Edited by: Nicolau Beckmann, Novartis Institutes for BioMedical Research, Switzerland
Reviewed by: Gerd Heusch, University of Duisburg-Essen, Germany; Matteo Sorge, University of Turin, Italy; Qun Chen, Virginia Commonwealth University, United States
*Correspondence: Leonardo Maciel,
This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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
Ischemic preconditioning (IPC) is a cardioprotective maneuver constituted by brief episodes of ischemia/reperfusion (I/R) before sustained severe myocardial I/R (
Mitochondria are listed as end-effectors of many cardioprotective maneuvers (
Recently we reported the presence of small, 5–10 kDa, peptides (5–10-sP) among the humoral factors released on coronary effluent from hearts submitted to IPC, as having cardioprotective effects (
Thus, the aims of the present study were (
All chemicals (analytical grade) were obtained from Sigma-Aldrich (USA) if not otherwise specified. All solutions were freshly prepared and filtrated (1.2 μm, Millipore). Wistar rats (male. 300–350 g, CCS-Central Animal Facility) were used following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th edition, 2011) and the local Institutional Animal Care and Use Committee (100/16).
The I/R experiments were performed on isolated rat hearts as described previously (
Protocol 1—Isolated heart experiment. IPC hearts were subjected to 3 cycles of 5 min global ischemia/5 min reperfusion. Coronary effluent perfusion (CE) or its fractions were perfused in naíve hearts for 15 min prior to ischemia/reperfusion. HSP10 was administered for 10 min before I/R. 5-hydroxydecanoate (5HD) or chelerythrine (Chel) were administered for 20 min, starting 5 min prior to 5–10 kDa fraction perfusion. Protocol 2—Hypoxia/reoxygenation on isolated mitochondria. The buffer was made hypoxic. Mitochondrial proteins (300 μg) were added to the hypoxic buffer. After 8 min of hypoxia, O2-saturated incubation buffer was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation or the respective normoxic time control (10 min), pyruvate and malate were given as substrates for complex I, mitochondria were stimulated with ADP and respiration was measured over 2 min. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
All hearts were submitted to 20 min of the baseline period.
Isolated perfused hearts were continuously perfused with saline KHB solution for 70 min.
Isolated perfused hearts were subjected to 30 min of global zero-flow ischemia and 10 min reperfusion.
Isolated perfused hearts were subjected to 3 cycles of 5 min/5 min global zero-flow ischemia/reperfusion immediately prior to 30 min of ischemia and 10 min of reperfusion. Coronary effluent was collected during the reperfusion episodes of the IPC maneuver and subsequently used (within 2 h).
Isolated perfused hearts were perfused with coronary effluent from IPC hearts for 15 min before 30 min of global ischemia followed by 10 min of reperfusion.
The coronary effluent was filtered using an ultrafiltration membrane (Amicon model 8200, Millipore) with a cut-off for 10 kDa. The retained fraction was collected and resuspended in 150 ml of Krebs–Henseleit buffer solution. The ultrafiltrated coronary effluent was perfused on the same day of the collection. Isolated perfused hearts were perfused with the >10 kDa peptides fraction for 15 min before 30 min of global ischemia followed by 10 min of reperfusion. The fraction was perfused within 2 h, including ultrafiltration and pH stabilization.
The coronary effluent was filtered using ultrafiltration membranes (Amicon model 8200, Millipore) with cut-offs for 5 and 10 kDa. respectively. The IPC coronary effluent was initially ultrafiltrated using the 10 kDa cut-off membrane. The permeated fluid was collected and again ultrafiltrated using the 5 kDa cut-off membrane. The retained was resuspended in 150 ml of Krebs–Henseleit buffer solution. Isolated perfused hearts were perfused with the 5–10 kDa small peptides fraction (5–10-sP) for 15 min before 30 min of global ischemia followed by 10 min of reperfusion. The fraction was perfused within 2 h, including ultrafiltration and pH stabilization.
Hydroxydecanoate (5HD) was added to the perfusate at a final concentration of 10 μmol·L−1. Isolated perfused hearts were treated with 5HD for 5 min before 5–10-sP infusion and together with 5–10-sP infusion. The protocol was otherwise identical to that for
Chelerythrine (Chel) was added to the perfusate at a final concentration of 10 μmol·L−1. Isolated perfused hearts were treated with Chel for 5 min before 5–10-sP infusion and together with 5–10-sP infusion. The protocol was otherwise identical to that for 5–10-sP infusion.
Isolated perfused hearts were perfused with HSP10 1 μmol·L−1 (
Mitochondria isolation was performed as described previously (
Mitochondrial respiration was measured with a Clark-type electrode (Strathkelvin, Glasgow, UK) at 37°C during magnetic stirring in incubation buffer containing in mmol·L−1: 125 KCl, 10 MOPS, 2 MgCl2, 5 KH2PO4, 0.2 EGTA with pyruvate (5 mmol·L−1), and malate (5 mmol·L−1) as substrates for complex I. The oxygen electrode was calibrated using a solubility coefficient of 217 nmol O2/ml at 37°C. For the measurement of complex I respiration, the mitochondria (corresponding to a mitochondrial protein amount of 100 µg) were added to 1 ml incubation buffer. After 2 min of incubation, 1 mmol·L−1 ADP was added, and ADP-stimulated respiration was measured for 2 min. Afterward, the mitochondria were used to either measure complex IV respiration, and maximal uncoupled oxygen uptake in the respiration chamber or incubation buffer containing the mitochondria was taken from the respiration chamber to measure ATP production or extramitochondrial ROS concentration, respectively. Complex IV respiration was stimulated by adding N,N,N,N′-tetramethyl-p-phenylenediamine (TMPD, 300 μmol·L−1) plus ascorbate (3 μmol·L−1), which donates electrons to cytochrome oxidase
The buffer (without pyruvate and malate or succinate) was made hypoxic by the introduction of purified nitrogen until the oxygen concentration was <15 nmol O2 ml−1. Mitochondria (200 μg) were added to 0.5 ml hypoxic buffer. After 8 min of hypoxia, the O2-saturated incubation buffer (0.5 ml) was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation or the respective normoxic time control (10 min), were added pyruvate (5 mmol·L−1) and malate (5 mmol·L−1) as substrates for complex I or succinate (5 mmol·L−1) as substrates for complex II. Mitochondria were stimulated with ADP and respiration was measured for 2 min. For the measurement of complex II respiration, after hypoxia/reoxygenation, Rotenone (1 µmol·L−1) was added. After 2 min of incubation, 1 mmol·L−1 ADP was added. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production. Simulated Hypoxia/Reoxygenation protocols on isolated mitochondria were performed as follows and shown in
Mitochondria were added to 0.5 ml hypoxic buffer. After 8 min of hypoxia, the O2-saturated incubation buffer (0.5 ml) was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation, the substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondria were added to 0.5 ml oxygenated buffer. After 8 min, O2-saturated incubation buffer (0.5 ml) was added for 2 min (time control 10 min), and substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 1 μmol·L−1 HSP10. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I or Rotenone and substrates for complex II were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 Chelerythrine. After 1 min, was added 1 μmol·L−1 HSP10. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 5HD. After 1 min, was added 1 μmol·L−1 HSP10. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve re-oxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 5–10-sP. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 5–10-sP, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I or Rotenone and substrates for complex II were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 Chelerythrine. After 1 min 5–10-sP was added. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 5–10-sP, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 5HD. After 1 min, 1 μmol·L−1 5–10-sP was added. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 5–10-sP, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 1 μmol·L−1 5HD or 10 μmol·L−1 Chelerythrine. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.
After the measurement of ADP-stimulated respiration, the incubation buffer containing mitochondria was taken from the respiration chamber and immediately supplemented with the ATP assay mix (diluted 1:5). Mitochondrial ATP production after each respiration measurement was determined immediately and compared with ATP standards using a 96-well white plate and a spectrofluorometer (SpectraMax® M3, Molecular Devices, EUA) at 560 nm emission wavelength.
The mitochondrial swelling and transmembrane potential were evaluated using a spectrofluorometer (SpectraMax® M3, Molecular Devices, EUA). The integrity of the mitochondrial membrane was assessed by osmotically induced volume changes of the mitochondria and spectrophotometric determination of the apparent absorption of the suspension at 540 nm. A mitochondrial suspension (100 μg/ml) was added to the respiration medium in the absence of respiratory substrates, at 37°C and under constant stirring. The mitochondrial turgor was stimulated with 100 nmol·L−1 calcium. The swelling was expressed as a percentage of the absorption of the solution containing mitochondria in the presence of cyclosporin A (0% of mitochondrial turgor), in relation to that absorbed after the addition of FCCP (100% of mitochondrial turgor). For mitochondrial transmembrane potential (Δ
The Amplex Red Hydrogen Peroxide Assay Kit (Life Technologies, Carlsbad, CA, USA) was used to determine extramitochondrial ROS concentration. Amplex Red reacts at 1:1 stoichiometry with peroxides under catalysis by horseradish peroxidase (HRP) and produces highly fluorescent resorufin. The incubation buffer containing mitochondria was removed from the respiration chamber and immediately supplemented with 50 µmol·L−1Amplex UltraRed and 2 U/ml HRP. The supernatant was collected after 120 min incubation in the dark. Extramitochondrial ROS concentration was determined and compared with H2O2 standards using a 96-well black plate and a spectrofluorometer (SpectraMax® M3, Molecular Devices, EUA) at 540 nm emission and 580 nm extinction wavelengths.
Data are presented as the mean ± standard error of the mean (SEM). Data were analyzed by Prism 6.0 software (GraphPad, San Diego, California, USA) using two-way ANOVA for repeated measures (Left ventricular developed pressure and coronary flow) and one-way ANOVA (Mitochondrial respiration, ATP production, mitochondrial swelling, mitochondrial transmembrane potential, and extramitochondrial ROS concentration). When a significant difference was detected, one-way ANOVA was followed by Bonferroni
The baseline left ventricular developed pressure was not different among the groups. Control hearts did not show LVDP oscillations during the 40-min normal perfusion. During the preischemic preconditioning, the LVDP values were similar among the groups. The preconditioned groups IPC, coronary effluent, 5–10-sP, and HSP10 presented a greater post-ischemic recovery of LVDP than I/R, > 10 kDa, 5–10-sP + 5HD and 5–10-sP + Chel (
Left ventricular developed pressure of isolated perfused rat hearts.
Time | Groups | ||||||||
---|---|---|---|---|---|---|---|---|---|
Control (n = 5) | I/R (n = 5) | IPC (n = 5) | Coronary effluent (n = 5) | >10 kDa (n = 5) | 5–10-sP (n = 5) | 5–10-sP+5HD (n = 5) | 5–10-sP+Chel (n = 5) | HSP10 (n = 5) | |
Baseline 30 min | 105.1 ± 13.3 | 101.5 ± 12.7 | 104.7 ± 13.8 | 99.8 ± 7.9 | 105.3 ± 11. 3 | 103.9 ± 15.8 | 104.5 ± 11.4 | 114.4 ± 15.3 | 105.4 ± 13.4 |
Normal flow 40 min | 104.0 ± 13.5 | ||||||||
Normal flow 50 min | 108.3 ± 14.6 | ||||||||
Normal flow 70 min | 103.5 ± 12.4 | ||||||||
After preconditioning | 97.6 ± 12.9 | 107.6 ± 10.7 | 95. 3 ± 15.9 | 97.5 ± 9.7 | 87.4 ± 9.6 | 106.6 ± 7.8 | 98.3 ± 9.8 | ||
Ischemia 5 min | 0.8 ± 0.5 | 1.0 ± 0.3 | 1.2 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.6 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
Ischemia 30 min | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 1.1 ± 0.90 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
Reperfusion 10 min | 14.6 ± 6.7 | 51.9 ± 19.6* | 46.7 ± 7.6* | 19.6 ± 11.8$ | 49.8 ± 8.6* | 8.3 ± 7.2& | 11.3 ± 9.6& | 47.2 ± 10.3* |
Developed left ventricular pressure (LVDP) was calculated as the difference between the systolic and the end diastolic pressure. LVDP values (mmHg) were measured at different time points: at end of stabilization period (baseline), at 40, 50 and 70 min of normal flow in Control hearts, after preconditioning (IPC, perfusion of coronary effluent, 5–10-sP, HSP10, 5HD or chelerythrine), at 5 and 30 min of ischemia, and at 10 min of reperfusion. Mean ± SEM. *P < 0.05 vs I/R, $P < 0.05 vs >10 kDa, &P < 0.05 vs 5–10 kDa.
ADP-stimulated complex I respiration was reduced after I/R compared to Control (
Adenosine diphosphate (ADP)-stimulated complex I respiration of isolated mitochondria from rat hearts.
Complex IV respiration with TMPD and ascorbate and maximal uncoupled oxygen uptake with FCCP of isolated mitochondria from rat hearts.
The complex II respiration did not show a difference between the baseline and the Time control (
Succinate and adenosine diphosphate (ADP)-stimulated complex II respiration of isolated mitochondria from rat hearts.
The mitochondrial ATP production of the I/R group was diminished compared to the Control group (
Adenosine triphosphate (ATP) production of isolated mitochondria from rat hearts.
Mitochondrial ROS production after I/R was higher compared to Control (
Reactive oxygen species (ROS) production of isolated mitochondria from rat hearts.
The mitochondrial Δψm was increased after I/R compared to Control (
Mitochondrial swelling and mitochondrial transmembrane potential (Δψm) of isolated mitochondria from rat hearts.
Mitochondrial swelling showed the same pattern as mitochondrial ΔΨ. The mitochondrial swelling was prevented by IPC, coronary effluent and 5–10-sP when compared with I/R (
To the best of our knowledge, this is the first study to describe direct actions of HSP10 on mitochondrial function preservation, since incubation of isolated mitochondria with HSP10 prior to hypoxia/reoxygenation improved the mitochondrial function. Additionally, 5–10-sP from preconditioned coronary effluent and HSP10 maintained mitochondrial function in isolated hearts submitted to I/R and in isolated fresh mitochondria submitted to hypoxia/reoxygenation. These data support the hypothesis that mitochondria are target organelles for humoral factors released during IPC in myocardial tissue.
Ischemic preconditioning is the most effective conditioning maneuver for heart protection and prior studies suggested that IPC is associated with improved mitochondrial respiration and infarct size reduction (
The present study showed that exogenous HSP10 or 5–10-sP induces mitochondrial function improvement by direct (in isolated mitochondria) and indirect (in perfused isolated rat hearts) exposure. Although, it remains unclear how HSP10 directly protects the mitochondria against hypoxia/reoxygenation injury, our data suggested that this effect could be mediated by KATP channels activation. HSP10 has been reported to modulate intracellular cardioprotective signaling such as BCL-2 (
The increased ATP production is an expected consequence of increased ADP-stimulated respiration (
Our previous study showed that cardioprotection elicited by 5–10-sP was mediated by PKC (
The lack of data using the PKC inhibitor (chelerythrine) and the blocker for KATP (5HD) in an isolated heart model treated with HSP10 represents a limitation in clarifying the pathways involved in mitochondrial protection conferred by HSP10. In fact, these experiments were not performed due to a large amount of HSP10 required for the execution of each experiment in an isolated heart model, since the synthesis, purification, and quantification of this amount of protein would take several months. The authors recognize a statistical limitation due to the repetition of some groups in different graphs regarding the assessment of mitochondrial function in hearts undergoing I/R.
In conclusion, our findings provide evidence that the improvement of mitochondrial function by preconditioned coronary effluent transfer is mediated by 5–10-sP humoral factors. This study also supports the mitochondria as an intracellular target of protection by 5–10-sP, and this protection is dependent on the PKC pathway and mitochondrial KATP channel activation. Finally, this study shows the direct and indirect actions of HS P10 on mitochondrial function maintenance against I/R or hypoxia/reoxygenation injuries.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by the Comissão de Ética no Uso de Animais (CEUA) in scientific experimentation of the Centro de Ciências da Saúde of the Federal University of Rio de Janeiro (100/16) and followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th edition, 2011).
LM, AC, and JN: conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents. DO and GM: acquisition of data, analysis, and interpretation of data. JN is the principal investigator. All authors discussed the results and commented on the manuscript.
This work was supported by the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) (grant 483639/2013-3 and grant INCT regenera 465656/2014-05) and the Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) (grant E-26/112.085/2012).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). AC and JN are research fellows from CNPq.
AKT, Protein kinase B; FCCP, Carbonyl cyanide-p-trifluoromethoxyphenyl-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-p-phenylenediamine; Δψ: Mitochondrial transmembrane potential; IPC, ischemic preconditioning; I/R, ischemia/reperfusion; RIPC, remote ischemic preconditioning; 5–10 kDa small peptides (5–10-sP); HSP10, 10 kDa-mitochondrial heat shock protein; LVP, left ventricular pressure; CE, Coronary effluent; Chel, Chelerythrine; 5HD, 5-hydroxydecanoate; PKC