Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell–cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.
Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca 2+ release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca 2+ on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [ ) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G +/− hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca 2+ release during systole, gradually overloading the sarcoplasmic reticulum with Ca 2+. The resultant Ca 2+ overload then randomly caused bursts of prolonged Ca 2+ release, activating electrogenic Na + -Ca 2+ exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.ryanodine receptor | heart | cardiac arrhythmias | CPVT | sarcoplasmic reticulum
Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca 2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca 2+ release probably due to Ca 2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2 A4860G+/− cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower. mitochondria | nanotunnels | CPVT | RyR2 | mitochondrial dynamics
Calstabin2 is a component of the cardiac ryanodine receptor (RyR2) macromolecular complex, which modulates Ca2+ release from the sarcoplasmic reticulum in cardiomyocytes. Previous reports implied that genetic deletion of Calstabin2 leads to phenotypes related to cardiac aging. However, the mechanistic role of Calstabin2 in the process of cardiac aging remains unclear. To assess whether Calstabin2 is involved in age-related heart dysfunction, we studied Calstabin2 knockout (KO) and control wild-type (WT) mice. We found a significant association between deletion of Calstabin2 and cardiac aging. Indeed, aged Calstabin2 KO mice exhibited a markedly impaired cardiac function compared with WT littermates. Calstabin2 deletion resulted also in increased levels of cell cycle inhibitors p16 and p19, augmented cardiac fibrosis, cell death, and shorter telomeres. Eventually, we demonstrated that Calstabin2 deletion resulted in AKT phosphorylation, augmented mTOR activity, and impaired autophagy in the heart. Taken together, our results identify Calstabin2 as a key modulator of cardiac aging and indicate that the activation of the AKT/mTOR pathway plays a mechanistic role in such a process.
As the most prototypical G protein-coupled receptor, -adrenergic receptor (AR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca 2؉ influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca 2؉ release flux via ryanodine receptors (RyRs). However, whether and how AR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca 2؉ imaging technology, we measured the response of a single RyR Ca 2؉ release unit, in the form of a Ca 2؉ spark, to its native trigger, the Ca 2؉ sparklet from a single LCC. We found that acute application of the selective AR agonist isoproterenol (1 M, <20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca 2؉ release flux underlying a Ca 2؉ spark to SR Ca 2؉ content indicated that AR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that AR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca 2؉ content. The PKA antagonists Rp-8-CPT-cAMP (100 M) and H89 (10 M) both eliminated these effects, indicating that AR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during AR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered AR effects in cardiovascular diseases.-adrenergic receptor ͉ calcium signaling ͉ excitation-contraction coupling ͉ isoproterenol ͉ protein kinase A T he ability of the heart to beat faster and stronger is central to the vertebrate survival instinct (1, 2). In the basal state, only a fraction of heart pumping power is used. During stress, -adrenergic receptor (AR)-mediated sympathetic modulation of heart function releases the functional reserve of the excitation-contraction (E-C) coupling to meet the increased demands of blood pumping power (3, 4). The cardiac E-C coupling operates as a coordinated system, and the degree of coordination/synchronization among its component organelles and molecules is a major determinant of the system output (2). The Ca 2ϩ transient that governs the cardiac cell contraction is generated via the Ca 2ϩ -induced Ca 2ϩ release (CiCR) mechanism (1, 5, 6), in which the Ca 2ϩ influx through L-type Ca 2ϩ channels (LCCs) during excitation activates the ryanodine receptor (RyR) Ca 2ϩ release from the sarcoplasmic reticulum (SR) (7,8). At the molecular level, individual LCC opening and local RyR Ca 2ϩ release, in the form of Ca 2ϩ spark firing, are both stochastic processes (8, 9). This stochastic behavior allows a wide dynamic range of the CiCR system, i.e., from firing random Ca 2ϩ sparks to generating global Ca 2ϩ transients.During AR stimulation, local Ca 2ϩ releases are ac...
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