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- W2012449435 abstract "ICa-gated release of Ca2+ from the SR is the dominant mechanism mediating cardiac E-C coupling. On the other hand, in the absence of Ca2+ entry, supplemental Ca2+ release may be activated mechanically, either from the SR secondary to nitrosylation of RyRs or from mitochondria as a direct effect of puff-induced shear force. Here we have probed the puff-induced Ca2+ release in voltage-clamped cardiomyocytes, where the Ca2+-indicator rhod-2 was targeted to mitochondria by: a) staining with the AM-form of the dye for 45 minutes, b) incubating without dye for 24-30 hours and c) dialyzing with dye-free internal solution through the voltage-clamp pipette for 20 minutes before initiating measurements. In such cells, which remained responsive for periods as long as 2 hours, we measured relatively slow (∼1s) puff-induced deceases in fluorescence suggestive of mitochondrial Ca2+ release as previously found in non-dialyzed and permeabilized cells (Belmonte and Morad, 2008, J Physiol 586:1376). To clarify the [Ca2+]i- signaling under these conditions, our experimental paradigm included activation of ICa both at the beginning and end of a 2 second priming interval where the cell was exposed to control solution, 10 mM Caffeine or zero Na+ (and high K+). The caffeine-induced Ca2+ signal was biphasic with internal solution containing 0.2 or 14mM EGTA, generating INaCa only in 0.2 but not in 14mM EGTA during the rapid initial rise of Ca2+, suggesting that the maintained component of the Ca2+ signal arises from a confined and most likely mitochondrial space, not detected by NCX. We conclude that patch clamped Rhod-2 loaded myocytes that were washed overnight and dialyzed for 1-2 hour with 14mM EGTA produced reliable mitochondrial Ca2+ signals supporting the finding that the PF-triggered Ca2+-transients are caused by mitochondrial Ca2+ release." @default.
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- W2012449435 date "2010-01-01" @default.
- W2012449435 modified "2023-09-30" @default.
- W2012449435 title "Pressure Puff Induced Calcium Signals in Voltage Clamped Cardiomyocytes" @default.
- W2012449435 doi "https://doi.org/10.1016/j.bpj.2009.12.2883" @default.
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