AIMS: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischemic cardiac injury of Kv7.4 channels have been investigated. METHODS AND RESULTS: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by qPCR. Western-blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly-isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with about 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immuno-gold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl+ influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca2+ levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/reoxygenation, and largely prevented the functional and morphological changes triggered by global ischemia/reperfusion (I/R) in Langendorff-perfused rat hearts. CONCLUSIONS: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury.

Expression and function of Kv7.4 channels in Rat cardiac mitochondria: possible targets for cardioprotection

SOLDOVIERI, Maria Virginia;AMBROSINO, Paolo;TAGLIALATELA, Maurizio
2016

Abstract

AIMS: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischemic cardiac injury of Kv7.4 channels have been investigated. METHODS AND RESULTS: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by qPCR. Western-blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly-isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with about 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immuno-gold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl+ influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca2+ levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/reoxygenation, and largely prevented the functional and morphological changes triggered by global ischemia/reperfusion (I/R) in Langendorff-perfused rat hearts. CONCLUSIONS: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11695/48446
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