequency of Ca2+ sparks, and decreased Ca2+ content in the sarcoplasmic reticulum. These dysregulations were improved by pretreatment with empagliflozin. Moreover, empagliflozin effectively inhibited doxorubicin-induced mitochondrial ROS production in isolated myocytes and rescued doxorubicin-induced oxidation of Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) and CaMKII-dependent phosphorylation of RyR2. Similarly, preincubation with 10 μmol/L Mito-TEMPO mimicked the protective effects of empagliflozin. Collectively, Empagliflozin ameliorated the doxorubicin-induced contraction malfunction and Ca2+-handling disorders. These findings suggest that empagliflozin alleviates Ca2+-handling disorders by improving ROS production in the mitochondria and alleviating the enhanced oxidative CaMKII signaling pathway induced by doxorubicin.

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.

Dantrolene inhibition of ryanodine channels (RyR2) in artificial lipid bilayers depends on FKBP12.6.

Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM. In this study, we tested the hypothesis that dantrolene inhibition of RyR2 in the presence of CaM is modulated by RyR2 phosphorylation at S2808 and S2814. Phosphorylation was altered by incubations with either exogenous phosphatase (PP1) or kinases; PKA to phosphorylate S2808 or endogenous CaMKII to phosphorylate S2814. We found that PKA caused selective dissociation of FKBP12.6 from the RyR2 complex and a loss of dantrolene inhibition. Rapamycin-induced FKBP12.6 dissociation from RyR2 also resulted in the loss of dantrolene inhibition. Subsequent incubations of RyR2 with exogenous FKBP12.6 reinstated dantrolene inhibition. These findings indicate that the inhibitory action of dantrolene on RyR2 depends on RyR2 association with FKBP12.6 in addition to CaM as previously found.

Chronic angiotensin II signaling drives sustained PIP2 deficits and altered sarcolemmal Cav1.2 expression and function in cardiomyocytes.

Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca2+ handling and elevated levels of angiotensin II (AngII). This hormone signals through Gq-coupled AT1 receptors, initiating hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Previous work revealed acute AngII signaling and PIP2 depletion destabilizes CaV1.2 clusters in cardiomyocytes, triggering their internalization, and subsequent reduction of whole-cell Ca2+ current (ICa) and contractility. However, the effects of chronically elevated AngII on the phospholipid landscape and associated effects on ICa remain unknown. We subjected mice to sustained delivery of AngII to test the impact on phosphoinositides, ICa, and excitation-contraction coupling. We hypothesized that chronic AngII signaling leads to sustained PIP2 and CaV1.2 deficits, contributing to the diminished functional output associated with disease development. Phospholipid mass spectrometry revealed extensive alteration of phosphoinositide species, including a ∼44% reduction in PIP2 and ∼37% reduction in cardioprotective PIP3 in AngII-infused hearts compared to controls. To study the effects of this phospholipid imbalance on CaV1.2, we examined ICa, finding similar current density in both groups. However, super-resolution imaging revealed a ∼54% reduction in t-tubular CaV1.2 expression and a ∼23% reduction in cluster area. Conserved ICa density despite reduced t-tubular CaV1.2 suggests that either the channels have relocated to the sarcolemmal crest, or their Po has increased. We examined crest CaV1.2 and observed no change with AngII, thus the Po of the remaining channels must increase to maintain ICa. Western blots revealed enhanced phosphorylation of CaV1.2, phosphorylation of RyR2, and an increased expression of the catalytic subunit of PKA, suggesting a compensatory sympathetic activation underlying conserved ICa. These findings support a model wherein chronic elevation of AngII disrupts phosphoinositide and CaV1.2 levels, elucidating the role of chronic AngII in disease progression.

3D spatial modeling of sinoatrial node cells reveals a critical role of subcellular ryanodine receptor distribution in pacemaker automaticity.

Ryanodine receptors (RyRs) represent a central component in sinoatrial node (SAN) pacemaker system, as dysregulated RyRs associated with catecholaminergic polymorphic ventricular tachycardia are linked with SAN dysfunction. Within each SAN cell (SANC), the subcellular distribution pattern of RyRs is region-dependent, varying from central SAN (mostly underneath the surface sarcolemma) to peripheral SAN (striated pattern). A similar distribution pattern is also reported for the PKA- and/or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of RyRs. The impact and functional implication of their distribution patterns on the SANC pacemaker activity have not been fully elucidated. Here, we employed our recently developed 3D spatial model of mouse SANCs to investigate how the subcellar distribution and phosphorylation of RyRs affect spontaneous firing. Our model that features a high surface-to-center gradient of RyR expression well recapitulates the coupled-clock mechanisms, and the biomarkers of simulated spontaneous action potentials agree well with those from previous experimental studies. When abolishing the gradient without changing the whole-cell expression of RyRs in the SANCs, the model predicts 15.0% slowing of the firing rate. Reversing the surface-to-center gradient of RyR distribution (i.e., higher density in the cell center vs surface sarcolemma) further reduced the firing rate (by 25.3%) while increasing the beat-to-beat variation in cycle length, due to disrupted coupling between local Ca2+ releases and the membrane clock. Likewise, preventing RyR phosphorylation caused a substantial slowing in the firing rate (17.6%) while also augmenting beat-to-beat variability. Our simulations mechanistically uncover the functional significance of subcellar RyR distribution and its phosphorylation in maintaining robust automaticity of SANCs. The modeling framework will be applied to systematically delineate the impact of Ca2+-clock protein phosphorylation, distribution, and their interplay with the membrane-clock proteins on the SANC automaticity.

Molecular mechanisms of cardiac complications associated with COVID-19.

The COVID-19 pandemic has had a devastating global impact, resulting in over 5,000,000 deaths. In the United States alone, over 1,000,000 individuals have died from COVID-19. Cardiovascular complications of COVID-19 include arrhythmias, heart failure, and myocardial infarction and COVID-19 has differentially impacted racial and ethnic groups. Ethnic minority groups, including African Americans and Hispanics, have a higher risk of COVID-19 hospitalization and death, independent of their socioeconomic, lifestyle and health-related factors. Our data indicate substantial proteomic remodeling of cardiac tissues from SARS-CoV-2 infected mice including upregulation of arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy and dilated cardiomyopathy pathways. Markers of collagen deposition were significantly enriched in the COVID-19 group and confirmed by Masson’s trichrome staining in the hearts of SARS-CoV-2 infected mice. Inflammatory cell infiltration, rupture of cardiomyocytes and significantly increased thrombotic events were also observed. Cardiac tissues of COVID-19 patients exhibited oxidative stress, inflammatory and adrenergic signaling, and calcium dyshomeostasis. Furthermore, we have observed posttranslational modifications of cardiac RyR2 calcium release channels from human COVID-19 hearts including increased PKA phosphorylation and oxidation of RyR2 known as the “leaky phenotype” of these channels. These biochemical changes correlated with the cardiomyopathic pathways activation identified by whole cell proteomic analyses in the hACE2 mouse model of COVID-19.

Cardiac Piezo1 Exacerbates Lethal Ventricular Arrhythmogenesis by Linking Mechanical Stress with Ca2+ Handling After Myocardial Infarction.

Ventricular arrhythmogenesis is a key cause of sudden cardiac death following myocardial infarction (MI). Accumulating data show that ischemia, sympathetic activation, and inflammation contribute to arrhythmogenesis. However, the role and mechanisms of abnormal mechanical stress in ventricular arrhythmia following MI remain undefined. We aimed to examine the impact of increased mechanical stress and identify the role of the key sensor Piezo1 in ventricular arrhythmogenesis in MI. Concomitant with increased ventricular pressure, Piezo1, as a newly recognized mechano-sensitive cation channel, was the most up-regulated mechanosensor in the myocardium of patients with advanced heart failure. Piezo1 was mainly located at the intercalated discs and T-tubules of cardiomyocytes, which are responsible for intracellular calcium homeostasis and intercellular communication. Cardiomyocyte-conditional Piezo1 knockout mice (Piezo1Cko) exhibited preserved cardiac function after MI. Piezo1Cko mice also displayed a dramatically decreased mortality in response to the programmed electrical stimulation after MI with a markedly reduced incidence of ventricular tachycardia. In contrast, activation of Piezo1 in mouse myocardium increased the electrical instability as indicated by prolonged QT interval and sagging ST segment. Mechanistically, Piezo1 impaired intracellular calcium cycling dynamics by mediating the intracellular Ca2+ overload and increasing the activation of Ca2+-modulated signaling, CaMKII, and calpain, which led to the enhancement of phosphorylation of RyR2 and further increment of Ca2+ leaking, finally provoking cardiac arrhythmias. Furthermore, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Piezo1 activation remarkably triggered cellular arrhythmogenic remodeling by significantly shortening the duration of the action potential, inducing early afterdepolarization, and enhancing triggered activity.This study uncovered a proarrhythmic role of Piezo1 during cardiac remodeling, which is achieved by regulating Ca2+ handling, implying a promising therapeutic target in sudden cardiac death and heart failure.

Phosphorylation of RyR2 at S2814 is a necessary step for the protective effect of β-adrenergic stimulation against Ca2+ alternans

Phosphorylation of RyR2 at S2814 is a necessary step for the protective effect of β-adrenergic stimulation against Ca2+ alternans

Spatial Distribution of Calcium Sparks Determines Their Ability to Induce Afterdepolarizations in Human Atrial Myocytes

Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF.

RYR2 phosphomutants show that the tetramers and the junctional sarcoplasmic reticulum form a structural syncytium.

We previously showed that RYR2 tetramers are distributed nonuniformly within ventricular dyads, and that physiological and pathological factors can alter their relative positions. Agents that decreased Ca2+ spark frequency, high Mg2+, and saturating concentrations of the immunophilins FKBP12 and FKBP12.6 drew the receptors together, minimizing their nearest-neighbor distance and reducing the size of the clusters. Activating kinases with a phosphorylation cocktail did the opposite. The purpose of this study is to test the hypothesis that phosphorylation of RYR2 is required for the structural changes we have observed. We measured junctional sarcoplasmic reticulum (jSR) lengths using 2-D transmission electron microscopy (TEM) and directly visualized RYR2 distribution using dual-tilt electron tomography in phosphomutant mice S2808A, S2814A, S2814D, and S2030A. Mouse hearts were hung on a Langendorff and treated with either saline or 300 nmol/liter isoproterenol (ISO) for 2 min before being fixed and sectioned for analysis. We found that (1) RYR2 distribution in mouse ventricles is comparable to that reported for rats and humans, (2) the response to ISO applied to an intact, beating heart is identical to a phosphorylation cocktail applied to isolated permeabilized myocytes, and (3) all of the mutations produced significant changes in the tetramer arrangements and/or NND relative to wild-type (WT) mice. Our 2-D TEM measurements showed that (1) in WT mice, ISO significantly increased the length of the jSR, (2) ISO significantly increased the jSR lengths of WT, S2814A, and S2808A mice, but not the S2030A mouse, and (3) the jSR length of the S2814D mouse was significantly greater than WT, but not WT + ISO or S2814D + ISO, indicating that a mutation of the RYR2 alone caused a significant change in the jSR length. These results indicate that the tetramers and the jSR form a structural syncytium.

Stabilizing Cardiac Ryanodine Receptor With Dantrolene Treatment Prevents Binge Alcohol–Enhanced Atrial Fibrillation in Rats

Binge drinking is a risk factor for cardiac arrhythmias, known as the holiday heart syndrome. Atrial fibrillation (AF) is the most frequently diagnosed arrhythmia in this condition. Recent reports indicated that cardiac ryanodine receptor (RyR2) dysfunction and Ca 2+ leak contribute to alcohol-enhanced AF. In this study, we investigated whether stabilizing RyR2 with dantrolene treatment can prevent alcohol-enhanced AF in rats. A binge drinking rat model was established with alcohol (2 g /kg, IP) delivered once every other day for 4 times. The study consisted of following 3 groups: control group (n = 9), binge alcohol group (n = 10), and binge alcohol + dantrolene (A+D) group (dantrolene, 10 mg/kg, IP before each alcohol injection, n = 9). Echocardiography, left ventricular hemodynamics, in vivo atrial electrophysiology and AF inducibility test, RyR2 phosphorylation level, and blood norepinephrine level were studied 24 hours after the last injection. Ca 2+ leak in isolated atrial myocytes from control and binge alcohol rats was examined. Binge alcohol significantly increased AF inducibility (1/9 in control vs. 8/9 in binge alcohol group, P < 0.05) and AF duration. Dantrolene treatment significantly reduced both AF inducibility (2/9 in dantrolene group, P < 0.05) and AF duration. Binge alcohol significantly increased Ca 2+ leak in isolated atrial myocytes, which was reduced by dantrolene treatment. Blood norepinephrine,7 RyR2 phosphorylation level, cardiac echocardiography, and left ventricular hemodynamics were not significantly affected 24 hours after binge drinking. In conclusion, stabilizing RyR2 with dantrolene treatment significantly attenuated binge drinking-enhanced AF, suggesting that therapeutic strategies stabilizing RyR2 could be a preventive measure to blunt binge drinking-enhanced AF arrhythmogenesis.