Modulation of focal and global Ca2+ release in calsequestrin-overexpressing mouse cardiomyocytes.
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. 3. Activation of Ca2+ current (ICa) 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 ICa 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 ICa by elevation of [Ca2+]o from 2 to 10 mM or addition of 3 microM isoproterenol (isoprenaline) failed to normalize the frequency of spontaneous Ca2+ sparks at rest or the pattern and the magnitude of ICa-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 ICa or Ca2+ transients in control myocytes, re-established spontaneous focal Ca2+ releases in CSQ-overexpressing cells, triggered large ICa-gated cellular Ca2+ transients, and strongly enhanced the kinetics of inactivation of ICa. 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 ICa, but was mostly restored in the presence of caffeine, which may alter the Ca2+ sensitivity of the ryanodine receptor.
Wang
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Georgetown University Medical Center, Department of Pharmacology, 3900 Reservoir Road, NW, Washington, DC 20007, USA.
Cleemann
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Jones
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LR
Morad
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D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002155:Calsequestrin / Q000096:biosynthesis* / Q000235:genetics; D002469:Cell Separation; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D004594:Electrophysiology; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology
D000318:Adrenergic beta-Agonists; D002120:Calcium Channel Agonists; D002155:Calsequestrin; D000697:Central Nervous System Stimulants; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D007545:Isoproterenol; D002118:Calcium