ABSTRACT
Systolic Ca2+ transients are shaped by the concerted summation of Ca2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca2+ release across the cell, although the triggers and consequences of this phenomenon remain unclear. Rat ventricular cardiomyocytes were paced at increasing rates. A discordance index (SD of local alternans ratios) was developed to quantify discordance in confocal recordings of Ca2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca2+ transient-frequency relationship. In addition, the largest local calcium transient in two consecutive transients was measured to build a potential "best release" profile, which quantitatively confirmed discordance-induced Ca2+ release impairment (DICRI). Diastolic Ca2+ homeostasis was also observed to be disrupted by discordance, as late Ca2+ release events elicited instability of resting Ca2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use.
Subject(s)
Calcium Channel Blockers , Calcium Signaling , Myocytes, Cardiac , Ryanodine Receptor Calcium Release Channel , Animals , Arrhythmias, Cardiac/metabolism , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Excitation Contraction Coupling , Myocytes, Cardiac/metabolism , Rats , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic ReticulumABSTRACT
Background Istaroxime is an inhibitor of Na+/K+ ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban-dependent inhibition of the sarcoplasmic reticulum Ca2+ ATPase. We have previously shown that pharmacologic Na+/K+ ATPase inhibition promotes calcium/calmodulin-dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na+/K+ ATPase inhibition versus istaroxime. Methods and Results Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi-inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin-dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin-dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca2+ release. In rat cardiomyocytes, istaroxime did not significantly increase Ca2+ spark and wave frequency but increased the proportion of aborted Ca2+ waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca2+ wave incidence observed with istaroxime remains present, suggesting that istaroxime-dependent relief on phospholamban-dependent sarcoplasmic reticulum Ca2+ ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug. Conclusions Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin-dependent kinase II-dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca2+ handling.
Subject(s)
Arrhythmias, Cardiac/drug therapy , Calcium/metabolism , Etiocholanolone/analogs & derivatives , Myocytes, Cardiac/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Arrhythmias, Cardiac/metabolism , Calcium Signaling/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cardiotoxicity , Etiocholanolone/pharmacology , Male , Mice , Ouabain/pharmacology , Rats , Rats, Wistar , Sarcoplasmic Reticulum Calcium-Transporting ATPases/drug effectsABSTRACT
BACKGROUND: It has been shown that carvedilol and its non ß-blocking analog, VK-II-86, inhibit spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The aim of this study is to determine whether carvedilol and VK-II-86 suppress ouabain-induced arrhythmogenic Ca2+ waves and apoptosis in cardiac myocytes. MethodsâandâResults: Rat cardiac myocytes were exposed to toxic doses of ouabain (50 µmol/L). Cell length (contraction) was monitored in electrically stimulated and non-stimulated conditions. Ouabain treatment increased contractility, frequency of spontaneous contractions and apoptosis compared to control cells. Carvedilol (1 µmol/L) or VK-II-86 (1 µmol/L) did not affect ouabain-induced inotropy, but significantly reduced the frequency of Ca2+ waves, spontaneous contractions and cell death evoked by ouabain treatment. This antiarrhythmic effect was not associated with a reduction in Ca2+ calmodulin-dependent protein kinase II (CaMKII) activity, phospholamban and ryanodine receptor phosphorylation or SR Ca2+ load. Similar results could be replicated in human cardiomyocytes derived from stem cells and in a mathematical model of human myocytes. CONCLUSIONS: Carvedilol and VK-II-86 are effective to prevent ouabain-induced apoptosis and spontaneous contractions indicative of arrhythmogenic activity without affecting inotropy and demonstrated to be effective in human models, thus emerging as a therapeutic tool for the prevention of digitalis-induced arrhythmias and cardiac toxicity.