The cardiac ryanodine receptor (RyR2) governs the release of Ca 2؉ from the sarcoplasmic reticulum, which initiates muscle contraction. Mutations in RyR2 have been linked to ventricular tachycardia (VT) and sudden death, but the precise molecular mechanism is unclear. It is known that when the sarcoplasmic reticulum store Ca 2؉ content reaches a critical level, spontaneous Ca 2؉ release occurs, a process we refer to as store-overload-induced Ca 2؉ release (SOICR). In view of the well documented arrhythmogenic nature of SOICR, we characterized the effects of disease-causing RyR2 mutations on SOICR in human embryonic kidney (HEK)293 cells and found that, at elevated extracellular Ca 2؉ levels, HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells. Using this cell model, we demonstrated that the RyR2 mutations linked to VT and sudden death, N4104K, R4496C, and N4895D, markedly increased the occurrence of SOICR. At the molecular level, we showed that these RyR2 mutations increased the sensitivity of single RyR2 channels to activation by luminal Ca 2؉ and enhanced the basal level of [ 3 H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca 2؉ activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca 2؉ activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca 2؉ overload-associated VT. V entricular tachycardia (VT) is the leading cause of sudden death in patients with heart failure (HF). Delayed afterdepolarizations (DAD) frequently occur in failing hearts and are a major cause of VT, but the reason for the increased incidence of DAD-associated VT in patients with HF is not completely clear. Abnormal Ca 2ϩ handling is believed to be involved in the pathogenesis of VT (1-4). In keeping with this view, mutations in Ca 2ϩ handling proteins, including the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2), have been linked to catecholaminergic polymorphic VT (CPVT), which is also thought to be DAD-based (5-10). These similarities suggest that CPVT and DAD-associated VT in patients with HF may share a common arrhythmogenic mechanism. Thus, knowledge gained from investigation of inherited CPVT should lead to a better understanding of the molecular basis of the more commonly occurring VT in patients with HF and other cardiac diseases.RyR2 is an intracellular Ca 2ϩ release channel located in the sarcoplasmic reticulum (SR) (11). It is a key component of excitation contraction (EC) coupling in cardiac muscle, which is believed to take place via a mechanism known as Ca A number of conditions, such as physical and emotional stresses, digitalis toxicity, elevated extracellular Ca 2ϩ , ischemia͞ reperfusion, etc., can lead to SR Ca 2ϩ overload and subsequent SOICR in cardiac cells (19), and SOICR can activate inward currents. These Ca...

Abstract-Local, rhythmic, subsarcolemmal Ca 2ϩ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na ϩ -Ca 2ϩ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of ␤-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca 2ϩ cycling and beating rate is also present in vivo, as the regulation of beating rate by local ␤-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca 2ϩ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca 2ϩ balance and spontaneous SR Ca 2ϩ cycling, ie, phospholamban and L-type Ca 2ϩ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na ϩ -Ca 2ϩ exchanger current and is crucial for both basal and reserve cardiac pacemaker function. R ecent studies have demonstrated that in sinoatrial (SA) nodal cells (SANC) generate local, rhythmic, subsarcolemmal Ca 2ϩ releases (LCRs) under basal conditions, ie, even in the absence of experimental Ca 2ϩ loading or stimulation of ␤-adrenergic receptors (␤-ARs). [1][2][3] In rabbit SANC, spontaneous, rhythmic LCRs occur during the late diastolic depolarization and activate Na ϩ -Ca 2ϩ exchanger (NCX) to generate an inward current that accelerates the depolarization rate, and, thus, LCRs are involved in control of spontaneous beating rate of SANC. 1 The mechanisms that permit SANC, but not ventricular myocytes, to generate rhythmic LCRs under basal conditions, however, have not been delineated.Spontaneous SR Ca 2ϩ release is facilitated by factors that increase the rate at which the SR can pump Ca 2ϩ , foremost among which are elevated cell Ca 2ϩ or elevated cAMP and its attendant protein kinase A (PKA)-dependent protein phosphorylation that results from intense ␤-AR stimulation. Whereas the cytosolic Ca 2ϩ concentration does not appreciably differ in rabbit ventricular cells and SANC, 2,4 the cAMP level of the intact SA node is high, 5 and it has been suspected that the basal cAMP level within SANC is elevated. 6,7 The SA node, however, is highly innervated, and neither the basal cAMP le...

The transition from juvenile to adult life is accompanied by programmed remodeling in many tissues and organs, which is key for organisms to adapt to the demand of the environment. Here we report a novel regulated alternative splicing program that is crucial for postnatnal heart remodeling in the mouse. We identify the essential splicing factor ASF/SF2 as a key component of the program, regulating a restricted set of tissue-specific alternative splicing events during heart remodeling. Cardiomyocytes deficient in ASF/SF2 display an unexpected hypercontraction phenotype due to a defect in postnatal splicing switch of the Ca(2+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) transcript. This failure results in mistargeting of the kinase to sarcolemmal membranes, causing severe excitation-contraction coupling defects. Our results validate ASF/SF2 as a fundamental splicing regulator in the reprogramming pathway and reveal the central contribution of ASF/SF2-regulated CaMKIIdelta alternative splicing to functional remodeling in developing heart.

Vascular proliferative disorders, such as atherosclerosis and restenosis, are the most common causes of severe cardiovascular diseases, but a common molecular mechanism remains elusive. Here, we identify and characterize a novel hyperplasia suppressor gene, named HSG (later re-named rat mitofusin-2). HSG expression was markedly reduced in hyper-proliferative vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rat arteries, balloon-injured Wistar Kyoto rat arteries, or ApoE-knockout mouse atherosclerotic arteries. Overexpression of HSG overtly suppressed serum-evoked VSMC proliferation in culture, and blocked balloon injury induced neointimal VSMC proliferation and restenosis in rat carotid arteries. The HSG anti-proliferative effect was mediated by inhibition of ERK/MAPK signalling and subsequent cell-cycle arrest. Deletion of the p21(ras) signature motif, but not the mitochondrial targeting domain, abolished HSG-induced growth arrest, indicating that rHSG-induced anti-proliferation was independent of mitochondrial fusion. Thus, rHSG functions as a cell proliferation suppressor, whereas dysregulation of rHSG results in proliferative disorders.

No abstract

Carvedilol is one of the most effective beta-blockers for preventing ventricular tachyarrhythmias (VTs) in heart failure (HF), but the mechanisms underlying its favorable anti-arrhythmic benefits remain unclear. Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), are known to evoke VTs in patients with HF. Here we show that carvedilol is the only beta-blocker that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol combined with its beta-blocking activity likely contributes to its favorable anti-arrhythmic effect. To allow individual and optimal titration of these beneficial activities, we developed a novel SOICR-inhibiting, minimally-beta-blocking carvedilol analogue VK-II-86. We found that VK-II-86 alone prevented stress-induced VTs in RyR2 mutant mice, and was more effective when combined with a selective beta-blocker metoprolol or bisoprolol. Thus, SOICR inhibition combined with optimal beta-blockade presents a new, promising and potentially patient-tailorable anti-arrhythmic approach.

Abstract-Hyperphosphorylation of the cardiac Ca 2ϩ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca 2ϩ dependence of [ 3 H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute ␤-adrenergic stimulation, and that

Luminal Ca 2؉ in the endoplasmic and sarcoplasmic reticulum (ER͞ SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca 2؉ entry, Ca 2؉ -induced Ca 2؉ release, and ER͞SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca 2؉ ], called ''Ca 2؉ blinks,'' within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca 2؉ release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca 2؉ , or Ca 2؉ sparks, but changes of [Ca 2؉ ] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s ؊1 , which is Ϸ6-fold faster than the recovery of local Ca 2؉ release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca 2؉ signaling thus broadens our understanding of cardiac store Ca 2؉ regulation and function and opens the possibility for local regulation of diverse store-dependent functions.calcium-induced calcium release ͉ calcium spark ͉ cardiac myocytes ͉ endoplasmic reticulum ͉ sarcoplasmic reticulum L ocal Ca 2ϩ releases from the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) in muscle have been shown to underlie neurosecretion, memory encoding, neurite growth, muscle contraction, and apoptosis (1-4). Whereas the ER͞SR serves primarily as the intracellular Ca 2ϩ store, luminal Ca 2ϩ plays an active role in many regulatory systems, including store-operated capacitative Ca 2ϩ entry (5, 6), Ca 2ϩ -induced Ca 2ϩ release (7-9), and ER͞SR stress-mediated cell death (10, 11). Over the last decade, the elementary Ca 2ϩ release events have been directly visualized as Ca 2ϩ ''sparks'' (12-15), ''puffs'' (16), ''syntillas'' (17), or the equivalent (18) in the cytoplasm of both excitable and nonexcitable cells. However, the reciprocal store depletion signals, which were speculated in various models of spark termination (refs. 7 and 19; see ref. 20 for a review), have not been seen experimentally. In theory, a rapid refilling of local store Ca 2ϩ from the bulk of ER͞SR might occur and prevent significant local Ca 2ϩ depletion (21,22). Alternatively, this failing could be due to lack of a means to probe Ca 2ϩ inside this delicate membrane-bound intracellular structure with the required sensitivity, resolution, and speed, given the extremely small release flux involved (Ϸ2⅐10 Ϫ19 mol of Ca 2ϩ ) (12, 17). Using confocal imaging, electron microscopy, and electrophysiological approaches, we investigated dynamic Ca 2ϩ regulation inside nanometer-sized SR structures during elementary Ca 2ϩ release events in intact heart muscle cells. Our results afforded insights into mechanisms underlying spark termination and refract...