Acute inhibition of excessive mitochondrial fission after myocardial infarction prevents long-term cardiac dysfunction

Abstract

Background: Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats.

Methods and results: We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28 ± 2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions.

Conclusions: Together, we show that excessive mitochondrial fission at reperfusion contributes to long-term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long-term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.

Keywords: Drp1; cardiac myocytes; heart; mitochondria; protein‐protein interaction inhibitor.

Figure 1.

Mitochondrial Drp1 translocation in cardiac myocytes under ischemia‐reoxygenation injury is blocked by P110. A, Primary culture cardiomyocytes were subjected to 2 hour of ischemia followed by 2 to 60 minutes of reoxygenation. Western blot analysis of Drp1 in total and mitochondrial fractions was determined by anti‐Drp1 antibodies. VDAC was used as a loading control. Quantitation of the levels of Drp1 is provided in a histogram. B, Cells were treated with peptide TAT_47‐57 and P110 (1 μmol/L) for 30 minutes prior 2 hours ischemia and during 30 minutes reoxygenation before analyzing Drp1 levels as above. P110 decreased Drp1 levels at the mitochondria. Enolase and VDAC were used as a loading controls and subcellular compartment controls. C, Cross‐linked proteins from total lysate cardiomyocytes after IRO treated with control or P110 peptide were immunoprecipitated using anti‐Fis1, anti‐Mff and anti‐MIEF1, respectively. Drp1 co‐immunoprecipitated was analyzed by Western blot analysis. Antibody control and 5% input is shown for respective proteins. The amounts of pulled‐down Drp1 are shown under the respective blots, normalized to the respective protein. Data are expressed as mean±SE of three independent experiments performed in duplicate for each group. (*P<0.05 vs normoxia, **P<0.05 vs IRO). Drp1 indicates dynamin related protein 1; Fis1, fission protein 1; IRO, ischemia and reoxygenation; VDAC, voltage‐dependent anion channel.

Figure 2.

Mitochondrial fragmentation and function in cardiac myocytes under IRO. A, Mitochondria of cardiac myocytes after 2 hours of ischemia followed by 2 hours of reoxygenation in the presence or absence of P110 are labeled with anti‐Tom20 antibody to determine the number of cells with mitochondria fragmentation. Bar scale=20 μm. Boxed area under each micrograph is enlarged to determine mitochondria fragmentation. B, Cytochrome c release was determined in the cytosolic fraction after IRO injury in the presence and absence of the peptide. Enolase and VDAC were used as cytosolic and mitochondrial loading control, respectively. C, P110 treatment decreased the number of TUNEL‐positive cells after IR. D and E, Cellular reactive oxygen species and mitochondria reactive oxygen species are measured in intact cells after IRO using a fluorescent plate reader. Data are expressed as mean±SE of three independent experiments performed in duplicate for each group. (*P<0.05 vs normoxia, **P<0.05 vs IRO). IR indicates ischemia and reperfusion; IRO, ischemia and reoxygenation; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 3.

Localization of Drp1 at the mitochondria and mitochondrial function on IR injury in an ex vivo Langendorff model. A, Isolated heart protocol of IR. B, Hearts subject to IR injury were treated with TAT or P110 at 1 μmol/L during equilibration period and for 20 minutes at reperfusion. P110 reduces mitochondrial fragmentation after IR injury. Mitochondrial morphology was analyzed by electron microscopy of the indicated groups. Mitochondrial size and arrangement is shown at ×4000 magnification of respective heart sections (bar=0.5 μm). C, Lower magnification (×800) of EM sections showed mitochondria arranged along the myofibrils after P110 treatment (bar=2 μm). D, Flow cytometry analysis (FACS) of isolated cardiac mitochondria size (forward scatter; FCS) in respective groups after IR. The mean of each group is shown in histogram, on the right (*P<0.05 vs normoxia, **P<0.05 vs IR). E, Translocation of Drp1 to the mitochondria upon IR is blocked by P110 peptide compared to normoxia and control (n=6). Quantitative data of the Western blot demonstrating Drp1 translocation is provided in histogram, on the right (*P<0.05 vs normoxia, **P<0.05 vs IR). Drp1 indicates dynamin related protein 1; EM, electron microscopy; IR, ischemia and reperfusion.

Figure 4.

Cardiac damage and mitochondrial functions in heart subjected to IR ex vivo. A, Infarct size was determined by TTC staining (insert). B, Measurement of mitochondrial H₂O₂ release was determined in mitochondrial fraction of hearts after IR injury (100 μg each) using Amplex Red oxidation as a fluorescent marker. C, Level of ATP was measured in total heart extract after IR injury. Cleaved caspase 3 was determined as a marker of apoptosis (D). Autophagy (E) and JNK phosphorylation (F) as markers of cell stress are apparent in ex vivo heart after IR injury, as measured by increase in LC3‐II (E) and p‐JNK (F) in total lysates of heart subjected to IR in an ex vivo model. The effect of treatment with P110 (1 μmol/L) before and after reperfusion decreased autophagy and JNK phosphorylation as compared with normoxia and IR control hearts. (*P<0.05 vs normoxia, **P<0.05 vs IR; n=6/group). IR indicates ischemia and reperfusion; JNK, Jun kinase; LC3‐II, microtubule‐associated protein 1 light chain 3; TTC, triphenyltetrazolium chloride.

Figure 5.

Cardiac function in myocardial infarction‐induced heart failure. A, Protocol of the treatment. B, Fractional shortening was measured in heart after LAD occlusion for 30 minutes followed by 3 days and 3 weeks of reperfusion in the presence of the respective peptides (0.5 mg/kg IP). Echocardiogram results are shown as individual rat heart results with respective P values between groups of control sham operated rats, and rats subjected to 30 minutes LAD occlusion and treated at the onset of reperfusion with cont (control peptide) or P110 using IP injection of 0.5 mg/kg. (n=7/control MI group, n=6/Normoxia and MI treated group). LAD indicates left anterior descending; MI, myocardial infarction.

Figure 6.

Mitochondrial bioenergetics in cardiac isolated mitochondria from post‐myocardial infarction dysfunction animals. Mitochondrial respiratory rates upon addition of ADP (state 3), oligomycin (state 4) and CCCP (uncoupling state) (A) and respiratory control rate (state 3/state 4) (B). Measurements were performed in cardiac isolated mitochondria after 3 weeks of in vivo rat acute myocardial infarction model. P110 treatment at the onset of reperfusion improved mitochondrial functions (*P<0.05 vs normoxia; **P<0.05 vs MI control peptide; n=6/normoxia group, n=7/MI control and treated group). CCCP indicates carbonyl cyanide m‐chlorophenyl hydrazone; MI; myocardial infarction.