Ouabain is a Pharmacomimic of Mutant RyR2 Ca2+ Release Dysfunction but is not a Serum-Borne Trigger of CPVT

Abstract

Mutation-linked RyR2 Ca2+ handling dysfunction is an intracellular driver for stress-evoked arrhythmias. Disease pathogenesis links augmented β-adrenergic stimulation to the acute dysfunction of mutant RyR2. However, the incomplete efficacy of β-blockade in clinical and experimental scenarios suggests that other serum-borne factors, in addition to catecholamines, contribute to Ca2+ release dysfunction. Cardiac electrical dysfunction in CPVT is remarkably similar to that elicited by digitalis toxicity and a related glycoside, ouabain, has been identified as an endogenous Ca2+-mobilising ‘hormone’. Circulating levels of adrenally-derived ouabain are reportedly elevated in heart failure and post-exercise. Here we investigated whether physiologic levels of ouabain could provoke mutant RyR2 channel dysfunction. In single HL-1 cardiomyocytes, ouabain (EC50 = 14.3nM) perturbed Ca2+ homeostasis in a manner strikingly similar to that determined in cells expressing CPVT-linked R4497C mutation. Moreover, dose-dependent ouabain-evoked Ca2+ dysfunction resulted in a left-shifted caffeine activation profile typical of mutant RyR2 channels. LC/MS/MS analysis determined equivalent levels of ouabain in serum obtained from CPVT- and normal subjects (500-800pM) and exercise stress-testing did not acutely elevate ouabain levels in either cohort. In cells expressing the R4497C mutation, which exhibited basal Ca2+ dysfunction, addition of ouabain at physiologic (1nM) and supra-physiologic (10-100nM) concentrations did not further exacerbate cellular Ca2+ abnormalities. In contrast, the addition of serum obtained from exercised normal and CPVT subjects (100ug protein equivalents; ∼2pM ouabain) evoked hallmark mutation-linked RyR2 dysfunction that was not completely abolished by β-blockade. These findings confirm that non-catecholaminergic factors contribute to RyR2 mutant dysfunction, but preclude the possibility that endogenous ouabain is a physiologically relevant trigger of CPVT. However, the hypothesis that endogenous ouabain may induce underlying changes in excitation-contraction coupling that increase the propensity for mutation-linked RyR2 dysfunction is being investigated.