Abstract
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that contributes a crucial role in protection against ischemia (ISC)-reperfusion (REP) injury by driving expression of anti-apoptotic and anti-oxidant genes. STAT3 is also present in the mitochondria, where it modulates the activity of the electron transport chain (ETC) and the permeability transition pore. Transgenic mice that overexpress a mitochondrial-targeted, transcriptionally inactive STAT3 in cardiomyocytes (MLS-STAT3E mice) exhibit a persistent, partial blockade of electron transfer through complex I that uniquely did not lead to tissue dysfunction at baseline, yet increased mitochondrial ischemic tolerance. The direct contribution of non-transcriptional, mitochondria-localized STAT3 to protection during ISC-REP remains to be established. We hypothesized that the enhanced mitochondrial tolerance to ischemia present in MLS-STAT3E mice would decrease cardiac injury during ISC-REP. In the isolated buffer-perfused heart model, MLS-STAT3E hearts exhibit a decreased infarct size compared to non-transgenic littermate hearts. Contractile recovery, expressed as a percent of LV developed pressure before ISC, is improved in MLS-STAT3E mice. Mitochondria isolated at the end of 60 min. of REP from MLS-STAT3E hearts show attenuated ROS release. The partial and persistent blockade of complex I present in MLS-STAT3E mice decreases cardiac injury during REP, in part via a persistent decrease in ROS production and attenuation of mitochondrial permeability transition pore opening at the onset of REP. In vivo, MLS-STAT3E hearts exhibit substantially higher postoperative survival rate and a substantial decrease in myocardial infarct size. STAT3 mediates cardioprotection not only via canonical action as a transcription factor, but also as a modulator of ETC activity directly in the mitochondria.
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Acknowledgments
This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to E.J.L.), the American Heart Association Postdoctoral Fellowship Award (to K.S.), the American Heart Association Scientist Development Grant (to Q.C.), American Heart Association Scientist Development Grant and Grant in Aid (to F.N.S.) and the Pauley Heart Center, Virginia Commonwealth University.
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395_2015_509_MOESM1_ESM.tif
Supplementary material 1 Supplemental Fig.S1 At later reperfusion, MLS-STAT3E does not alter PAR polymers formation. Equal amounts of cytosolic extracts from ISC-REP and time control hearts from WT and MLS-STAT3E mice were resolved by SDS-PAGE, transferred to PVDF membrane and probed for PAR (Merck Millipore, Billerica, MA). GAPDH was used as protein loading control. Densitometry was measured using ImageJ (NIH, Bethesda) and expressed as ratio of probed protein signal to GAPDH signal and normalized to time control set as 1. Results are mean ± SEM, n = 6. *P < 0.05 vs. corresponding time control. The dotted bars reflect the data obtained under ISC-REP conditions. (TIFF 465 kb)
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Supplementary material 2 Supplemental Fig.S2 STAT3 serine 727 phosphorylation status in the mitochondria from MLS-STAT3E and WT hearts subjected to 35 min ISC and 60 min REP. 30 μg of mitochondrial protein from WT hearts and 5 μg of mitochondrial protein from MLS-STAT3E hearts were separated by SDS-PAGE, transferred to PVDF membrane and probed for phosphorylated STAT3 serine 727 (p-Ser727-STAT3) and total STAT3. Porin was used as protein loading control. A representative immunoblot of three independent experiments is shown. (TIFF 1688 kb)
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Supplementary material 3 Supplemental Fig.S3 MLS-STAT3E attenuates MPTP during early REP. MLS-STAT3E and WT hearts were subjected to ex vivo Langendorff model of 35 min ISC and 10 or 60 min REP followed by mitochondria isolation and assessment of Calcium Retention Capacity (CRC). CRC was evaluated in mitochondria by sequential pulses of calcium (5 nmol) in a presence of calcein green indicator. Results are mean ± SEM, n = 4. *P < 0.05 vs. 10 min REP, #P < 0.05 vs. WT. (TIFF 483 kb)
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Supplementary material 4 Supplemental Fig.S4 MLS-STAT3E attenuates cytochrome c release into cytosol during early REP. Equal amounts of cytosolic extracts from WT and MLS-STAT3E hearts subjected to ex vivo Langendorff model of 35 min ISC and 10 REP were resolved by SDS-PAGE, transferred to PVDF membrane and probed for cytochrome c. GAPDH was used as protein loading control. Densitometry was measured using ImageJ (NIH, Bethesda) and expressed as ratio of probed protein signal to GAPDH signal. Results are mean ± SEM, n = 4. *P < 0.05 vs. WT. (TIFF 776 kb)
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Szczepanek, K., Xu, A., Hu, Y. et al. Cardioprotective function of mitochondrial-targeted and transcriptionally inactive STAT3 against ischemia and reperfusion injury. Basic Res Cardiol 110, 53 (2015). https://doi.org/10.1007/s00395-015-0509-2
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DOI: https://doi.org/10.1007/s00395-015-0509-2