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Rationale: Synchronized release of Ca²⁺ into the cytosol during each cardiac cycle determines cardiomyocyte contraction., Objective: We investigated synchrony of cytosolic [Ca²⁺] decay during diastole and the impact of cardiac remodeling., Methods and Results: Local cytosolic [Ca²⁺] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²⁺] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²⁺ release. Stimulation of sarcoplasmic reticulum Ca²⁺ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²⁺] decay (TAUglobal). Na⁺/Ca²⁺ exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²⁺ uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density., Conclusions: In cardiomyocytes, cytosolic [Ca²⁺] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²⁺] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI)., Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI., Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca(2+) transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca(2+) release, F>F(50) within 20 ms) or their absence (delayed areas). Spontaneous Ca(2+) release events during diastole, Ca(2+) sparks, reflecting RyR activity and properties, were subsequently assigned to either category., Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na(+)/Ca(2+) exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca(2+) influx via Ca(2+) channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca(2+) content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca(2+) removal by NCX at the membrane was significantly lower in MI., Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca(2+) loss and raise SR Ca(2+) content, but may promote Ca(2+) waves.

In ventricular cardiac myocytes, T-tubule density is an important determinant of the synchrony of sarcoplasmic reticulum (SR) Ca2+ release and could be involved in the reduced SR Ca2+ release in ischemic cardiomyopathy. We therefore investigated T-tubule density and properties of SR Ca2+ release in pigs, 6 weeks after inducing severe stenosis of the circumflex coronary artery (91+/-3%, N=13) with myocardial infarction (8.8+/-2.0% of total left ventricular mass). Severe dysfunction in the infarct and adjacent myocardium was documented by magnetic resonance and Doppler myocardial velocity imaging. Myocytes isolated from the adjacent myocardium were compared with myocytes from the same region in weight-matched control pigs. T-tubule density quantified from the di-8-ANEPPS (di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate) sarcolemmal staining was decreased by 27+/-7% (P<0.05). Synchrony of SR Ca2+ release (confocal line scan images during whole-cell voltage clamp) was reduced in myocardium myocytes. Delayed release (ie, half-maximal [Ca2+]i occurring later than 20 ms) occurred at 35.5+/-6.4% of the scan line in myocardial infarction versus 22.7+/-2.5% in control pigs (P<0.05), prolonging the time to peak of the line-averaged [Ca2+]i transient (121+/-9 versus 102+/-5 ms in control pigs, P<0.05). Delayed release colocalized with regions of T-tubule rarefaction and could not be suppressed by activation of protein kinase A. The whole-cell averaged [Ca2+]i transient amplitude was reduced, whereas L-type Ca2+ current density was unchanged and SR content was increased, indicating a reduction in the gain of Ca2+-induced Ca2+ release. In conclusion, reduced T-tubule density during ischemic remodeling is associated with reduced synchrony of Ca2+ release and reduced efficiency of coupling Ca2+ influx to Ca2+ release.