A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/-) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/- mice are not susceptible to CPVT. Ex3-del+/- cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/- mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/- mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/- mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.

The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

Rationale

We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca²⁺) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca²⁺-ATPase), essential in maintaining SR Ca²⁺ content cycling during each heartbeat, is completely unknown.

Objective

To test the hypothesis that JNK2 increases SERCA2 activity SR Ca²⁺ content and exacerbates an arrhythmic SR Ca²⁺ content leak-load relationship.

Methods and results

We used confocal Ca²⁺ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca²⁺/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca²⁺ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca²⁺ affinity. Unlike the CaMKII (Ca²⁺/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca²⁺ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca²⁺ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca²⁺ leak present, the JNK2-enhanced SR Ca²⁺ uptake limited leak-induced reduction in SR Ca²⁺, normalizing Ca²⁺ transient amplitude, but at a higher arrhythmogenic SR Ca²⁺ leak. JNK2-specific inhibition completely normalized SR Ca²⁺ handling, attenuated arrhythmic Ca²⁺ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts.

Conclusions

We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca²⁺ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca²⁺ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Objectives

Carisbamate (CRS) is a novel monocarbamate compound that possesses antiseizure and neuroprotective properties. However, the mechanisms underlying these actions remain unclear. Here, we tested both direct and indirect effects of CRS on several cellular systems that regulate intracellular calcium concentration [Ca²⁺ ]_i .

Methods

We used a combination of cellular electrophysiologic techniques, as well as cell viability, Store Overload-Induced Calcium Release (SOICR), and mitochondrial functional assays to determine whether CRS might affect [Ca²⁺ ]_i levels through actions on the endoplasmic reticulum (ER), mitochondria, and/or T-type voltage-gated Ca²⁺ channels.

Results

In CA3 pyramidal neurons, kainic acid induced significant elevations in [Ca²⁺ ]_i and long-lasting neuronal hyperexcitability, both of which were reversed in a dose-dependent manner by CRS. Similarly, CRS suppressed spontaneous rhythmic epileptiform activity in hippocampal slices exposed to zero-Mg²⁺ or 4-aminopyridine. Treatment with CRS also protected murine hippocampal HT-22 cells against excitotoxic injury with glutamate, and this was accompanied by a reduction in [Ca²⁺ ]_i . Neither kainic acid nor CRS alone altered the mitochondrial membrane potential (ΔΨ) in intact, acutely isolated mitochondria. In addition, CRS did not affect mitochondrial respiratory chain activity, Ca²⁺ -induced mitochondrial permeability transition, and Ca²⁺ release from the ER. However, CRS significantly decreased Ca²⁺ flux in human embryonic kidney tsA-201 cells transfected with Ca_v 3.1 (voltage-dependent T-type Ca²⁺ ) channels.

Significance

Our data indicate that the neuroprotective and antiseizure activity of CRS likely results in part from decreased [Ca²⁺ ]_i accumulation through blockade of T-type Ca²⁺ channels.

Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.