Autophagy protects cardiomyocytes from the myocardial ischaemia-reperfusion injury through the clearance of CLP36 - PubMed (original) (raw)

doi: 10.1098/rsob.160177.

Chao Liu 2, Lei Gu 3, Lina Wang 2, Yongliang Shang 2, Qiong Liu 4, Junyi Wan 4, Jian Shi 2, Fang Wang 2, Zhiliang Xu 2, Guangju Ji 5, Wei Li 6

Affiliations

Autophagy protects cardiomyocytes from the myocardial ischaemia-reperfusion injury through the clearance of CLP36

Shiguo Li et al. Open Biol. 2016 Aug.

Abstract

Cardiovascular disease (CVD) is the leading cause of the death worldwide. An increasing number of studies have found that autophagy is involved in the progression or prevention of CVD. However, the precise mechanism of autophagy in CVD, especially the myocardial ischaemia-reperfusion injury (MI/R injury), is unclear and controversial. Here, we show that the cardiomyocyte-specific disruption of autophagy by conditional knockout of Atg7 leads to severe contractile dysfunction, myofibrillar disarray and vacuolar cardiomyocytes. A negative cytoskeleton organization regulator, CLP36, was found to be accumulated in Atg7-deficient cardiomyocytes. The cardiomyocyte-specific knockout of Atg7 aggravates the MI/R injury with cardiac hypertrophy, contractile dysfunction, myofibrillar disarray and severe cardiac fibrosis, most probably due to CLP36 accumulation in cardiomyocytes. Altogether, this work reveals autophagy may protect cardiomyocytes from the MI/R injury through the clearance of CLP36, and these findings define a novel relationship between autophagy and the regulation of stress fibre in heart.

Keywords: Atg7; CLP36; autophagy; myocardial ischaemia-reperfusion injury; stress fibre.

© 2016 The Authors.

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Figures

Figure 1.

Figure 1.

Cardiomyocyte-specific knockout of Atg7 in mice. (a) The ATG7 protein level was dramatically reduced and the autophagic flux was impaired in cardiomyocyte-specific _Atg7_-deficient mouse hearts. Immunoblotting analysis of ATG7, SQSTM1 and LC3 were performed in vehicle or tamoxifen-treated Atg7flox/flox and Atg7flox/flox;Cre mouse hearts. GAPDH served as a loading control. (b) LC3 punctate structures disappeared in tamoxifen-treated Atg7flox/flox;Cre mouse hearts. Immunofluorescence analysis using LC3 (green) was performed in vehicle or tamoxifen-treated Atg7flox/flox and Atg7flox/ flox;Cre mouse hearts. Nuclei were stained with DAPI (blue). (c) SQSTM1 was accumulated and could not be sorted into the lysosome in cardiomyocyte-specific _Atg7_-deficient mouse hearts. Immunofluorescence analysis using SQSTM1 (red) and LAMP2 (green) were performed in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. Nuclei were stained with DAPI (blue).

Figure 2.

Figure 2.

The knockout of Atg7 in cardiomyocytes causes severe contractile dysfunction. (a) Representative trans-thoracic M-mode echocardiograms recorded from the parasternal short axis on the level of the papillary muscles of the left ventricle (LV) in each group. (b–f) The parameters of echocardiographic measurements in groups shown in (a). (b) LVDd, diastolic left ventricle internal dimension; (c) LVDs, systolic left ventricle internal dimension; (d) FS, fractional shortening of left ventricle dimension; (e) LVPWd, diastolic left ventricle posterior wall thickness; (f) LVEF, left ventricular ejection fraction. Values represent the mean ± s.e.m. of data from three to six mice in each group. *p < 0.05 versus all other groups. (g) The histology of the heart from vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mice using hematoxylin and eosin (H&E) staining. Arrows indicate disordered myofibre, triangles indicate vacuoles in the cross-section of cardiomyocytes. (h) The TEM analysis of the heart from vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mice. Asterisks indicate vacuoles in the cross-section of cardiomyocytes, triangles indicate disorganized myofibre, M indicates mitochondria.

Figure 3.

Figure 3.

CLP36 is accumulated in _Atg7_-deficient cardiomyocytes. (a) The protein level of CLP36 was accumulated in _Atg7_-deficient cardiomyocytes. Immunoblotting analysis of CLP36, SQSTM1 and LC3 were performed in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. GAPDH served as a loading control. (b) Relative mRNA level of Clp36 in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. (c) CLP36 was accumulated and localized on the Z-disc of the sarcomere in _Atg7_-deficient cardiomyocytes. Immunofluorescence analysis using phalloidin (green, labelled by FITC) and CLP36 (red) were performed in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. Nuclei were stained with DAPI (blue). (d–f) The protein level of α-actinin and palladin was accumulated in _Atg7_-deficient cardiomyocytes. (d) Immunoblotting analysis of α-actinin and palladin were performed in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. GAPDH served as a loading control. (e) Immunofluorescence analysis using phalloidin (green, labelled by FITC) and α-actinin (red) were performed in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. (f) Immunofluorescence analysis using phalloidin (green, labelled by FITC) and palladin (red) was performed in vehicle or tamoxifen-treated Atg7flox/flox, Atg7flox/flox;Cre mouse hearts. Nuclei were stained with DAPI (blue).

Figure 4.

Figure 4.

The cardiomyocyte-specific knockout of Atg7 could also impair the autophagic flux and cause CLP36 accumulation after myocardial ischaemia-reperfusion treatment. (a) The CLP36 was accumulated and the autophagic flux was disrupted in ischaemia-reperfusion-treated _Atg7_-deficient mice. After myocardial ischaemia-reperfusion treatment, immunoblotting analysis of CLP36, SQSTM1 and LC3 was performed in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts. p97 served as a loading control. (b) LC3 punctate structures disappeared in ischaemia-reperfusion-treated _Atg7_-deficient cardiomyocytes. Immunofluorescence analysis using LC3 (green) was performed in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts. Nuclei were stained with DAPI (blue). (c) Relative mRNA level of Clp36 in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts. (d) Immunofluorescence analysis using phalloidin (green, labelled by FITC) and CLP36 (red) were performed in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts after ischaemia-reperfusion treatment. Nuclei were stained with DAPI (blue). (e–g) The protein level of α-actinin and palladin was accumulated in _Atg7_-deficient cardiomyocytes after ischaemia-reperfusion treatment. (e) Immunoblotting analysis of α-actinin and palladin were performed in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts after ischaemia-reperfusion treatment. GAPDH served as a loading control. (f) Immunofluorescence analysis using phalloidin (green, labelled by FITC) and α-actinin (red) were performed in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts after ischaemia-reperfusion treatment. (g) Immunofluorescence analysis using phalloidin (green, labelled by FITC) and palladin (red) was performed in vehicle or tamoxifen-treated Atg7flox/flox;Cre mouse hearts after ischaemia-reperfusion treatment. Nuclei were stained with DAPI (blue).

Figure 5.

Figure 5.

The cardiomyocyte-specific disruption of Atg7 aggravates the myocardial ischaemia-reperfusion injury with cardiac hypertrophy and contractile dysfunction. (a) Representative trans-thoracic M-mode echocardiograms recorded from the parasternal short axis on the level of the papillary muscles of the left ventricle (LV) in each group before or after ischaemia-reperfusion treatment. (b–f) The parameters of echocardiographic measurements in (a). (b) LVDd, diastolic left ventricle internal dimension; (c) LVDs, systolic left ventricle internal dimension; (d) FS, fractional shortening of left ventricle dimension; (e) LVPWd, diastolic left ventricle posterior wall thickness; (f) LVEF, left ventricular ejection fraction. *p < 0.05 versus all other groups.

Figure 6.

Figure 6.

The cardiomyocyte-specific disruption of Atg7 aggravates the myocardial ischaemia-reperfusion injury with myofibrillar disarray and severe cardiac fibrosis. (a) The histology of the heart from vehicle or tamoxifen-treated Atg7flox/flox;Cre mice after ischaemia-reperfusion treatment using hematoxylin and eosin (H&E) staining. Arrows indicate interstitial fibrosis. (b) Cardiac fibrosis detected by using Sirius-red staining in vehicle or tamoxifen-treated Atg7flox/flox;Cre mice after ischaemia-reperfusion treatment. (c) The TEM analysis of the heart from vehicle or tamoxifen-treated Atg7flox/flox;Cre mice after ischaemia-reperfusion treatment. Asterisks indicate vacuoles in cardiomyocytes, arrows indicate Z-disc, M indicates mitochondria, Nu indicates nucleus.

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