Protection against cardiac injury by small Ca2+-sensitive K+ channels identified in guinea pig cardiac inner mitochondrial membrane

Abstract

We tested if small conductance, Ca2+-sensitive K+ channels (SKCa) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O2–derived free radicals are required to initiate protection via SKCa channels, and, importantly, if SKCa channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O2•−), and m[Ca2+] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SKCa and IKCa channel opener DCEBIO (DCEB) was given for 10 min ending 20 min before IR. Either TBAP, a dismutator of O2•−, NS8593, an antagonist of SKCa isoforms, or other KCa and KATP channel antagonists, was given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O2•− and m[Ca2+], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SKCa channels to mitochondria and IMM was evidenced by a) identification of purified mSKCa protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca2+]–dependence of mSKCa channels in planar lipid bilayers, and d) matrix K+ influx induced by DCEB and blocked by SKCa antagonist UCL1684. This study shows that 1) SKCa channels are located and functional in IMM, 2) mSKCa channel opening by DCEB leads to protection that is O2•− dependent, and 3) protection by DCEB is evident beginning during ischemia.

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

“…The cardioprotective roles of SK Ca [14], IK Ca , [10], and BK Ca (big) [15,16] channels in IR and HR injury have been reported previously by our lab and others. We tested the functionality of SK Ca channel opening [14] by perfusing isolated hearts transiently before ischemia with the IK Ca [1,4,17] and SK2 and SK3 [18-21] channel opener DCEBIO (DCEB).…”

“…In the present report we have extended our prior work [14] to further characterize the mSK Ca channel, including its cardioprotective mechanism, and to identify the SK3 gene products in guinea pig, rat, and human heart. Our aims were: a) to localize and identify SK3 isoforms in guinea pig and human cardiac mitochondria; b) to identify the SK3 mitochondrial splice variant(s); c) to determine if the SK3 Cterminus downstream of the calmodulin binding domain is required for mitochondrial targeting; d) to furnish evidence that the Ca 2+ sensor of SK3 faces the matrix; e) to determine if increased Ca 2+ triggers K + uptake into mitochondria in HL-1 cells overexpressing SK3.1; f) to assess for cytoprotection against HR injury in HL-1 cells overexpressing SK3.1; and g) to examine for exacerbation of HR injury in SK3.1 silenced HL-1 cells.…”

“…We tested the functionality of SK Ca channel opening [14] by perfusing isolated hearts transiently before ischemia with the IK Ca [1,4,17] and SK2 and SK3 [18-21] channel opener DCEBIO (DCEB). We found [14] that DCEB elicited pharmacologic pre-conditioning (PPC) protection in a manner similar to that of the BK Ca channel opener NS1619 [16]; both the specific SK Ca antagonist NS8593 [22,23] and the ROS scavenger MnTBAP (TBAP), a dismutator of superoxide anion (O 2 · − ), abolished the cardioprotection [14]. Because excess reactive oxygen species (ROS) generation, depressed mitochondrial bioenergetics, and mCa 2+ overload, are major factors underlying IR injury [24], this suggested SK Ca channels may play a role in mitochondrial function.…”

“…At about the same time, we furnished novel evidence for the presence of SK2 and SK3 isoforms in the inner mitochondrial membrane (IMM) of guinea pig isolated ventricular mitochondria [14]. Based on multiple evidence including mass spectroscopy analysis and immuno-gold electron microscopy, we found that SK Ca channels are located in ventricular mitochondria, most likely within the IMM [14].…”

“…At about the same time, we furnished novel evidence for the presence of SK2 and SK3 isoforms in the inner mitochondrial membrane (IMM) of guinea pig isolated ventricular mitochondria [14]. Based on multiple evidence including mass spectroscopy analysis and immuno-gold electron microscopy, we found that SK Ca channels are located in ventricular mitochondria, most likely within the IMM [14]. Functionally, we found that the SK Ca channel opener DCEB, when given briefly before cardiac ischemia, reduced mitochondrial injury by reducing mitochondrial [Ca 2+ ] overload and excess O 2 · − emission, and by improving the redox state during reperfusion [14].…”