Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion
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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 (STAT3-KO) 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 (CsA). However, CsA reduced infarct size to a similar extent in wildtype and STAT3-KO mice in vivo. Thus, STAT3 possibly contributes to cardioprotection by stimulation of respiration and inhibition of MPTP opening.
KeywordsCardioprotection Ischemia Mitochondrial permeability transition pore Mitochondrion Reperfusion STAT
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 . 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 , 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 . 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 . Recently, STAT3 was identified in mouse cardiomyocyte mitochondria, where it regulates complex I activity and oxygen consumption . 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 cardiomyocyte-restricted deletion of STAT3 (STAT3-KO), which were generated by breeding STAT3-floxed mice (STAT3flox/flox) with α-myosin heavy chain promoter/Cre recombinase transgenic mice (αMHC-Cretg/−)  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.
For respiration and calcium handling, subfractionation, immunoprecipitation, western blot analysis, and confocal laser scan microscopy, subsarcolemmal mitochondria (SSM) were isolated, as described previously . 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 . 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 μmol/L Stattic (STAT3 inhibitory compound, 6-Nitrobenzo[b]thiophene 1,1-dioxide), a concentration previously shown to reduce STAT3 phosphorylation in isolated mouse hearts , 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.
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 μmol/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 μmol/L ADP . Complex 4 respiration was determined after the addition of 1.8 μM antimycin A to inhibit complex 3 and 300 μM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) and 3 mM ascorbate—which donates electrons to cytochrome oxidase via the reduction of cytochrome c. Maximal respiration was determined after the subsequent addition of 1.3 μmol/L oligomycin to inhibit the ATP synthase and 30 nmol/L FCCP.
Calcium retention capacities of 0.1 mg/mL rat SSM or 0.075 mg/mL mouse SSM were measured in 2 ml incubation buffer without EGTA at 25°C using 5 mmol/L glutamate and 2.5 mmol/L malate as substrates for complex 1 or 5 mmol/L succinate with 2 μmol/L rotenone as substrate for complex 2. Extramitochondrial calcium was detected using 0.5 μmol/L calcium green 5 N (Invitrogen, Carlsbad, CA, USA) with a Cary Eclipse spectrophotometer at excitation and emission wavelengths of 500 and 530 nm, respectively. Experiments were performed in the presence and absence of 40 μmol/L ADP and 1.2 mmol/L MgCl2. Six nmol (mouse SSM without ADP and MgCl2) or 10 nmol (all other groups) CaCl2 were added once per minute until an increase in calcium green fluorescence was detected, reflecting MPTP opening.
Subfractionation of mitochondria
Mitochondria were subfractionated following a modified protocol . Freshly isolated percoll-purified rat LV SSM were resuspended in 150 mmol/L KCl supplemented with 100 μM digitonin and protease inhibitors (Complete, Roche, Basel, Switzerland). After incubation for 10 min on ice, the mitochondria were centrifuged at 10,000g for 10 min at 4°C. The supernatant contained the proteins of the intermembrane space. The sediment was resuspended in isolation buffer supplemented with 200 μmol/L digitonin. An equal volume of sunflower oil was added, and the mitochondria were vigorously mixed for 5 min. After centrifugation for 5 min at 14,000g, the lower phase containing the matrix was separated from the interphase containing the membrane proteins.
Mitochondrial swelling was induced by incubation in hypoosmotic solution (5 mmol/L sucrose, 5 mmol/L Hepes, 1 mmol/L EGTA, pH 7.2) for 10 min before normalizing the osmolarity by adding hyperosmotic solution .
Immunoprecipitation and western blot analysis
Mitochondria or right ventricles were lysed in 1× Cell lysis buffer [Cell Signaling, Beverly, MA, USA; containing in mmol/L: Tris (pH 7.5) 20, NaCl 150, EDTA 1, EGTA 1, sodium pyrophosphate 2.5, β-glycerolphosphate 1, Na3VO4 1, Triton X-100 1%, Leupeptin 1 μg/mL, supplemented with 1× Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland)], and subsequently centrifuged at 13,000g for 10 min. The protein concentration of the supernatant was determined using the Dc protein assay (BioRad, Hercules, CA, USA).
For immunoprecipitation, 400 μg rat mitochondrial proteins were incubated with antibodies against total or phosphorylated STAT3 (Tyr705 or Ser727), Tom20, anti-rabbit or anti-mouse horseradish peroxidase conjugated IgGs for 1 h at 4°C. Protein A Agarose (Santa Cruz) was added to each sample followed by overnight incubation at 4°C. Protein A Agarose beads were washed three times with 500 μl PBS supplemented with 1× Complete Protease Inhibitors. After adding sample buffer, the samples were boiled for 5 min at 95°C.
Immunoprecipitated proteins, 5 μg subfractionated mitochondrial proteins or 25 μg total mitochondrial or right ventricular proteins were electrophoretically separated on 10% SDS gels and transferred to nitrocellulose membranes. Protein transfer was controlled by staining of membranes with Ponceau S. After blocking, the membranes were incubated with primary antibodies and then washed with the respective secondary antibodies. Immunoreactivities were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA) and densitometrically quantified with Scion Image software (Frederick, ML, USA).
Ischemia/reperfusion injury in vivo
Female STAT3-KO mice or their WT littermates were anesthetized with pentobarbital sodium (80 mg/kg ip). The temperature of the animals was kept stable between 36.6 and 37.4°C using heating pads, and the electrocardiogram was monitored continuously. After intubation (polyethylene-60 tubing), the animals were ventilated with a stroke rate of 130/min and a tidal volume of 1 mL. A midline thoracotomy and pericardiotomy were performed. The left anterior descending coronary artery (LAD) was occluded 2–3 mm distal to the tip of the left auricle using a 7.0 silk suture and a small tube to form a snare for 30 min. 5 min before reperfusion, 10 mg/kg cyclosporine A or 0.9% NaCl as vehicle were administered intravenously. Hearts were then reperfused for 120 min. 1 U/g heparin was given intraperitoneally 20 min before the end of reperfusion. At the end of the protocol, the LAD was re-occluded, 1 mL of 5% Evans Blue was injected into the left ventricle (LV), and mice were killed with a KCl bolus. Hearts were excised, and atrial and right ventricular tissues were removed. LV tissue was washed, frozen for 30 min at −20°C, cut into 5–6 transverse slices, and incubated for 20 min at 37°C in 2% TTC (2,3,5-triphenyl tetrazolium chloride) and for 12 h in 10% formalin. Tissue sections were photographed and infarct size was calculated as percent of the area at risk by planimetry.
The following antibodies were used in the present study: rabbit polyclonal anti-mouse phosphorylated STAT3 (Tyr705, Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-mouse phosphorylated STAT3 (Ser727, Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-mouse total STAT3 (Cell Signaling, Beverly, MA, USA), mouse monoclonal anti-mouse total STAT3 (Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-rat total connexin43 (Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-rat sodium/potassium (Na+/K+)-ATPase (Upstate, Waltham, MA, USA), mouse monoclonal anti-dog sarcoplasmic calcium (SERCA2)-ATPase (Sigma, Saint Louis, MO, USA), rabbit monoclonal anti-human histone deacetylase 2 (HDAC2, Abcam, Cambridge, UK), mouse monoclonal anti-rabbit GAPDH (Hytest, Turku, Finland), rabbit polyclonal anti-human translocase of the outer membrane 20 (Tom20, Santa Cruz, Santa Cruz, CA, USA), rabbit polyclonal anti-human VDAC (voltage-dependent anion channel, Abcam, Cambridge, UK), mouse monoclonal anti-human ATP synthase α (BD Transduction, San Diego, CA, USA), mouse monoclonal anti-rat cytochrome c (Santa Cruz, Santa Cruz, CA, USA), rabbit polyclonal anti-human cyclophilin D (Acris, Hiddenhausen, Germany), rabbit polyclonal anti-human MnSOD (manganese superoxide dismutase, Upstate, Lake Placid, NY, USA), or mouse monoclonal anti-rat cyclophilin D (MitoSciences, Eugene, OR, USA).
Data are presented as mean values ± SEM. Western blot data of subfractionated mitochondria and infarct size data were compared by two-way repeated measures ANOVA and Fisher’s LSD. Western blot data on the expression and phosphorylation level of STAT1, STAT3, and Tom20 in SSM/IFM and on STAT3 or Cx43 in young/aged mitochondria, as well as data on mitochondrial respiration were compared by unpaired Student’s t test. A p < 0.05 indicated a significant difference.
Since only STAT3 and STAT5 have an established role in endogenous cardioprotection, but only STAT3 is present in mitochondria, we subsequently focussed our investigations on mitochondrial STAT3.
Total STAT3 was immunoprecipitated from rat LV SSM. The specificity of the immunoprecipitation was demonstrated by the lack of STAT3 immunoreactivity in the control IgG precipitation and lack of manganese superoxide dismutase (MnSOD) and cytochrome c signals in the STAT3 precipitation (Fig. 2a). Both serine-phosphorylated and total STAT3 co-immunoprecipitated with the presequence receptor Tom20 (translocase of the outer membrane 20), which is part of the mitochondrial import machinery, in rat LV protein extracts (Fig. 2c). Also, mitochondrial STAT3 co-immunoprecipitated with cyclophilin D, which is involved in the regulation of MPTP opening (Fig. 2d).
Using substrates for complex 2, MPTP opening in the presence of ADP and MgCl2 was induced at similar calcium concentrations in WT and STAT3-KO mitochondria (in nmol CaCl2/mg protein, WT: 1,164 ± 47, n = 7 vs. STAT3-KO: 1,264 ± 56, n = 7, p = ns). However, Stattic treatment of rat mitochondria again accelerated MPTP opening (in nmol CaCl2/mg protein, DMSO: 1,357 ± 67, n = 7 vs. Stattic: 629 ± 39, n = 7, p < 0.05).
The present study demonstrates the presence of STAT1 and STAT3, but not of STAT5 in cardiomyocyte mitochondria. Our data confirm the previously described localization of STAT3 in cardiomyocyte mitochondria  and extend these prior data in that the mitochondrial localization of STAT proteins is not restricted to STAT3 but also true for STAT1. The lack of STAT5 in cardiomyocyte mitochondria under basal conditions does not exclude a possible mitochondrial import of STAT5 following ischemia/reperfusion; alternatively, the STAT5-mediated cardioprotection by ischemic preconditioning  works independently from mitochondrial signal transduction cascades.
In the present study, STAT1 and STAT3 were found in SSM and IFM, mitochondrial populations which differ in their protein composition as well as in their respiratory function and calcium retention capacity [7, 29, 35]. Therefore, the opposing roles of STAT1 and STAT3 in myocardial ischemia/reperfusion—STAT1 is associated with enhanced and STAT3 with reduced cardiomyocyte death after ischemia/reperfusion (for review see )—cannot be attributed to diverging localizations in mitochondrial subpopulations under physiological conditions. However, whether or not the distribution of STAT1 and STAT3 in mitochondrial subpopulations is changed after ischemia/reperfusion is currently unknown. The residual STAT3 immunoreactivity detected by western blot analysis in mitochondria from STAT3-KO hearts is presumably due to the presence of mitochondria from endothelial cells or smooth muscle cells—i.e. other STAT3 expressing cell types in the heart which are not affected by the cardiomyocyte-specific STAT3 knockout, but are included in the mitochondrial preparation.
STAT3 is encoded in the nucleus and therefore has to be imported into the mitochondria. Both phosphorylated and total STAT3 co-immunoprecipitated with the mitochondrial presequence receptor Tom20, suggesting an import of STAT3 via a Tom20-dependent pathway.
The transcriptional activity of STAT3 is regulated by post-translational modification, i.e. phosphorylation and dephosphorylation. Two major phosphorylation sites are known: phosphorylation at tyrosine 705 via Janus kinases which induces dimerization and translocation into the nucleus, and phosphorylation at serine 727 which plays a role in modulating transcriptional activity . Mitochondrial STAT3 was phosphorylated at both tyrosine 705 and serine 727. The phosphorylation of STAT3 at serine 727 appears to be more pronounced. Our data do not allow to distinguish whether or not STAT3 is phosphorylated prior to its import or within the mitochondria. Subfractionated mitochondria were characterized to identify the submitochondrial localization of STAT3, and STAT3 was predominantly detected in the mitochondrial matrix. However, the degree of phosphorylation of STAT3 in different mitochondrial compartments remains unclear. Our data are in accordance to and extend the data by Wegrzyn et al. , who have previously excluded the presence of STAT3 in the outer membrane of liver mitochondria. STAT3 as a transcriptional activator in the mitochondrial matrix may influence the transcription of genes encoded in the chondriome. However, Wegrzyn et al.  found no differences in the transcript levels of mitochondrially encoded genes in wildtype and STAT3-deficient pro-B cells. These results suggest that mitochondrial STAT3 serves a function different from transcriptional activation. Previous data propose that the serine 727 phosphorylation is important for the effect of STAT3 on mitochondrial respiratory complex 1 and 2 activities [27, 44].
In the present study, mitochondria isolated from STAT3-KO hearts had reduced ADP-stimulated complex 1 respiration compared to mitochondria isolated from WT hearts, confirming previous results . However, when measuring complex 2- or complex 4-mediated respiration and uncoupled respiration, only a trend towards a decreased ADP-stimulated respiration was detected. STAT3-deficiency was associated with decreased complex 5 activity and reduced ATP contents in H-RasV12-transformed cells ; however, the present study did not characterize complex 5 activity and ATP-production of isolated cardiomyocyte mitochondria.
The investigation of mitochondria isolated from STAT3-KO hearts does not allow to distinguish whether the effects of STAT3 on respiration are caused directly by mitochondrial STAT3 or indirectly by changing the transcription of nuclear-encoded mitochondrial target genes. We therefore also studied respiration in rat mitochondria, which were incubated with the STAT3-specific inhibitor Stattic. The concentration of 100 μmol/L Stattic has previously been shown to effectively reduce the tyrosine phosphorylation of STAT3 in isolated mouse hearts . In the present study, Stattic also decreased mitochondrial STAT3 tyrosine 705 and tended to reduce STAT3 serine 727 phosphorylation. In Stattic-treated mitochondria, ADP-stimulated complex 1 respiration was reduced to about 50% of control. Using complex 2 substrates, Stattic reduced ADP-stimulated respiration again to a lesser extent than with complex 1 substrates. Taken together, genetic reduction and pharmacological inhibition of STAT3 did not alter basal respiration, but decreased ADP-stimulated respiration, notably that of complex 1. Whereas STAT3 has been demonstrated to be associated with complex 1 and possibly with complex 2 , a recent study calculated the ratio of complexes 1 and 2 over STAT3 to be in the order of 105 , making a direct protein–protein interaction between STAT3 and complexes 1 and 2 unlikely. However, STAT3 may influence respiration indirectly via modulation of the activity of protein kinases, e.g. by facilitating the docking of kinases to target proteins .
The MPTP, a voltage-dependent, high conductance mitochondrial membrane channel, opens when exposed to high concentrations of reactive oxygen species and calcium at normal intracellular pH. The well-established inhibitory effects of ADP and MgCl2 on calcium-induced MPTP opening [9, 34] were confirmed in the present study, since the amounts of calcium needed to induce MPTP opening were higher in the presence of ADP and MgCl2. Opening of the MPTP releases cytochrome c and induces cardiomyocyte apoptosis and necrosis. Inhibition of MPTP opening is important for cardiomyocyte survival and the cardioprotection by ischemic pre- and postconditioning [8, 15, 19, 21]. Calcium-induced MPTP opening occurred at lower calcium concentrations in STAT3-KO mitochondria than in WT mitochondria respiring on complex 1 only in the presence of ADP and MgCl2. The more pronounced MPTP opening of mitochondria respiring on complex 1 with ADP and MgCl2 compared to basal conditions was also confirmed in Stattic-treated rat mitochondria. Stattic enhanced calcium-induced MPTP opening already under basal conditions in rat mitochondria, but not in mouse STAT3-KO mitochondria. Such accelerated MPTP opening in STAT3-KO mitochondria, especially in the presence of ADP and MgCl2, may contribute to the loss of endogenous cardioprotection in STAT3-deficient mice. Whether or not the impact of STAT3 on MPTP opening is due to changes in the mitochondrial membrane potential is unknown at present.
The more pronounced effect of Stattic on respiration and MPTP opening compared to that of STAT3-KO mitochondria may be due to the fact that Stattic inhibits all STAT3-containing mitochondria, whereas in mitochondrial preparations from mice with a cardiomyocyte-specific deletion of STAT3, a certain amount of STAT3 containing mitochondria from endothelial cells, vascular smooth muscle cells or invading blood cells is still present.
STAT3 co-immunoprecipitated with cyclophilin D, a matrix protein which facilitates MPTP opening when bound to the inner mitochondrial membrane . CsA prevents binding of cyclophilin D to the inner mitochondrial membrane, thereby delays MPTP opening and contributes to infarct size reduction after ischemia/reperfusion injury in animals [1, 16, 21, 40] and man . In the present study, CsA delayed calcium-induced MPTP opening in isolated mitochondria; however, this delay was more pronounced in control mitochondria than in STAT3-KO or Stattic-treated mitochondria. Whereas these in vitro data might suggest that infarct size reduction by CsA in vivo might be more pronounced in WT than in STAT3-KO mice, the present study demonstrated in fact a similar infarct size reduction by CsA in both genotypes. The magnitude of infarct size reduction by CsA was comparable to that achieved by ischemic postconditioning in WT mice . Therefore, inhibition of cyclophilin D by CsA appears to be rather downstream of STAT3 in the signaling cascade of cardioprotection, or STAT3 and CsA interaction with different domains of cyclophilin D. The reduction of reperfusion injury by maneuvers such as ischemic postconditioning is dependent on STAT3 [2, 22], whereas the reduction of reperfusion injury by inhibition of the STAT3 co-precipitating protein cyclophilin D is apparently downstream of STAT3.
The detection of total and phosphorylated STAT1 and STAT3 in mitochondria by western blot analysis and confocal laser scan microscopy is dependent on the affinities of the respective antibodies towards their target epitopes. Therefore, an accurate quantification of the amounts of total mitochondrial STAT1 and STAT3 is not possible without using purified proteins as standard. Whereas our immunoprecipitation and confocal laser scan microscopy data suggest that the phosphorylation of STAT3 is more prominent at Ser727 than at Tyr705, we cannot quantify the ratio of serine over tyrosine phosphorylated mitochondrial STAT3. The STAT3 inhibitor Stattic has been described to block the phosphorylation of STAT3 at Tyr705, and we confirmed this result in isolated mitochondria. However, the phosphorylation of STAT3 at Ser727 also tended to be decreased by Stattic. We can therefore not distinguish whether the decreased APD-stimulated respiration and the enhanced calcium-induced MPTP opening in Stattic-treated mitochondria are due to the inhibition of the STAT3 phosphorylation at residue Ser727, Tyr705 or both. One could speculate that the enhanced calcium-induced MPTP opening in STAT3-deficient mitochondria would be paralleled by enhanced infarct size in STAT3 KO mice after ischemia/reperfusion injury in vivo. However, similar infarct sizes were measured in WT and STAT3 KO mice. In contrast to the unaltered infarct size with ischemia/reperfusion per se, endogenous cardioprotection by ischemic pre- and postconditioning was abolished in STAT3 KO mice. These findings suggest that STAT3 deficiency can be compensated during ischemia/reperfusion, but that these compensatory mechanisms are not sufficient to mediate endogenous cardioprotection by ischemic pre- and postconditioning.
Potential clinical relevance
The reduced mitochondrial STAT3 level in aged hearts may be important for the loss of cardioprotection in aged mice [2, 5, 6]. Apart from aging, STAT3 expression is decreased in rats with diabetes  and in patients with end-stage heart failure , pathophysiological conditions also associated with loss of endogenous cardioprotection. The targeting of STAT3 to the mitochondria promotes Ras-dependent oncogenic transformation . However, the present data also point to beneficial roles of mitochondrial STAT3 for maintaining mitochondrial function and thereby enhancing cell survival. Mitochondrial STAT3 possibly contributes to cardioprotection by augmentation of ADP-stimulated respiration and inhibition of MPTP opening, but not through interaction with CsA binding to cyclophilin D. Our data suggest that patients with reduced contents of STAT3 would benefit from CsA therapy in acute myocardial infarction.
Taken together, STAT1 and STAT3 are localized in the matrix of cardiomyocyte mitochondria. Mitochondrial STAT3 stimulates respiration and inhibits calcium-induced MPTP opening, thereby potentially contributing to cardioprotection.
We thank Dr. Sabine Stahlhofen and Astrid Büchert, Elvira Ungefug, Ina Konietzka, Anita van de Sand, and Petra Gres for excellent technical assistance. K.B. (BO 2955/1-1) and G.H. (He1320/18-1) are recipients of grants from the Deutsche Forschungsgemeinschaft.
Conflict of interest
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