Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion

Kerstin Boengler 0 1 Denise Hilfiker-Kleiner 0 1 Gerd Heusch 0 1 Rainer Schulz 0 1 0 D. Hilfiker-Kleiner Molekulare Kardiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 1 K. Boengler G. Heusch R. Schulz (&) Institut fur Pathophysiologie , Zentrum fur Innere Medizin, Universitatsklinikum Essen , Hufelandstr. 55, 45122 Essen, Germany The signal transducer and activator of transcription 3 (STAT3) contributes to cardioprotection by ischemic pre- and postconditioning. Mitochondria are central elements of cardioprotective signaling, most likely by delaying mitochondrial permeability transition pore (MPTP) opening, and STAT3 has recently been identified in mitochondria. We now characterized the mitochondrial localization of STAT3 and its impact on respiration and MPTP opening. STAT3 was mainly present in the matrix of subsarcolemmal and interfibrillar cardiomyocyte mitochondria. STAT1, but not STAT5 was also detected in mitochondria under physiological conditions. ADP-stimulated respiration was reduced in mitochondria from mice with a cardiomyocyte-specific deletion of STAT3 (STAT3KO) versus wildtypes and in rat mitochondria treated with the STAT3 inhibitor Stattic (STAT3 inhibitory compound, 6-Nitrobenzo[b]thiophene 1,1-dioxide). Mitochondria from STAT3-KO mice and Stattic-treated rat mitochondria tolerated less calcium until MPTP opening occurred. STAT3 co-immunoprecipitated with cyclophilin D, the target of the cardioprotective agent and MPTP inhibitor cyclosporine A S. Lecour, Cape Town, South Africa, served as guest editor for the manuscript and was responsible for all editorial decisions, including the selection of reviewers. The policy applies to all manuscripts with authors from the editor's institution. - Signal transducer and activator of transcription (STAT) proteins transduce stress signals from the plasma membrane to the nucleus, thereby acting as signaling molecules and transcriptional regulators [4]. Several STAT proteins are expressed in the heart, among them are STAT1, 3, and 5. Even though STAT proteins are highly homologous, their functions differ substantially, notably in ischemia/ reperfusion: STAT1 enhances whereas STAT3 and STAT5 reduce cardiomyocyte death [4, 23]. STAT3 and 5 are involved in the reduction of myocardial injury by ischemic pre- and postconditioning [10, 24, 39, 43], i.e. endogenous cardioprotective phenomena which have been identified in all species so far, including man [17, 28, 42]. Deletion of STAT3 (STAT3-KO) or STAT5 abolishes the infarct size reduction by ischemic preconditioning in mice hearts [41, 46]; the infarct size reduction by ischemic postconditioning is also attenuated in STAT3-KO mice [2, 22]. Since the cardioprotection by classical ischemic pre- and postconditioning is not dependent on increased mRNA levels [26, 38], it is unlikely that STATs exert their protective effect via regulation of target gene transcription [14], although increased nuclear translocation and DNA-binding of STAT1 and STAT3 have been observed with ischemic preconditioning [45, 46]. Mitochondria are centrally involved in the signal transduction of ischemic pre- and postconditioning [18]. Opening of the mitochondrial permeability transition pore (MPTP) is a major determinant of cardiomyocyte death and inhibition of MPTP opening is central to protection by ischemic pre- and postconditioning [8, 15, 21], most likely also in humans [32]. Recently, STAT3 was identified in mouse cardiomyocyte mitochondria, where it regulates complex I activity and oxygen consumption [44]. The present study confirms these findings and extends them to further address (1) the presence of STATs in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria [7, 29, 35]; (2) the interaction of STAT3 with mitochondrial respiration and MPTP opening; (3) the interaction of STAT3 with other proteins; and finally (4) the importance of STAT3 for cardioprotective strategies. Materials and methods The present study was performed with approval by the Bioethical Committee of the State of Nordrhein-Westfalen, Germany. It conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). In the present study, female mice with a cardiomyocyterestricted deletion of STAT3 (STAT3-KO), which were generated by breeding STAT3-floxed mice (STAT3flox/flox) with a-myosin heavy chain promoter/Cre recombinase transgenic mice (aMHC-Cretg/-) [20] and their wild type (WT) littermates, male and female young (8 weeks, 9 females, 1 male) and aged (21 months, 9 females, 2 males) mice (C57/Bl6 background) and 3-month-old Lewis rats were studied. Mitochondria isolation For respiration and calcium handling, subfractionation, immunoprecipitation, western blot analysis, and confocal laser scan microscopy, subsarcolemmal mitochondria (SSM) were isolated, as described previously [3]. In brief, left ventricular (LV) tissue was minced in isolation buffer (in mmol/L: sucrose 250; HEPES 10; EGTA 1, pH 7.4). The LVs were homogenized (Ultra Turrax, 3 steps of *5 s each, low speed) and then centrifuged at 600g for 10 min. The supernatant was centrifuged at 10,780g for 10 min. The resulting sediment was resuspended in isolation buffer and centrifuged at 7,650g for 10 min. To analyze STAT1 and STAT3 content in SSM and interfibrillar mitochondria (IFM), SSM and IFM were isolated from rat ventricles, as previously described [7]. For mitochondrial subfractionation, immunoprecipitation, and western blot analysis, mitochondria were further purified by percoll-gradient ultracentrifugation. Mitochondria were layered on top of a 30% percoll solution in isolation buffer and centrifuged at 35,000g for 30 min. The lower mitochondrial band was collected and washed twice in isolation buffer by centrifugation at 8,000g for 5 min. Pharmacological inhibition of STAT3 Rat LV SSM were incubated with 100 lmol/L Stattic (STAT3 inhibitory compound, 6-Nitrobenzo[b]thiophene 1,1-dioxide), a concentration previously shown to reduce STAT3 phosphorylation in isolated mouse hearts [11], or DMSO as vehicle for 1 h at 4 C and subsequently analyzed for STAT3 phosphorylation at tyrosine 705 and serine 727 by immunoprecipitation (percoll-purified mitochondria). Non-purified mitochondria were used to study mitochondrial oxygen consumption and calcium-induced MPTP opening. Mitochondrial function The respiration of 0.1 mg/mL SSM proteins was measured in a Clark-type oxygen electrode (Strathkelvin, Glasgow, UK) at 25 C in incubation buffer using 5 mmol/L glutamate and 2.5 mmol/L malate as substrates for complex 1 or 5 mmol/L succinate (in combination with 2 lmol/L rotenone to inhibit complex 1) as substrate for complex 2. After recording of basal oxygen consumption, respiration was determined after the addition of 40 lmol/L ADP [7]. Complex 4 respiration was determined after the addition of 1.8 lM antimycin A to inhibit (...truncated)