Modulation of focal and global Ca2+ release in calsequestrin-overexpressing mouse cardiomyocytes

Abstract

1. Focal and global Ca2+ releases were monitored in voltage-clamped control and hypertrophied calsequestrin (CSQ)-overexpressing mouse cardiomyocytes, dialysed with fluo-3, using rapid (120-240 frames s-1) two-dimensional confocal imaging. 2. Spontaneous focal Ca2+ releases (Ca2+ sparks) were absent or significantly reduced in frequency in hypertrophied myocytes of CSQ-overexpressing mice compared to their age-matched controls. Sporadic Ca2+ sparks seen in CSQ-overexpressing myocytes had intensities and durations similar to those of controls although quantitative analysis showed a trend towards more diffuse focal releases. Activation of Ca2+ current (I(Ca)) failed to produce the typical sarcomeric Ca2+ striping pattern consistently seen in control myocytes. Instead, focal Ca2+ releases appeared as a disorganized patchwork of diffuse or 'woolly' fluorescence signals, resulting in slowly developing and reduced global Ca2+ transients. 4. Although the density of I(Ca) in CSQ-overexpressing myocytes was only slightly smaller than that of controls, the inactivation kinetics of the current were greatly reduced, consistent with the much smaller rate of rise of cytosolic Ca2+. 5. Enhancement of I(Ca) by elevation of [Ca2+](o) from 2 to 10 mM or addition of 3 μM isoproterenol (isoprenaline) failed to normalize the frequency of spontaneous Ca2+ sparks at rest or the pattern and the magnitude of I(Ca)-gated Ca2+ transients. Isoproterenol was somewhat more effective than elevation of [Ca2+](o). 6. In sharp contrast, low (0.5 mM) caffeine concentrations that produced no measurable effects on I(Ca) or Ca2+ transients in control myocytes, re-established spontaneous focal Ca2+ releases in CSQ-overexpressing cells, triggered large I(Ca)-gated cellular Ca2+ transients, and strongly enhanced the kinetics of inactivation of I(Ca). 7. Our data suggest that impaired Ca2+ signalling in CSQ-overexpressing myocytes results from reduced co-ordination and decreased frequency of Ca2+ sparks. The impaired Ca2+ signalling could not be restored by procedures that increased I(Ca), but was mostly restored in the presence of caffeine, which may alter the Ca2+ sensitivity of the ryanodine receptor.