DJ-1 protects against cell death following acute cardiac ischemia-reperfusion injury

Abstract

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia-reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia-reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1(L166P) and DJ-1(Cys106A) mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.

Figure 1

Assessment of the role of DJ-1 in vitro. Modulation of DJ-1 protein in vitro in HL-1 cells by transfection with WT and MitoDJ-1 and non-functional mutant DJ-1 (L166P and Cys106A). (a) Cell survival in response to simulated IRI, n=5/group. (b) MPTP opening in response to laser-induced oxidative stress, n≥5/group. (c) Representative images of TMRM-loaded mitochondria: (i) Control, (ii) WT-DJ (more elongated mitochondria), (iii) Cys106A and (iv) MitoDJ-1 (more elongated mitochondria). (d) Mitochondrial morphology, n≥5/group. Significance assessed by one-way ANOVA; significance indicated against comparison with control (white bars). *P<0.05, **P<0.01

Figure 2

Echocardiographic phenotyping of DJ-1 WT and KO animals. (a–d) Isoproterenol (4 ng/g) treatment significantly increased the heart rate and fractional shortening in both DJ-1 WT and KO mice (n≥5/group; significance assessed by one-way ANOVA comparing WT Basal with WT ISO and KO Basal with KO ISO, where **P<0.01 and ***P<0.001). There were no significant differences in any cardiac parameter between DJ-1 WT and KO mice at baseline or in response to isoproterenol challenge (n≥5/group, significance assessed by one-way ANOVA comparing WT and KO, ^NSP>0.05)

Figure 3

Susceptibility of DJ-1 KO to in vivo IRI. Infarct size following in vivo IRI in DJ-1 WT and KO mice. IS normalized to myocardial AAR to give IS/AAR%. (a) IS/AAR% in DJ-1 WT and KO mice subjected to standard IRI model of 45 min ischemia and 24 h reperfusion, n≥7/group. (b and c) IS/AAR% where standard IPC protocol of 5 min ischemia and 5 min reperfusion applied before main ischemic insult, n≥7/group. Significance assessed by unpaired t-test, *P<0.05, ***P<0.001. No statistical significance indicated as ^NSP>0.05

Figure 4

Calcium-induced mitochondrial swelling in isolated mitochondria. Mitochondria isolated from DJ-1 WT and KO hearts were subjected to calcium-induced mitochondrial swelling. Extent of mitochondrial swelling was assessed by optical density using spectrophotometer. (a) Baseline. (b) Calcium chloride (CaCl₂) 600 μM. (c) CaCl₂ 800 μM. (d) CsA pre-treatment at 1 μM for 15 min before swelling assay using 600 μM CaCl₂. No apparent differences in mitochondrial swelling between DJ-1 WT and KO mitochondria, n=6/group

Figure 5

Mitochondrial morphology in DJ-1 WT and KO hearts at baseline. Mitochondrial morphology in DJ-1 WT and KO hearts was assessed using EM of hearts isolated at baseline. (a) Representative EM images showing mitochondrial morphology in DJ-1 WT (i) and KO (ii) hearts at baseline; scale bar=2 μM. (b) Assessment of mitochondrial morphology by quantification of the proportion of mitochondria<1 sarcomere in length, equivalent to 1 sarcomere in length and >1 sarcomere in length, n≥3/group. Significance assessed by one-way ANOVA; significance indicated against comparison with DJ-1 WT control (white bars), *P<0.05, **P<0.01, ***P<0.001

Figure 6

Functional characterization of DJ-1 WT and KO hearts. (a) Myocardial ATP assay in DJ-1 WT and KO hearts. ATP assessed using luminescence assay and normalized to DJ-1 WT baseline, n≥5/group. No significant difference in ATP levels between DJ-1 WT and KOs; significance assessed by unpaired t-test, ^NSP>0.05. (b) Mitochondrial respiration assessed by oxygen flux readings for individual respiratory complexes in mitochondria isolated from DJ-1 WT and KO hearts, n≥5/group. No significant difference in mitochondrial respiratory function; significance assessed by one-way ANOVA, significance indicated against comparison with DJ-1 WT control (white bars), ^NSP>0.05. Characterization of ATP levels and mitochondrial respiration in DJ-1 WT hearts is shown in Supplementary Figure S4