DJ-1 protects the heart against ischemia-reperfusion injury by regulating mitochondrial fission

Abstract

Recent data indicates that DJ-1 plays a role in the cellular response to stress. Here, we aimed to examine the underlying molecular mechanisms mediating the actions of DJ-1 in the heart following myocardial ischemia-reperfusion (I/R) injury. In response to I/R injury, DJ-1 KO mice displayed increased areas of infarction and worsened left ventricular function when compared to WT mice, confirming a protective role for DJ-1 in the heart. In an effort to evaluate the potential mechanism(s) responsible for the increased injury in DJ-1 KO mice, we focused on SUMOylation, a post-translational modification process that regulates various aspects of protein function. DJ-1 KO hearts after I/R injury were found to display enhanced accumulation of SUMO-1 modified proteins and reduced SUMO-2/3 modified proteins. Further analysis, revealed that the protein expression of the de-SUMOylation enzyme SENP1 was reduced, whereas the expression of SENP5 was enhanced in DJ-1 KO hearts after I/R injury. Finally, DJ-1 KO hearts were found to display enhanced SUMO-1 modification of dynamin-related protein 1, excessive mitochondrial fission, and dysfunctional mitochondria. Our data demonstrates that the activation of DJ-1 in response to myocardial I/R injury protects the heart by regulating the SUMOylation status of Drp1 and attenuating excessive mitochondrial fission.

Keywords: DJ-1; Mitochondrial fission; Myocardial ischemia; SUMOylation.

Figure 1

Representative immunoblots of DJ-1 protein expression in (A) heart homogenates and (B) isolated cardiomyocytes from adult C57BL/6J mice and in heart homogenates from adult DJ-1 deficient (DJ-1 KO) mice. Immunohistochemical identification of DJ-1 (red), α-sarcomeric actin (green) and DAPI (blue) in isolated cardiomyocytes from adult C57BL/6J mice. Scale bar denotes 100 μm. (C) Representative immunoblots of the cardiac expression of the full length and cleaved form of DJ-1 following 45 minutes of myocardial ischemia and different periods of reperfusion. The cleaved form is shown in the long exposure image. Densitometric analysis of the (D) full-length form of DJ-1, (E) the cleaved form of DJ-1, and (F) cleaved DJ-1 protein as a percentage of the total level of DJ-1 proteins (full-length DJ-1 plus cleaved DJ-1). (G) Representative immunoblots of the expression of the full length and cleaved form of DJ-1 in mitochondrial fractions following 45 minutes of myocardial ischemia and 4 hours of reperfusion. Densitometric analysis of the (H) full-length form of DJ-1 and (I) cleaved DJ-1 protein as a percentage of the total level of DJ-1 proteins Numbers inside bars indicates sample size. Values are means ±SEM. N.D., not detected. *p<0.05, **p<0.01 and ***p<0.001 vs. Sham.

Figure 2

(A) Representative mid-ventricular photomicrographs of hearts from Wild-Type (WT) and DJ-1 KO mice showing the degree of infarction following 45 minutes of left coronary artery occlusion and 24 hours or reperfusion. (B) Myocardial area-at-risk (AAR) as a percentage (%) of total left ventricle (LV) and infarct size (INF) as a percentage of area-at-risk (AAR) and LV. (C) Circulating Troponin-I levels (ng/mL) in each group. (D) Left Ventricular (LV) Ejection Fraction, and (E) Fractional Shortening measured in separate groups of mice using echocardiography images and invasive hemodynamics 1 week following myocardial ischemia and reperfusion (1 wk Post). Values are means ±SEM. *p<0.05 and ***p<0.001 vs. WT or Baseline.

Figure 3

Representative immunoblots and densitometric analysis of (A–B) SUMO-1 modified and (C–D) SUMO-2/3 modified proteins in heart homogenates from WT and DJ-1 KO mice. Representative immunoblots and densitometric analysis of (E) free SUMO-1 and (F) free SUMO-2/3. All samples were prepared from WT and DJ-1 KO hearts collected following 45 minutes of ischemia and 4 hours of reperfusion. Values are means ±SEM. *p<0.05 and **p<0.01 vs. WT Sham.

Figure 4

(A) Representative immunoblots and densitometric analysis of (B) Ubc9, (C) PIAS2 and PIAS4 and in heart homogenates from WT and DJ-1 KO mice. (D) Representative immunoblots and densitometric analysis of (E) SENP1 and (F) SENP5 in heart homogenates from WT and DJ-1 KO mice. (G) Representative immunoblots and densitometric analysis of the interaction of DJ-1 with (H) SENP1 and (I) SENP5 in heart homogenates prepared from WT mice. All samples were collected following 45 minutes of ischemia and 4 hours of reperfusion. Values are means ±SEM. *p<0.05, **p<0.01, and ***p<0.001 vs. WT Sham.

Figure 5

(A) Representative immunoblots and (B–C) densitometric analysis of the SUMO status of Drp1. (D) Ratio of SUMO-2/3 to SUMO-1 modified Drp1. (EF) Representative immunoblots and densitometric analysis of Drp1 in mitochondrial fractions. All samples were prepared from WT and DJ-1 KO hearts collected following 45 minutes of ischemia and 4 hours of reperfusion. Values are means ±SEM. *p<0.05 and **p<0.01 vs. WT Sham.

Figure 6

(A) Representative electron microscopy images of cardiac mitochondria from different groups of WT and DJ-1 KO mice following 45 minutes of ischemia and 4 hours of reperfusion. Mice were administered vehicle (Veh) or the mitochondrial division inhibitor-1 (mdivi-1) 15 minutes prior to the onset of ischemia. Scale bar equals 2.5 μm. (B) Number of mitochondria per μm² in each field of view. Summary of mitochondria (C) perimeter and (D) area measurements (μm²). (E) Percentage of mitochondria in a given field that fell into three size categories based on area: <0.6 μm², 0.6 μm² – 1.0 μm², and >1.0 μm². (F–G) Representative images and quantification of dihydroethidium (DHE) staining of heart sections from WT and DJ-1 KO mice. Scale bar denotes 100 μm. (H) Caspase-3 activity. Values are means ±SEM. *p<0.05, **p<0.01 and ***p<0.001 vs. WT Sham.

Figure 7

(A) Representative immunoblots of the full length and cleaved form of DJ-1 in isolated rat neonatal cardiomyocytes subjected to hypoxia/reoxygenation. Densitometric analysis of the (B) full-length form of DJ-1, (C) the cleaved form of DJ-1, and (D) cleaved DJ-1 protein as a percentage of the total level of DJ-1 proteins. Cells were transfected with either siRNA to DJ-1 (siRNA-DJ1) or control siRNA (siRNA-scr). (E) Representative images of cells stained with antibodies to TOM20 to visualize mitochondrial networks following H/R (x100 magnification). Scale bar equals 5 μm. Cells were transfected with either siRNA-scr, siRNA-DJ1, siRNA to SENP5 (siRNA-SEPN5) or a combination of siRNA to DJ-1 and SENP5 (siRNA-DJ1/SENP5). (F) Quantification of mitochondrial interconnectivity (area/perimeter). (G) Intracellular reactive oxygen species levels (mean fluorescence intensity of DCFH oxidation) following H/R in cells treated with vehicle or MitoTempo (10 μM). (H) Cell viability following H/R as determined by the Cell Titer Blue assay in cells transfected with siRNA-scr, siRNA-DJ1, siRNA-SENP5, or siRNA-DJ1/SENP5. Values are means±SEM of three independent experiments of three biological replicates. ***p<0.001 vs. siRNA-scr Normoxia.