Abstract 1286: Increased K+ Flux into Mitochondria Confers Cardioprotection from Hypoxia
Increased mitochondrial K+ influx through various types of mitochondrial K+ channels has been implicated in cardioprotection during stress conditions. However, current evidence for this protective mechanism is inferred mostly from pharmacological studies that have used the putative mitochondrial K+ channel agonists and antagonists. In the present study, we hypothesized that selective mitochondrial expression of a K+ channel protects cardiomyocytes from injury by diminishing Ca2+ overload. Targeted K+ channel expression was accomplished by cloning the Kir6.2 gene in pCMV/mito/GFP vector. The proper trafficking to mitochondria of transfected HL-1 and HEK293 cell lines was confirmed by colocalization studies, immunoblot-ting and patch clamping. The rate of K+ uptake was measured by the K+-sensitive fluorescent indicator, PBFI AM. The impact of increased mitochondrial K+ permeability on cellular viability was assessed by the measurement of LDH release after exposing the cells to hypoxia/ reoxygenation. Mitochondrial Ca2+ homeostasis and transmembrane potential were monitored with mitochondrial Ca2+-sensitive indicator rhod-2 and potentiometric dye TMRE, respectively. The K+ uptake was significantly increased in the cells with mitochondrial overexpression of K+ channels which indicated a functional incorporation of the channel into mitochondria. Also, the cells tolerance to stress was significantly improved (LDH release increased to 162±7% of baseline compared to 239±15% in the cells without K+ channel overexpression). This was paralleled by substantially blunted mitochondrial Ca2+ loading (relative rhod-2 fluorescence increased to 168±15% in mitochondria expressing Kir6.2 vs. 249±13% for the control mitochondria). This observed effect was presumably due to a partial mitochondrial depolarization that was detected in the mitochondria overexpressing the active K+ channel, which reduces the driving force for excessive Ca2+ accumulation. In conclusion, this study provides the first nonpharmacological evidence that an increased K+ influx to mitochondria confers cardioprotection against hypoxic stress, most likely by preventing detrimental effects of mitochondrial Ca2+ overload.
Supported in part by AHA (0515460Z to MLJ).