Abstract
Humoral factors released during ischemic preconditioning (IPC) protect the myocardium against ischemia/reperfusion (I/R) injury. We have recently identified 10 kDa-heat shock protein (HSP10) and a fraction of small 5–10 kDa peptides (5–10-sP) in the coronary effluent of IPC-treated hearts and demonstrated their cardioprotective potential. We here used our isolated mitochondria model to characterize the impact of exogenous HSP10 and 5–10-sP on mitochondria function from myocardium subjected to I/R injury. Isolated perfused rat hearts were submitted to 30-min global ischemia and 10-min reperfusion. Before ischemia, isolated hearts were infused with saline or 5–10-sP, with or without a mitochondrial ATP-sensitive-K+-channel blocker (5HD 10 μmol·L−1) or PKC inhibitor (chelerythrine 10 μmol·L−1), before I/R. HSP10 (1 µmol·L−1) was infused into isolated hearts before I/R without blockers. At 10-min reperfusion, the mitochondria were isolated and mitochondrial function was assessed. In a subset of experiments, freshly isolated mitochondria were directly incubated with HSP10 or 5–10-sP with or without 5HD or chelerythrine before in vitro hypoxia/reoxygenation. Infusion of 5–10-sP (n = 5) and HSP10 (n = 5) into isolated hearts before I/R improved mitochondrial ADP-stimulated respiration, ATP production and prevented mitochondrial ROS formation compared to the I/R group (n = 5); this effect was abrogated by 5HD and chelerythrine. In freshly isolated mitochondria with in vitro hypoxia/reoxygenation, HSP10 (n = 16) and 5–10-sP (n = 16) incubation prevented reductions of mitochondrial ADP-stimulated respiration (91.5 ± 5.1 nmol O2/min/mg PTN), ATP production (250.1 ± 9.3 μmol ATP/200μg PTN), and prevented mitochondrial ROS production (219.7 ± 9.0 nmol H2O2/200μg PTN) induced by hypoxia/reoxygenation (n = 12, 51.5 ± 5.0 nmol O2/min/mg PTN; 187 ± 21.7 μmol ATP/200 μg PTN; 339.0 ± 14.3 nmol H2O2/200 μg PTN, p < 0.001, respectively). 5HD reduced the ADP-stimulated respiration in the HSP10 group (65.84 ± 3.3 nmol O2/min/mg PTN), ATP production (193.7 ± 12.1 μmol ATP/200μg PTN) and increased ROS in the 5–10-sP group (274.4 ± 21.7 nmol H2O2/200 μg PTN). Mitochondria are a target of the cardioprotection induced by 5–10-sP and HSP10. This protection is dependent of PKC and mKATP activation. HSP10 can act directly on mitochondria and protects against hypoxia/reoxygenation injury by mKATP activation.
Highlights
Ischemic preconditioning (IPC) is a cardioprotective maneuver constituted by brief episodes of ischemia/reperfusion (I/R) before sustained severe myocardial I/R (Murry et al, 1986; Hausenloy et al, 2016)
We showed the activation of PKC and mitochondrial ATPdependent potassium channel (KATP) by 5–10 kDa small peptides fraction (5–10-sP) (Maciel et al, Abbreviations: AKT, Protein kinase B; FCCP, Carbonyl cyanide-ptrifluoromethoxyphenyl-hydrazone; KHB, Krebs–Henseleit buffer; LVDP, Left Ventricular Developed Pressure; LVEDP, Left Ventricular End-Diastolic Pressure; MPTP, Mitochondrial permeability transition pore; PKC, Protein kinase C; ROS, Reactive Oxygen Species; TMPD, N,N,N,N-tetramethyl-pphenylenediamine; Dy: Mitochondrial transmembrane potential; IPC, ischemic preconditioning; I/R, ischemia/reperfusion; RIPC, remote ischemic preconditioning; 5–10 kDa small peptides (5–10-sP); HSP10, 10 kDamitochondrial heat shock protein; LVP, left ventricular pressure; CE, Coronary effluent; Chel, Chelerythrine; 5HD, 5-hydroxydecanoate; PKCε, Epsilon-isoform PKC; PTN, Protein
Chelerythrine did not abolish the effects of HSP10 or 5–10-sP despite having a nonsignificant trend to reduce the maintenance of the respiration generated by HSP10 or 5–10-sP. 5HD reduced the respiration maintenance induced by HSP10 but showed a nonsignificant trend to reduce in the 5–10-sP treatment (Figures 2E, F)
Summary
Ischemic preconditioning (IPC) is a cardioprotective maneuver constituted by brief episodes of ischemia/reperfusion (I/R) before sustained severe myocardial I/R (Murry et al, 1986; Hausenloy et al, 2016). IPC can be induced directly in the heart and at distance in remote tissues and organs (remote ischemic preconditioning, RIPC) (Heusch et al, 2015a; Kleinbongard et al, 2017). Changes in mitochondrial function by IPC or RIPC have been shown in several studies: mitochondrial respiration was improved in rat hearts (Heusch, 2015b; Kleinbongard et al, 2018) and in the right atrium of patients undergoing cardiac surgery (Heusch et al, 2015b). Infarct size reduction, induced by RIPC, was associated with improved mitochondrial function at early reperfusion, supporting the concept that the mitochondria are an intracellular target of protection by RIPC (Heusch et al, 2010). Despite the mitochondria being a target of the cardioprotective pathways activated by RIPC, signaling pathways for mitochondrial protection remain unclear (Gedik et al, 2017)
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