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

Excitation-contraction coupling involves the faithful conversion of electrical stimuli to mechanical shortening in striated muscle cells, enabled by the ubiquitous second messenger, calcium. Crucial to this process are ryanodine receptors (RyRs), the sentinels of massive intracellular calcium stores contained within the sarcoplasmic reticulum. In response to sarcolemmal depolarization, RyRs release calcium into the cytosol, facilitating mobilization of the myofilaments and enabling cell contraction. In order for the cells to relax, calcium must be rapidly resequestered or extruded from the cytosol. The sustainability of this cycle is crucially dependent upon precise regulation of RyRs by numerous cytosolic metabolites and by proteins within the lumen of the sarcoplasmic reticulum and those directly associated with the receptors in a macromolecular complex. In addition to providing the majority of the calcium necessary for contraction of cardiac and skeletal muscle, RyRs act as molecular switchboards that integrate a multitude of cytosolic signals such as dynamic and steady calcium fluctuations, β-adrenergic stimulation (phosphorylation), nitrosylation and metabolic states, and transduce these signals to the channel pore to release appropriate amounts of calcium. Indeed, dysregulation of calcium release via RyRs is associated with life-threatening diseases in both skeletal and cardiac muscle. In this paper, we briefly review some of the most outstanding structural and functional attributes of RyRs and their mechanism of regulation. Further, we address pathogenic RyR dysfunction implicated in cardiovascular disease and skeletal myopathies.

Rationale Most cardiac ryanodine receptor (RyR2) mutations associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) are postulated to cause one distinctive form of Ca2+ release dysfunction. Considering the spread distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity was also feasible. Objective To determine the molecular and cellular mechanism(s) by which a novel RyR2-V2475F mutation associated with CPVT in humans triggers Ca2+-dependent arrhythmias in whole hearts and intact mice. Methods and Results Recombinant channels harboring CPVT-linked RyR2 mutations were functionally characterized using [3H]ryanodine binding and single channel recordings. Homologous recombination was used to generate a knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice heterozygous for the mutation (RyR2-V2475F+/−) and their wild-type (WT) littermates were Ca2+-imaged by confocal microscopy under conditions that mimic stress. The propensity of WT and RyR2-V2475F+/− mice to develop arrhythmias was tested at the whole heart level and in intact animals. Recombinant RyR2-V2475F channels displayed a) increased cytosolic Ca2+ activation, b) abnormal PKA phosphorylation, and c) increased activation by luminal Ca2+. The RyR2-V2475F mutation appears embryonic lethal in homozygous mice, but heterozygous mice have no alterations at baseline. Spontaneous Ca2+ release (SCR) events were more frequent and had shorter latency in isoproterenol-stimulated cardiomyocytes from RyR2-V2475F+/− hearts, but their threshold was unchanged with respect to WT. Adrenergically-triggered tachyarrhythmias were more frequent in RyR2-V2475F+/− mice. Conclusions The mutation RyR2-V2475F is phenotypically strong among other CPVT mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living mice, this mutation appears too severe to be harbored in all RyR2 channels, but remains undetected under basal conditions if expressed at relatively low levels. β-adrenergic stimulation breaks the delicate Ca2+ equilibrium of RyR2-V2475F+/− hearts and triggers life-threatening arrhythmias.