Deficit in PINK1/PARKIN-mediated mitochondrial autophagy at late stages of dystrophic cardiomyopathy

Abstract

Aims: Duchenne muscular dystrophy (DMD) is an inherited devastating muscle disease with severe and often lethal cardiac complications. Emerging evidence suggests that the evolution of the pathology in DMD is accompanied by the accumulation of mitochondria with defective structure and function. Here, we investigate whether defects in the housekeeping autophagic pathway contribute to mitochondrial and metabolic dysfunctions in dystrophic cardiomyopathy.

Methods and results: We employed various biochemical and imaging techniques to assess mitochondrial structure and function as well as to evaluate autophagy, and specific mitochondrial autophagy (mitophagy), in hearts of mdx mice, an animal model of DMD. Our results indicate substantial structural damage of mitochondria and a significant decrease in ATP production in hearts of mdx animals, which developed cardiomyopathy. In these hearts, we also detected enhanced autophagy but paradoxically, mitophagy appeared to be suppressed. In addition, we found decreased levels of several proteins involved in the PINK1/PARKIN mitophagy pathway as well as an insignificant amount of PARKIN protein phosphorylation at the S65 residue upon induction of mitophagy.

Conclusions: Our results suggest faulty mitophagy in dystrophic hearts due to defects in the PINK1/PARKIN pathway.

Keywords: Autophagy; Cardiomyopathy; Dystrophin; Mitochondria; PARKIN; PINK1.

Figure 1

Abnormal mitochondrial morphology and impaired ATP synthesis in dystrophic cardiac myocytes. (A) Typical transmission electron micrographs of heart from 12 months and older WT (left panels) and mdx (right panels) mice. Arrows indicate mitochondria with a loss of cristae. (B) Percent of defective mitochondria in cardiac tissue isolated from 3 to 4 months and 12+ months old mdx (gray bars) and WT (white bars) mice. Samples from N = 4 (WT) or N = 3 (mdx) hearts were probed in each experimental cluster. About 10–23 random fields with 50–100 mitochondria were analysed in each experimental group. (C) Total ATP content in hearts from WT (white bars) and mdx (gray bars) mice. Heart samples from 3 to 4 months old (N = 9, mdx and WT) and 12+ (N = 3, both mdx and WT) months old mice were probed. (*P < 0.05, **P < 0.01, ANOVA test).

Figure 2

Autophagy markers indicate augmented autophagy in mdx hearts. Cardiac tissue was collected from 12 months and older mdx and WT mice under control conditions or after their treatment with chloroquine. Samples were immunoblotted with anti-LC3 (A), anti-SQSTM1 (B), anti-ULK1 (C), anti-phospho-ULK1(Ser555) (D), and anti-GAPDH (all panels) antibodies to determine protein levels of LC3-I, LC3-II, SQSTM1, ULK1, and phosphorylated ULK1. Samples from N = 4–8 senescent hearts were tested in each experimental group. Data obtained from mdx tissue are shown as gray bars whereas white bars illustrate data acquired from WT tissue. (*P < 0.05, **P < 0.01, ANOVA test).

Figure 3

LC3 puncta are more prominent in resting mdx cardiomyocytes. (A) Left panel shows typical overlay image of cardiomyocyte stained with anti-LC3 antibodies and then with Alexa Fluor 488-congugated secondary antibodies (green puncta on the image) and with nuclear fluorescent label DAPI (blue staining). Right panel shows binary image of the cardiomyocyte, where pixels with the numerical values above the preset threshold are marked in white. (B) Representative binary images of myocytes isolated from mdx and WT animals injected with either vehicle or rapamycin. (C) Pooled data of LC3 puncta area relative to area occupied by a cell in mdx and WT cardiomyocytes isolated from hearts of 12+ months old WT (N = 3, n = 14 and N = 3, n = 19) and mdx (N = 3, n = 15 and N = 3, n = 17) animals with and without rapamycin treatment, respectively. Data obtained from mdx tissue are shown as gray bars whereas white bars illustrate data acquired from WT tissue. (*P < 0.05, **P < 0.01, ANOVA test).

Figure 4

Mitophagy is compromised in dystrophic cardiomyocytes isolated from hearts of 12+ months old mice (evidence from immunocytochemistry). (A) Representative overlay images of WT and mdx cardiomyocytes incubated with anti-LC3 antibodies and then with Alexa Fluor 488-conjugated secondary antibodies (green puncta on the image) and MitoTracker^® Orange (red staining) without (top panels) and with (bottom panels) CCCP treatment. (B) Graph compares area occupied by LC3 puncta per cell area in myocytes with and without treatment with CCCP. Number of cells analysed was for WT (N = 3, n = 15 and N = 3, n = 24) and mdx hearts (N = 3, n = 15 and N = 3, n = 11) without and with incubation with CCCP, respectively. Data indicate enhanced autophagic activity in WT. Arrows on the images point to the ‘circle-like’ LC3 structures, which are likely to be associated with mitochondria. (C) Magnified images of cardiomyocytes containing the mitochondria associated LC3 circles. (D) Graph compares numbers of LC3 circles per cell area in WT (N = 3, n = 11 and N = 3, n = 21) and mdx (N = 3, n = 11 and N = 3, n = 20) myocytes with and without treatment with CCCP, respectively. Only LC3 puncta/circles associated with mitochondrial structures were considered for the analysis. This was ensured by taking additional images within ∼2 μm above and below the initial focal plane. Right panels in (C) show images of three representative mitochondria surrounded by LC3 puncta in three different projections (xy, xz, and yz). Data indicate that mitochondrial uncoupling induced substantially more mitochondria-associated LC3 circles in WT cardiomyocytes. Data obtained from mdx tissue are shown as gray bars whereas white bars illustrate data acquired from WT tissue. (*P < 0.05, **P < 0.01, ANOVA test).

Figure 5

Mitophagy is compromised in dystrophic cardiomyocytes isolated from hearts of 12+ months old mice (evidence from EM studies). (A) Images of TMRE loaded WT cardiomyocyte before (left panel) and after (right panel) DNP treatment. Application of 2 μM DNP resulted in depolarization of some mitochondria (seen as black spots). (B) Typical transmission electron micrographs of heart slices from 12+ months old WT (left panel) and mdx (right panel) mice in vivo treated with DNP. Enlarged images below are taken from WT tissue and show mitochondria included in autophagosomes (indicated by arrows). (C) Left panel shows percent of defective mitochondria in cardiac tissue isolated from mdx and WT mice treated with DNP. Right panel represents percent of mitochondria sequestered in autophagosomes. Samples from N = 5 hearts were analysed in each experimental cluster. About 26–32 random fields with 50–100 mitochondria were analysed in each experimental group. Data obtained from mdx tissue are shown as gray bars whereas white bars illustrate data acquired from WT tissue. (*P < 0.05, **P < 0.01, ANOVA test).

Figure 6

Mitophagy is compromised in dystrophic cardiomyocytes (evidence from biochemical studies). Mitochondrial fractions were isolated from hearts of 12+ months old mice under control conditions or after their treatment with DNP. Samples were immunoblotted with anti-LC3, anti-SQSTM1, anti-PINK1, anti-PARKIN, anti-Tom20, and anti-COXIV antibodies to determine mitochondria-associated levels of LC3-II, SQSTM1, PINK1, and PARKIN proteins. Samples from N = 4 hearts were tested in each experiments group. Data obtained from mdx tissue are shown as gray bars whereas white bars illustrate data acquired from WT tissue. (*P < 0.05, **P < 0.01, ANOVA test).

Figure 7

Defects in PINK1/PARKIN-mediated mitophagy in dystrophic hearts. (A, B) Expression of PARKIN. Cardiac tissue was collected from mice treated with either vehicle (control) or DNP. qPCR was used to determine level of gene encoding PARKIN (left panel on A). Samples were also immunoblotted with anti-PARKIN (A), anti-PARKIN-S65P (B), and anti-GAPDH antibodies to determine levels of PARKIN protein and PARKIN protein phosphorylated at S65 residue (summarized at the graphs on the right panels). (C) Expression of PINK1. Cardiac tissue was collected from mice under control conditions. Graph on the left represents levels of expression of gene encoding PINK1. Samples were also immunoblotted with anti-PINK1 and anti-VDAC antibodies to determine protein levels of PINK1 (middle and right panels). (D) Expression of Mfn2 protein, which is involved in mitochondrial mitophagy and mitochondrial dynamics. Overall, samples from N = 3–8 12+ months old mdx and WT hearts were tested. Experiments were carried out on whole tissue homogenates. There were three replicates for each sample during qPCR measurements. Data obtained from mdx tissue are shown as gray bars whereas white bars illustrate data acquired from WT tissue. (*P < 0.05, **P < 0.01, ANOVA test).