Intracellular Dyssynchrony of Diastolic Cytosolic [Ca 2+ ] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

Rationale: Synchronized release of Ca 2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective : We investigated synchrony of cytosolic [Ca 2+ ] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca 2+ ] transients (1 µm intervals) were recorded in murine, porcine and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca 2+ ] decay based on the local time constants of decay (TAU local ). The standard deviation of TAU local (SD TAU ) as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca 2+ release. Stimulation of SERCA with forskolin or istaroxime accelerated, its inhibition with cyclopiazonic acid slowed TAU local significantly more in slowCaR, thus altering the relationship between SD TAU and global [Ca 2+ ] decay (TAU global ). NCX-inhibitor SEA0400 prolonged TAU local similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca 2+ uniporter blocker Ru360. Variation in TAU local was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAU local correlated with local sarcomere relengthening. In mice with myocardial hypertrophy following trans-aortic constriction (TAC), in pigs with chronic myocardial ischemia and in end-stage human heart failure, variation in TAU local was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions : In cardiomyocytes, cytosolic [Ca 2+ ] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca 2+ ] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction. rate of Ca 2+ removal from the cytosol determines the rate of Ca 2+ diffusion from the myofilaments and cardiomyocyte relaxation. The activity of the Ca 2+ transporters SERCA and NCX is regulated in functional and structural microdomains, involving kinases, phosphatases and regulatory proteins 10-12 . Local regulation of cytosolic Ca 2+ removal may result in cytosolic diastolic Ca 2+ gradients due to intracellular variation of the rate of [Ca 2+ ] decay. Mitotracker -dependent along in a In cardiomyocytes was quantified SR Ca 2+ reuptake via SERCA and NCX1-mediated Ca 2+ extrusion are the main pathways of Ca 2+ removal from the cytosol in cardiac myocytes. Sarcolemmal NCX1 is closely associated with phosphatases, kinases, and regulatory proteins, most importantly phospholemman 14 , which form a potential basis for local regulation. NCX1 is distributed along the sarcolemmal membrane and the T-tubules. We have shown previously in pig cardiomyocytes that regions with early and delayed Ca 2+ release, reflect regions with high and low T-tubule density, respectively 1 , 5 . In the present study, the kinetics of Ca 2+ release was not related to the kinetics of local [Ca 2+ ] decay, suggesting that the distribution of T-tubules as a scaffold for NCX1 did not have a large influence on the regional variation in the rate of local cytosolic [Ca 2+ ] decay in healthy cardiomyocytes. Furthermore, our findings of similarly prolonged Ca 2+ decay in all cell regions with NCX-inhibition (in healthy and also in TAC mice with higher NCX activity) and the unaltered variability of Ca 2+ decay kinetics in NCX1 heterozygous mice argue against a role for the sarcolemmal NCX in the observed intracellular variation of the cytosolic [Ca 2+ ] decay rate. However, as NCX1 activity in human myocardium has been reported to contribute to an even larger extend to cytosolic Ca 2+ removal 15 , the role of NCX for dyssynchrony in human myocardium remains to be investigated.