Chloride Transport Inhibition Causes Calcium-Dependent Arrhythmic Activity in Isoproterenol-Treated Rabbit Cardiomyocytes

Abstract

During s-adrenergic stimulation, chloride outward current mediated by CFTR has been proposed to aid repolarization and shorten action potential duration. With sustained stimulation and absent a Cl- extrusion mechanism, CFTR-mediated outward current may result in intracellular Cl- accumulation and collapse of the Cl- electrochemical gradient, leading to arrhythmias. Recently, we identified robust expression of an electroneutral K-Cl cotransporter (KCC) in vertebrate cardiomyocytes and have proposed that it plays a crucial role in Cl- homeostasis by countering channel-mediated Cl- accumulation during s-adrenergic stimulation. We tested the hypothesis that both CFTR and KCC activity are critical during s-adrenergic stimulation in paced (1Hz) acutely isolated adult rabbit cardiomyocytes. Application of novel inhibitors of either CFTR (10µM CFTR Inh-172) or KCC (2µM 11k) did not appreciably alter the regular Ca transients during steady state pacing in rabbit cardiomyocytes. Addition of 300nM isoproterenol increased Ca transient amplitude and accelerated [Ca]i decline (as expected). However, in this state, the application of either CFTR or KCC inhibitor induced prominent aftercontractions, indicative of cellular Ca overload and arrhythmogenic activity. We hypothesized that these two inhibitors elicit arrhythmic activity via distinct mechanisms: CFTR inhibition may acutely prolong action potentials directly contributing to Ca loading (independent of altered [Cl-]i), whereas KCC inhibition might allow CFTR current to dissipate the [Cl-]o/[Cl-]i gradient and thus indirectly reduce CFTR-mediated outward current (by reduced driving force). We are currently testing this working hypothesis using a novel ratiometric fluorescent protein-based Cl- sensor.