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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.

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Sarcoplasmic reticulum (SR) Ca 2 transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca 2 transport. Intracellular Ca 2 is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca 2 ] i transient were reduced in HF. Ca 2 uptake assays showed 448% reduction of V max in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 305% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P o of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor-adrenergic stimulation of Ca 2 release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca 2 uptake, more than Ca 2 release, contributing to the depressed and slow Ca 2 transient characteristic of HF. (Circ Res. 2002;91:1015-1022.) Key Words: heart failure ryanodine receptor sarcoplasmic reticulum protein kinase A

Abstract-Increased phosphorylation of the cardiac ryanodine receptor (RyR)2 by protein kinase A (PKA) at the phosphoepitope encompassing Ser2808 has been advanced as a central mechanism in the pathogenesis of cardiac arrhythmias and heart failure. In this scheme, persistent activation of the sympathetic system during chronic stress leads to PKA "hyperphosphorylation" of RyR2-S2808, which increases Ca 2ϩ release by augmenting the sensitivity of the RyR2 channel to diastolic Ca 2ϩ . This gain-of-function is postulated to occur with the unique participation of RyR2-S2808, and other potential PKA phosphorylation sites have been discarded. Although it is clear that RyR2 is among the first proteins in the heart to be phosphorylated by ␤-adrenergic stimulation, the functional impact of phosphorylation in excitation-contraction coupling and cardiac performance remains unclear. We used gene targeting to produce a mouse model with complete ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Whole-heart and isolated cardiomyocyte experiments were performed to test the role of ␤-adrenergic stimulation and PKA phosphorylation of Ser2808 in heart failure progression and cellular Ca 2ϩ handling. We found that the RyR2-S2808A mutation does not alter the ␤-adrenergic response, leaves cellular function almost unchanged, and offers no significant protection in the maladaptive cardiac remodeling induced by chronic stress. Moreover, the RyR2-S2808A mutation appears to modify single-channel activity, although modestly and only at activating [Ca 2ϩ ]. Taken together, these results reveal some of the most important effects of PKA phosphorylation of RyR2 but do not support a major role for RyR2-S2808 phosphorylation in the pathogenesis of cardiac dysfunction and failure.

Abstract-Despite

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The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/ 6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/ Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca 2+ ] i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca 2+ ] i , as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.-Kalbitz, M., Grailer, J. J., Fattahi, F., Jajou, L., Herron, T. J., Campbell, K. F., Zetoune, F. S., Bosmann, M., Sarma, J. V., Huber-Lang, M., Gebhard, F., Loaiza, R., Valdivia, H. H., Jalife, J., Russell, M. W., Ward, P. A. Role of extracellular histones in the cardiomyopathy of sepsis. FASEB J. 29, 2185-2193 (2015). www.fasebj.org