| Popis: |
Cardiac EC-coupling is mainly controlled by Ica-gated Ca-release, but the extent to which mitochondria contribute to the overall Ca-signaling remains controversial. To examine the possible role of mitochondria in Ca-signaling, we used a lower Ca-affinity mitochondrial probe, mitycamE31Q (300MOI, 48-72h) in conjunction with TMRE and Fura-2AM in cultured neonatal rat and adult feline cardiomyocytes using a dual wavelength TIRF imaging system (< 150nm penetration, 10Hz). Calcium was simultaneously measured in cytosol and mitochondria using mitycamE31Q and Fura-2. TMRE was used to monitor mitochondrial membrane potential. MitycamE31Q and TMRE staining of feline cardiomyocytes showed fluorescent longitudinal bands. Caffeine application caused a rapid decline in Fura-2 signal (increased Ca2+) that recovered slowly while mitycamE31Q signal initially increased (loss of [Ca2+]m or [H+]m) before decaying back to or below baseline ([Ca2+]m or [H+]m uptake). Simultaneously measured TMRE signal showed mitochondrial depolarization and repolarization. Low Na+ slowly decreases both Fura-2AM and mitycamE31Q signals, consistent with rise of cytosolic and mitochondrial Ca2+. FCCP produced little changes in the Fura-2AM signal, but increased the rate of spontaneous Ca-waves as it increased mitochondrial Ca2+ and depolarized mitochondrial membrane potential (increase TMRE signal). In contrast, single mitochondrial signals demonstrated heterogeneous populations of mitochondrial activity. Our findings suggest that Ca2+ in the mitochondria not only shows transients that are generally delayed and attenuated compared to the cytosolic Ca2+ signals, but at intervals can also be released locally and spontaneously. When synchronized, the local mitochondrial Ca2+ releases generate large cellular cytosolic Ca2+ releases that may interfere with normal Ca2+ signaling. (Support: NIH, RO1-HL16152, RO1-HL107600). |
