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. 2013 Jan 11;288(2):915-26.
doi: 10.1074/jbc.M112.411363. Epub 2012 Nov 14.

Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction

Dieter A Kubli  1 , Xiaoxue Zhang, Youngil Lee, Rita A Hanna, Melissa N Quinsay, Christine K Nguyen, Rebecca Jimenez, Susanna Petrosyan, Anne N Murphy, Asa B Gustafsson
Affiliations

Parkin protein deficiency exacerbates cardiac injury and reduces survival following myocardial infarction

Dieter A Kubli et al. J Biol Chem. .

Abstract

It is known that loss-of-function mutations in the gene encoding Parkin lead to development of Parkinson disease. Recently, Parkin was found to play an important role in the removal of dysfunctional mitochondria via autophagy in neurons. Although Parkin is expressed in the heart, its functional role in this tissue is largely unexplored. In this study, we have investigated the role of Parkin in the myocardium under normal physiological conditions and in response to myocardial infarction. We found that Parkin-deficient (Parkin(-/-)) mice had normal cardiac function for up to 12 months of age as determined by echocardiographic analysis. Although ultrastructural analysis revealed that Parkin-deficient hearts had disorganized mitochondrial networks and significantly smaller mitochondria, mitochondrial function was unaffected. However, Parkin(-/-) mice were much more sensitive to myocardial infarction when compared with wild type mice. Parkin(-/-) mice had reduced survival and developed larger infarcts when compared with wild type mice after the infarction. Interestingly, Parkin protein levels and mitochondrial autophagy (mitophagy) were rapidly increased in the border zone of the infarct in wild type mice. In contrast, Parkin(-/-) myocytes had reduced mitophagy and accumulated swollen, dysfunctional mitochondria after the infarction. Overexpression of Parkin in isolated cardiac myocytes also protected against hypoxia-mediated cell death, whereas nonfunctional Parkinson disease-associated mutants ParkinR42P and ParkinG430D had no effect. Our results suggest that Parkin plays a critical role in adapting to stress in the myocardium by promoting removal of damaged mitochondria.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of Parkin−/− mice. A, Western blot for Parkin in heart and brain tissue (asterisk indicates nonspecific band detected by the Parkin antibody). B–E, WT and Parkin−/− mouse body weight (B), heart weight (C), heart weight/body weight (HW/BW) ratio (D), and lung weight/body weight (LW/BW) ratio (E). Mean ± S.E. (n = 7–11, *, p < 0.05, **, p < 0.01 versus WT).
FIGURE 2.
FIGURE 2.
Mitochondrial respiration is normal in Parkin−/− mouse hearts at 3 months of age. A and B, state 3 and state 4 respiration rates of mitochondria isolated from WT or Parkin−/− mouse hearts with substrates for complex I (pyruvate/malate) (A) or complex II (succinate/rotenone) (B). C, respiratory control (RCR) ratios for complex I and complex II substrates (n = 4). Pyr/Mal, pyruvate/malate; Succ/Rot, rotenone/succinate. D, maximal mitochondrial respiration rates in isolated intact WT and Parkin−/− adult mouse myocytes (n = 3). Mean ± S.E. No significant differences were observed. E, swelling of isolated mitochondria in the presence of 150 μm calcium (n = 3). F, the degree and rate of swelling (amplitude and Vmax, respectively) were not significantly different between WT and Parkin−/− mitochondria. Base-line absorbance values for Parkin−/− mitochondria were significantly higher than WT. Mean ± S.E. (n = 3, *, p < 0.05 versus WT).
FIGURE 3.
FIGURE 3.
Parkin-deficient hearts have disorganized and smaller mitochondria. A, representative transmission electron micrographs of heart sections from 3-month-old mice. Arrows signify smaller mitochondria. B, quantitation of mean mitochondrial area ± S.E. in WT and Parkin−/− hearts (*, p < 0.05 versus WT). C, Western blot of mitochondrial fusion proteins Mfn1, Mfn2, and Opa1. D, Western blot of mitochondrial fission proteins Drp1 and Fis1.
FIGURE 4.
FIGURE 4.
Parkin−/− mice have increased susceptibility to MI. A, Kaplan-Meyer survival curve (p < 0.05). B, representative Masson's trichrome staining of WT and Parkin−/− hearts 7 days after MI. C, representative images of left ventricular remodeling 7 days after MI. D, quantitation of left ventricular remodeling (n = 10, *, p < 0.05 versus WT). E, representative Western blot demonstrating rapid up-regulation of Parkin expression in the border zone of WT mice after MI (Post-MI). Quantitation of Parkin/GAPDH ratio is shown (*, **,***, p < 0.05 versus 0 h, n = 5). AU, arbitrary units. F, representative Western blot of Parkin expression in the remote zone of WT mice after MI (n = 5). Data are mean ± S.E.
FIGURE 5.
FIGURE 5.
Echocardiography of WT and Parkin−/− mice 7 days after MI. A, representative M-mode echocardiograms of WT and Parkin−/− hearts prior to MI and 7 days after MI (Post-MI). B–F, echocardiographic analysis revealed reduced fractional shortening (FS) (B) and ejection fraction (EF) (C), as well as enlarged left ventricular end diastolic dimension (LVEDD) (D), left ventricular end systolic dimension (LVESD) (E), and LV volume (F) in Parkin−/− hearts. Mean ± S.E. (WT, n = 13; Parkin−/−, n = 10, *, p < 0.05, **, p < 0.01 versus WT).
FIGURE 6.
FIGURE 6.
Mitophagy is impaired in Parkin-deficient hearts. A and B, induction of autophagy in the border zone (A) and remote zone (B) 4 h after MI. Western blots and quantitation analyses for LC3II/I levels in the border zone at base-line (Con) in the ventricle and in the border zone 4 h after MI are shown. Mean ± S.E. (*, p < 0.05 versus control, n = 8) AU, arbitrary units; n.s., not significant. C, Western blots for Parkin and LC3 in the mitochondrial fractions of the border zones of WT and Parkin−/− mice 4 h after MI. D, Western blots for ubiquitinated proteins in the cytosolic and mitochondrial fractions of the border zones of WT and Parkin−/− mice 4 h after MI. E, Western blots for Parkin and LC3 in the mitochondrial fractions of the remote zones of WT and Parkin−/− mice 4 h after MI. F, Western blots for ubiquitinated proteins in the cytosolic and mitochondrial fractions of the remote zones of WT and Parkin−/− mice 4 h after MI.
FIGURE 7.
FIGURE 7.
Accumulation of damaged mitochondria in Parkin-deficient hearts after MI. A, ultrastructural analysis by transmission electron microscopy of WT and Parkin−/− myocytes in the border zone of the infarct 4 h after MI. Black arrows indicate autophagosomes, and white arrows indicate disrupted contractile elements. B and C, oxygen consumption rates (OCR) of mitochondria in the remote zone (RZ) and border zone (BZ) 4 h after MI with pyruvate/malate (B) or succinate/rotenone (C) as substrates. Mean ± S.E. (n = 5 for WT, n = 6 for Parkin−/−).
FIGURE 8.
FIGURE 8.
Rotenone treatment fails to induce mitophagy in Parkin−/− myocytes. A, representative images of WT and Parkin−/− myocytes. Cells infected with LC3-GFP were treated with DMSO or 40 μm rotenone for 1 h. After fixation, mitochondria were stained with anti-COXIV. B, quantitation of autophagosomes co-localizing with mitochondria. Mean ± S.E. (n = 3; *, p < 0.05 versus WT control, **, p > 0.05 versus Parkin−/− control). C, quantitation of the mean number of LC3-GFP positive autophagosomes per cell in WT and Parkin−/− myocytes. Mean ± S.E. (n = 3; *, p < 0.05 versus WT control, **, p > 0.05 versus Parkin−/− control). D, adult myocytes from WT or Parkin−/− mice were infected with β-Gal or Parkin for 24 h prior to 4 h of hypoxia and quantitation of cell death. Mean ± S.E. (n = 3, *, p < 0.05 versus Normoxia, **, p < 0.05 versus hypoxia).
FIGURE 9.
FIGURE 9.
Overexpression of Parkin protects myocytes against hypoxia-mediated cell death. A, representative images of adult rat cardiac myocytes infected with mCherry-Parkin and stained for mitochondrial COXIV. Yellow lines represent line scans. B, quantitation of myocytes with mCherry-Parkin translocation. Mean ± S.E. (*, p < 0.01 versus normoxia, n = 4). C, representative line-scan analyses demonstrating co-localization of Parkin (red line) with mitochondrial COXIV (green line). AU, arbitrary units. D, adult rat cardiomyocytes were infected with adenoviruses encoding mCherry, Parkin, ParkinR42P (R42P), or ParkinG430D (G430D) for 24 h prior to 8 h of hypoxia and quantitation of cell death. Mean ± S.E. (n = 3–5, *, p < 0.05 versus normoxia + mCherry, **, p < 0.05 versus hypoxia + mCherry). E, Western blot analysis for endogenous Parkin in myocytes after exposure to 4 or 8 h of hypoxia.
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