Paradoxical Loss of Sarcoplasmic Reticulum Calcium Release Refractoriness Caused by Overexpressing Calsequestrin in Cardiac Muscle

Abstract

L-type Ca current (ICa) triggers Carelease from the sarcoplasmic reticulum (SR) by activating RyR2 Ca release channels. Here we probe the role of the SR protein calsequestrin in the regulation of SR Ca2+ release. Voltage-clamped cardiomyocytes loaded with Fluo-4 were stimulated with 1 Hz train (S1 stimuli). Ca2+ release was activated with ICa tail currents to maintain constant trigger. Application of premature extra stimuli (S2) at successively shorter S1-S2 coupling interval resulted in significant depression of Ca2+ release during S2 stimuli in wild-type myocytes. In calsequestrin knockout myocytes (Casq2 KO), the amplitudes of S2 and S1 were the same even at shortest S1-S2 intervals (Figure). This result suggests that calsequestrin is the primary regulator of release refractoriness. To test this hypothesis further, we next used myocytes from calsequestrin overexpressing mice (Casq2 OverX). Casq2 OverX cardiomyocytes exhibit significantly depressed Ca release during S1 transients despite increased SR Ca2+ content. Surprisingly, Ca release amplitudes during the premature S2 stimulus exceeded S1 at short S1-S2 intervals (Figure). Thus, either too little or too much calsequestrin causes loss of SR Ca release refractoriness. Supported by NIH-RO1-HL88635.View Large Image | View Hi-Res Image | Download PowerPoint Slide