Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

Abstract

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the Nterminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload.

Endogenous and Agonist-induced Opening of Mitochondrial Big Versus Small Ca2+-sensitive K+ Channels on Cardiac Cell and Mitochondrial Protection

“…In the present study, evidence that the protective effects of DCEB and NS1619 are mediated by signaling O 2 •− is again indicated by reversal of the protection in the presence of TBAP as shown previously for SK Ca 18, 26 and BK Ca 4 channel agonists. Enhanced transfer of electrons by uncoupling before ischemia may minimize respiratory inefficiency; i.e., by reducing matrix contraction and improving respiration on reperfusion.…”

“…4, 18, 26, 50–53 Allowing for possible non-specific effects of the agonists and antagonists, our data furnish support that pharmacologic opening of either BK Ca or SK Ca channels is cardioprotective. Moreover, intrinsic activation of BK Ca or SK Ca channels, and especially when activated together, may be essential for endogenous protection against IR injury.…”

“…18 These studies established that K Ca channel opening initiates a cascade of O 2 •− -dependent cardiac protective mechanisms that are based primarily in cardiac myocyte mitochondria. In a recent extension of this research, 26 we: a) confirmed SK Ca protein localization in cardiac ventricular mitochondria of rats and humans, in addition to guinea pigs; b) identified mitochondrial splice variants of SK3 in guinea pig and human cardiac mitochondria; and c) found that overexpression or silencing of SK3 reduced or enhanced, respectively, stress induced damage in two cardiac cell lines, HL-1 and H9c2.…”

“…This suggests a translational value for potential intravenous treatment of K Ca channel agonists during cardiac IR injury in humans. In fact, we have recently identified splice variants of the SK Ca channel in human ventricular myocytes, 26 which warrants development of SK Ca -targeted therapeutic strategies for human cardiac IR injury.…”

“…This is because the respiratory complexes of the mitochondrial electron transport chain are most likely the major source of signaling ROS for cytoprotection and deleterious excess ROS production during IR injury. 1 Indeed, both BK Ca 50 and SK Ca 18, 26 channels are located in cardiac cell IMM rather than in the sarcolemmal membrane, and drug-induced opening of the BK Ca channel in isolated mitochondria leads to mild ROS production without significantly affecting ΔΨ m . 6 We believe that this small amount of O 2 •− emitted is important for signaling mechanisms that confer protection during treatment with a BK Ca or SK Ca agonist.…”