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•Ca2+ /Calmodulin (CaM) binds the CaM dependent kinase II inhibitor (CaMKII) KN93.•KN93 modifies NaV1.5 and RyR2 function independent of CaMKII.•KN93 disrupts a high affinity ...interaction between NaV1.5 and CaM.•When evaluating KN93 data, targets other than CaMKII need to be considered.
Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM–protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.
Atrial fibrillation (AF) is the most common arrhythmia and is associated with inflammation. AF patients have elevated levels of inflammatory cytokines known to promote vascular leak, such as vascular ...endothelial growth factor A (VEGF). However, the contribution of vascular leak and consequent cardiac edema to the genesis of atrial arrhythmias remains unknown. Previous work suggests that interstitial edema in the heart can acutely promote ventricular arrhythmias by disrupting ventricular myocyte intercalated disk (ID) nanodomains rich in cardiac sodium channels (Na
1.5) and slowing cardiac conduction. Interestingly, similar disruption of ID nanodomains has been identified in atrial samples from AF patients. Therefore, we tested the hypothesis that VEGF-induced vascular leak can acutely increase atrial arrhythmia susceptibility by disrupting ID nanodomains and slowing atrial conduction. Treatment of murine hearts with VEGF (30-60 min, at clinically relevant levels) prolonged the electrocardiographic P wave and increased susceptibility to burst pacing-induced atrial arrhythmias. Optical voltage mapping revealed slower atrial conduction following VEGF treatment (10 ± 0.4 cm/s vs. 21 ± 1 cm/s at baseline, p < 0.05). Transmission electron microscopy revealed increased intermembrane spacing at ID sites adjacent to gap junctions (GJs; 64 ± 9 nm versus 17 ± 1 nm in controls, p < 0.05), as well as sites next to mechanical junctions (MJs; 63 ± 4 nm versus 27 ± 2 nm in controls, p < 0.05) in VEGF-treated hearts relative to controls. Importantly, super-resolution microscopy and quantitative image analysis revealed reorganization of Na
1.5 away from dense clusters localized near GJs and MJs to a more diffuse distribution throughout the ID. Taken together, these data suggest that VEGF can acutely predispose otherwise normal hearts to atrial arrhythmias by dynamically disrupting Na
1.5-rich ID nanodomains and slowing atrial conduction. These data highlight inflammation-induced vascular leak as a potential factor in the development and progression of AF.
Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration ...(APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.
The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) ...required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodelling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.
Cardiac manifestations are common in severe COVID-19. This study compared the histologic, viral, and molecular findings in cardiac tissue in fatal COVID-19 (n = 11) and controls (n = 11). In situ ...hybridization (SARS-CoV2 RNA) and immunohistochemistry for viral proteins and the host response were quantified for the samples and compared with qRTPCR and Western blot data. Control hearts showed a high resident population of macrophages that had variable ACE2 expression. Cardiac ACE2 expression was 10× greater in the heart tissues of cases and controls with obesity or type II diabetes. Multifocal endothelial cell swelling and degeneration, perivascular edema plus microvascular thrombi were unique to the cases. SARS-CoV2 RNA and nucleocapsid protein were rarely detected in situ in any COVID-19 heart. However, in each case abundant SARS-CoV-2 spike protein was evident. Co-expression experiments showed that the spike protein localized mostly to the ACE2+ interstitial macrophages/pericytes that were activated as evidenced by increased IL6 and TNFα expression. Western blots confirmed the presence of the viral spike protein, but not the nucleocapsid protein, in the cardiac homogenates. The intercalated disc proteins connexin 43, the primary cardiac gap junction protein, and NaV1.5, the predominant cardiac sodium channel, each showed marked lateral migration in the myocytes in the cases, which would increase the risk of reentrant arrhythmias. It is concluded that the viral spike protein, endocytosed by macrophages/pericytes, can induce a myocarditis with the possibility of conduction dysfunction due to abnormal localization of key intercalated disc proteins.
•Cardiac disease in fatal COVID-19 is associated with viral spike protein, but not the infectious virus.•The viral spike protein is endocytosed in interstitial macrophages and induces a myocarditis.•The histologic findings show perivascular edema, endothelial cell damage, and microthrombi.
Background
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with ...mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral‐mediated delivery to alleviate arrhythmias in non–CaM‐related CPVT.
Methods and Results
To that end, we have designed a CaM protein (GSH‐M37Q; dubbed as therapeutic CaM or T‐CaM) that exhibited a slowed N‐terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T‐CaM was introduced to the heart of R33Q mice through recombinant adeno‐associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T‐CaM reduced isoproterenol‐promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T‐CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines.
Conclusions
Our results suggest that gene transfer of T‐CaM by adeno‐associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin‐associated CPVT model, thus supporting the potential of a CaM‐based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.
Key points
Atrial fibrillation is often initiated and perpetuated by abnormal electrical pulses repetitively originating from regions outside the heart's natural pacemaker.
In this study we examined ...the causal role of abnormal calcium releases from the sarcoplasmic reticulum in producing repetitive electrical discharges in atrial cells and tissues.
Calsequestrin2 is a protein that stabilizes the closed state of calcium release channels, i.e. the ryanodine receptors. In the atria from mice predisposed to abnormal calcium releases secondary to the absence of calsequestrin2, we observed abnormal repetitive electrical discharges that may lead to atrial fibrillation.
Here, we report a novel pathological rhythm generator. Specifically, abnormal calcium release leads to electrical activation, which in turn results in another abnormal calcium release. This process repeats itself and thus sustains the repetitive electrical discharges.
These results suggest that improving the stability of ryanodine receptors might be useful to treat atrial fibrillation.
Aberrant diastolic calcium (Ca) release due to leaky ryanodine receptors (RyR2s) has been recently associated with atrial fibrillation (AF) and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, it remains unclear how diastolic Ca release contributes to the rising of rapid repetitive focal activity, which is considered as a common AF triggering mechanism. To address this question, we conducted simultaneous voltage/Ca optical mapping in atrial tissue and one‐/two‐dimensional confocal imaging in atrial tissue and myocytes from wild‐type (WT, n = 15) and CPVT mice lacking calsequestrin 2 (Casq2−/−, n = 45), which promotes diastolic Ca release. During β‐adrenergic stimulation (100 nm isoproterenol), only Casq2−/− atrial myocytes showed pacing‐induced self‐sustained repetitive activity (31 ± 21 s vs. none in WT). Importantly, in atrial tissue, this repetitive activity could translate to Ca‐dependent focal arrhythmia. Ectopic action potential (AP) firing during repetitive activity occurred only when diastolic Ca release achieved a sufficient level of synchronization. The AP, in turn, synchronized subsequent diastolic Ca release by temporally aligning multiple sources of Ca waves both within individual myocytes and throughout the atrial tissue. This alternating interplay between AP and diastolic Ca release perpetuates the self‐sustaining repetitive activity. In fact, pharmacological disruption of synchronized diastolic Ca release (by ryanodine) prevented aberrant APs; and vice versa, the inhibition of AP (by TTX or 0 Na, 0 Ca solution) de‐synchronized diastolic Ca release. Taken together, these results suggest that a cyclical interaction between synchronized diastolic Ca release and AP forms a pathological rhythm generator that is involved in Ca‐dependent atrial arrhythmias in CPVT.
Excitation–contraction coupling is the bridge between cardiac electrical activation and mechanical contraction. It is driven by the influx of Ca2+ across the sarcolemma triggering Ca2+ release from ...the sarcoplasmic reticulum (SR) – a process termed Ca2+‐induced Ca2+ release (CICR) – followed by re‐sequestration of Ca2+ into the SR. The Na+/Ca2+ exchanger inextricably couples the cycling of Ca2+ and Na+ in cardiac myocytes. Thus, influx of Na+ via voltage‐gated Na+ channels (NaV) has emerged as an important regulator of CICR both in health and in disease. Recent insights into the subcellular distribution of cardiac and neuronal NaV isoforms and their ultrastructural milieu have important implications for the roles of these channels in mediating Ca2+‐driven arrhythmias. This review will discuss functional insights into the role of neuronal NaV isoforms vis‐à‐vis cardiac NaVs in triggering such arrhythmias and their potential as therapeutic targets in the context of the aforementioned structural observations.
Top: Schematic diagram showing the protein machinery of cardiac Na+–Ca2+ cycling showing a t‐tubule and associated junctional SR. Microfolds in t‐tubule are depicted based on recent findings. (Hong et al. 2014; Lavorato et al. 2015) Note the close proximity of neuronal sodium channels (nNaVs) to ryanodine receptor channels (RyRs) and the Na+–Ca2+ exchanger (NCX). Differential shading of the interstitial space within the t‐tubule and the cytoplasm within the dyadic cleft indicates local differences in ionic concentrations, particularly with respect to the bulk interstitium and cytosol respectively. Bottom left: During β‐adrenergic stimulation in healthy hearts, Na+ influx is enhanced secondary to CaMKII‐mediated phosphorylation of nNaVs. This augments Ca2+ influx via reverse mode NCX, and in turn, to enhanced SR Ca2+ release via RyRs. Inset shows normal electrocardiogram resulting from normal Na+–Ca2+ cycling. Bottom right: In diseased hearts, pathologically elevated Na+ influx via nNaVs results in a larger Ca2+ influx via reverse mode NCX. This, particularly in the presence of elevated diastolic Ca2+ levels or RyR leak, can trigger arrhythmogenic diastolic Ca2+ release. Inset electrocardiogram shows premature beats and arrhythmias triggered by diastolic Ca2+ releases.
Store-operated Ca
entry (SOCE), a major Ca
signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca
signaling in cardiac myocytes. Though it has been reported to play a ...role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca
entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca
waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.
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