Alterations in Ca2+ Sparks and T-Tubules Promote Slowed, Dyssynchronous Ca2+ Release in Failing Cardiomyocytes

Abstract

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca2+ transient which contributes to the impaired contractility in this condition. We investigated the underlying mechanisms in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by left coronary artery ligation, and at 10 weeks post-MI, mice exhibited symptoms of CHF including reduced cardiac function and increased lung weight. Cardiomyocytes were isolated from viable regions of the septum, and septal myocytes from SHAM-operated mice served as controls. Confocal line-scan imaging revealed a slowed rate of rise of Ca2+ transients (fluo-4 AM, 1 Hz) in CHF cells, which largely resulted from spatially non-uniform Ca2+ release. Ca2+ sparks recorded in resting myocytes were also slower to peak in CHF than SHAM (11.5±0.6 ms vs 9.5±0.6 ms, P<0.05) and longer lasting (FWHM=24.5±0.7 ms vs 21.6±1.0 ms, P<0.05). The mean increase in these measurements resulted from a sub-population of sparks in CHF cells with very long rise times but small amplitudes. Local Ca2+ transients (width=2 μm) measured at the same coordinates as these sparks were also slow to rise, indicating that altered Ca2+ spark kinetics contributed to the dyssynchronous Ca2+ release pattern in CHF. As well, di-8-ANEPPS staining revealed disorganized T-tubular structure in failing myocytes, which is also known to promote Ca2+ release dyssynchrony. Specifically, we observed irregular gaps between adjacent tubules where Ca2+ release was markedly delayed, occurring only after Ca2+ diffusion from regions where tubules were present. Thus, slowed and dyssynchronous Ca2+ release in failing myocytes results from a combination of altered ryanodine receptor function and T-tubule disorganization. We suggest that the sub-population of slow, small amplitude Ca2+ sparks in CHF may represent ryanodine receptors which are functionally uncoupled from their neighbours.