HNO Protects the Myocardium against Reperfusion Injury, Inhibiting the mPTP Opening via PKCε Activation

Abstract

Donors of nitroxyl (HNO), the one electron-reduction product of nitric oxide (NO^.), positively modulate cardiac contractility/relaxation while limiting ischemia-reperfusion (I/R) injury. The mechanisms underpinning HNO anti-ischemic effects remain poorly understood. Using isolated perfused rat hearts subjected to 30 min global ischemia/1 or 2 h reperfusion, here we tested whether, in analogy to NO^., HNO protection requires PKCε translocation to mitochondria and K_ATP channels activation. To this end, we compared the benefits afforded by ischemic preconditioning (IPC; 3 cycles of I/R) with those eventually granted by the NO^. donor, diethylamine/NO, DEA/NO, and two chemically unrelated HNO donors: Angeli's salt (AS, a prototypic donor) and isopropylamine/NO (IPA/NO, a new HNO releaser). All donors were given for 19 min before I/R injury. In control I/R hearts (1 h reperfusion), infarct size (IS) measured via tetrazolium salt staining was 66 ± 5.5% of the area at risk. Both AS and IPA/NO were as effective as IPC in reducing IS [30.7 ± 2.2 (AS), 31 ± 2.9 (IPA/NO), and 31 ± 0.8 (IPC), respectively)], whereas DEA/NO was significantly less so (36.2 ± 2.6%, p < 0.001 vs. AS, IPA/NO, or IPC). IPA/NO protection was still present after 120 min of reperfusion, and the co-infusion with the PKCε inhibitor (PKCV1-2500 nM) prevented it (IS = 30 ± 0.5 vs. 61 ± 1.8% with IPA/NO alone, p < 0.01). Irrespective of the donor, HNO anti-ischemic effects were insensitive to the K_ATP channel inhibitor, 5-OH decanoate (5HD, 100 μM), that, in contrast, abrogated DEA/NO protection. Finally, both HNO donors markedly enhanced the mitochondrial permeability transition pore (mPTP) ROS threshold over control levels (≅35-40%), an action again insensitive to 5HD. Our study shows that HNO donors inhibit mPTP opening, thus limiting myocyte loss at reperfusion, a beneficial effect that requires PKCε translocation to the mitochondria but not mitochondrial K⁺ channels activation.

Keywords: KATP channels; PKCε; mitochondrial permeability transition pore (mPTP); myocardial reperfusion injury; nitric oxide (NO.); nitroxyl (HNO).

Figure 1

Experimental protocols. All hearts were allowed to stabilize for 30′, after which they were assigned to one of the following experimental condition: Control—no treatment, 30′ global ischemia, 1 h reperfusion; IPC, 3–5′ cycles of preconditioning ischemia, 30′ global ischemia, 1 h reperfusion; Donors (IPA/NO, AS, DEA/NO)—treatment for 19′, 10’ washout, 30′ global ischemia, 1 h reperfusion; Donors + 5-HD, treatment for 19′ with donor and 5-HD, 10′ washout—30′ global ischemia, 1 h reperfusion; IPA/NO—treatment for 19′, 10′ washout, 30′ global ischemia, 2 h reperfusion; IPA/NO + PKCε inhibitor—treatment for 19′ with IPA/NO + inhibitor, 10′ washout, 30′ global ischemia, 2 h reperfusion.

Figure 2

(a) Infarct size expressed as percentage of the area at risk (left ventricle mass). Donors (IPA/NO, AS, and DEA/NO) are compared to the classical ischemic preconditioning (IPC). All donors are confirmed to grant a substantial protection against the I/R injury provoked by a 30′ ischemia and 1 h of reperfusion. Data are expressed as Average ± Standard Deviation. * p < 0.001, ns: non-significant. (b) Infarct size expressed as a percentage of the area at risk (left ventricle mass). IPC and donors (IPA/NO, AS, and DEA/NO) show a significant (p < 0.0001) reduction in infarct size compared to the control group, while the use of 5HD differentially affects AS and IPA/NO compared to DEA/NO^. No significant differences are observed among the sole donors. n = 6 for each experimental group. Data are expressed as Average ± Standard Deviation. * p < 0.001, ns: non-significant. (c) Myocardial damage expressed by LDH release during reperfusion. Perfusate samples were collected at 0′, 10′, 20′, 30′, 40′, 50′, and 1 h after restarting perfusion. LDH was quantified for each sample and summed. n = 6 for each experimental group. Data are expressed as Average ± Standard Deviation. * p < 0.001, ** p < 0.05, ns = p > 0.05.

Figure 3

(a) Infarct size expressed as a percentage of the area at risk (left ventricle mass). Neither the CTRL nor IPA/NO-treated heart showed a significant difference in terms of necrosis after 2 h of reperfusion when compared to 1 h. n = 6 for each experimental group. Data are expressed as Average ± Standard Deviation. * p < 0.001, ns: non-significant. (b) Infarct size expressed as a percentage of the area at risk (left ventricle mass). IPA/NO protection is abolished by co-infusion of a specific inhibitor of PKC translocation. N = 6 for each experimental group. Data are expressed as Average ± Standard Deviation. * p < 0.001, ns = p > 0.05.

Figure 4

(A) Quantification of membrane PKCε in myocardial tissue of ischemic hearts. Isolated hearts perfused with DEA/NO, IPA/NO, AS, or preconditioned with IPC. Membrane PKCε is higher in all treatments when compared to the CTRL group. All readings have been performed in triplicate maintaining the same ROI. (B) Quantification of cytosolic PKCε in myocardial tissue of ischemic hearts. Isolated hearts perfused with DEA/NO, IPA/NO, AS, or preconditioned with IPC. Cytosolic PKCε is lower in all treatments when compared to the CTRL group. All readings have been performed in triplicate maintaining the same ROI. (C) Ratio of membrane/cytosolic PKCε in myocardial tissue of ischemic hearts. Isolated hearts perfused with DEA/NO, IPA/NO, AS, or preconditioned with IPC. The ratio is significantly increased in all groups compared to the CTRL (p < 0.0001). All donors also show a significantly higher PKCε translocation when compared to IPC (* p < 0.001, ns = p > 0.05). (D) Representative images of Western blot membraned incubated with specific antibodies and revealed in chemiluminescence.

Figure 5

Visualization of PKCε translocation induced by the novel compound IPA/NO^. After treatment, hearts were processed to obtain thin slices to be probed with a PKCε antibody. Images were taken by confocal microscopy at 63×. In the left panel, a representative image on untreated, myocardial tissue, in the right panel a IPA/NO treated heart.

Figure 6

Effect of IPA/NO and Angeli’s Salt on mPTP ROS threshold in isolated cardiac myocytes. Mitochondria loaded with TMRM were laser line-scanned until mPTP induction. The average time required for the standardized photoproduction of ROS to cause mPTP induction (t_mPTP), normalized to that value under Control conditions, is taken as the index of the ROS threshold in that cell. Both, 1 and 10 µM IPA/NO exerted significant protection against the mPTP induction by ROS. Angeli’s Salt enhanced mPTP ROS threshold in a dose-dependent manner with the maximum protection at 10 µM. All experiments were performed at least in triplicate, with cell numbers greater than 12 in each independent experiment. The data are mean ± SEM.