Impaired Autophagy Contributes to Adverse Cardiac Remodeling in Acute Myocardial Infarction

<div><p>Objective</p><p>Autophagy is activated in ischemic heart diseases, but its dynamics and functional roles remain unclear and controversial. In this study, we investigated the dynamics and role of autophagy and the mechanism(s), if any, during postinfarction cardiac remodeling.</p><p>Methods and results</p><p>Acute myocardial infarction (AMI) was induced by ligating left anterior descending (LAD) coronary artery. Autophagy was found to be induced sharply 12–24 hours after surgery by testing LC3 modification and Electron microscopy. P62 degradation in the infarct border zone was increased from day 0.5 to day 3, and however, decreased from day 5 until day 21 after LAD ligation. These results indicated that autophagy was induced in the acute phase of AMI, and however, impaired in the latter phase of AMI. To investigate the significance of the impaired autophagy in the latter phase of AMI, we treated the mice with Rapamycin (an autophagy enhancer, 2.0 mg/kg/day) or 3-methyladenine (3MA, an autophagy inhibitor, 15 mg/kg/day) one day after LAD ligation until the end of experiment. The results showed that Rapamycin attenuated, while 3MA exacerbated, postinfarction cardiac remodeling and dysfunction respectively. In addition, Rapamycin protected the H9C2 cells against oxygen glucose deprivation <i>in vitro</i>. Specifically, we found that Rapamycin attenuated NFκB activation after LAD ligation. And the inflammatory response in the acute stage of AMI was significantly restrained with Rapamycin treatment. <i>In vitro</i>, inhibition of NFκB restored autophagy in a negative reflex.</p><p>Conclusion</p><p>Sustained myocardial ischemia impairs cardiomyocyte autophagy, which is an essential mechanism that protects against adverse cardiac remodeling. Augmenting autophagy could be a therapeutic strategy for acute myocardial infarction.</p></div

The effect of autophagy in the cardiac function and remodeling after AMI.

<p><b>A</b>, Rapamycin induced LC3 modification in the heart tissue (n = 4, *<i>P</i><0.05 <i>vs</i> CTL). <b>B</b>, the representative images and analysis results of echocardiographic assessment of hearts subjected to LAD ligation. (n = 8, *<i>P</i><0.05 <i>vs</i> Sham; #<i>P</i><0.05 <i>vs</i> CTL). <b>C</b>, the representative images and analysis results of TTC staining assessment of the hearts subjected AMI (n = 5, *<i>P</i><0.05 <i>vs</i> Sham; #<i>P</i><0.05 <i>vs</i> CTL). <b>D</b>, the representative images of Masson’s trichrome staining assessment of the infarct border zone at different time points after LAD ligation. Sham, mice without LAD ligation; CTL, LAD ligation with saline; Rapa, LAD ligation with Rapamycin treatment; 3MA, LAD ligation with 3MA treatment.</p

Rapamycin attenuated the inflammatory cell infiltration after LAD ligation.

<p>The representative images and statistical result of immunohistochemical assay of the infarct border zone of different time points after LAD ligation to test the infiltration of CD45⁺ leukocytes, including CD68⁺ macrophages (n = 5, *<i>P</i><0.05 <i>vs</i> Sham; #<i>P</i><0.05 <i>vs</i> CTL). Sham, mice without LAD ligation; CTL, LAD ligation with saline; Rapa, LAD ligation with Rapamycin treatment; 3MA, LAD ligation with 3MA treatment.</p

The effect of autophagy against oxygen glucose deprivation in the H9C2 cells <i>in vitro</i>.

<p><b>A</b>, Rapamycin induced LC3 modification in the H9C2 cells after OGD tested by Western blotting (n = 4). MTT assay (<b>B</b>) and trypan blue staining (<b>C</b>) to test the effect of different concentrations of Rapamycin on the cell survival after OGD for 24 hours (n = 8, *<i>P</i><0.05 <i>vs</i> OGD without Rapamycin). <b>D</b>, MTT assay to test the effect of Rapamycin and 3MA on the cell survival after OGD for different time (n = 8, *<i>P</i><0.05 <i>vs</i> OGD). <b>E</b>, The protein expression of ATG5 and LC3 after transfection of si<i>ATG5</i> in the H9C2 cells tested by Western blotting (n = 5, *<i>P</i><0.05 <i>vs</i> NC). <b>F</b>, The effect of si<i>ATG5</i> on the cell survival after OGD for different time in the H9C2 cells tested by MTT assay (n = 8, *<i>P</i><0.05 <i>vs</i> NC). <b>G</b>, The representative images and the analysis result of TUNEL staining in the H9C2 cells to test the effect of Rapamycin on cell apoptosis after OGD (n = 6, *<i>P</i><0.05 <i>vs</i> CTL, #<i>P</i><0.05 <i>vs</i> OGD). CTL, normal control; OGD, oxygen glucose deprivation; C+Rapa, normal control with Rapamycin treatment; O+Rapa, OGD with Rapamycin treatment. C+3MA, normal control with 3MA treatment; O+3MA, OGD with 3MA treatment. NC, negative control.</p

Autophagy is induced sharply and however impaired after myocardial ischemia stimuli <i>in vivo</i> and <i>in vitro</i>.

<p><b>A</b>, autophagy-associated protein marker LC3 and Beclin 1 were examined by Western blotting in the infarct border zone of different time points after LAD ligation (n = 5, *<i>P</i><0.05 <i>vs</i> Sham). <b>B</b>, The representative images of the electron microscopic analysis of the infarct border zone of different time points after LAD ligation. Rapamycin (2 mg/kg/day) was administered intraperitoneally for 21 days as a positive control. The arrows indicated autophagosomes. <b>C</b>, The protein expression of LC3 and Beclin 1 was examined in the H9C2 cells at different time points of OGD. (n = 8, *<i>P</i><0.05 <i>vs</i> CTL). <b>D</b>, GFP-LC3 modification in the H9C2 cells at different time points of OGD (n = 5, *<i>P</i><0.05 <i>vs</i> CTL). The H9C2 cells were treated with Rapamycin (100 nM) for 2 hours as a positive control. AMI, acute myocardial infarction; OGD, oxygen glucose deprivation; CTL, normal control.</p

Rapamycin inhibited NFκB activation after myocardial ischemia and inhibition of NFκB activated autophagy in return.

<p><b>A</b>, Rapamycin inhibited NFκB phosphorylation in the border zone of ischemic hearts tested by Western blotting (n = 5, *<i>P</i><0.05 <i>vs</i> Sham; #<i>P</i><0.05 <i>vs</i> CTL). <b>B</b>, The effect of Rapamycin on nuclear p65 and cytoplasm IκBα of the H9C2 cells after OGD tested by Western blotting (n = 5, *<i>P</i><0.05 <i>vs</i> CTL; #<i>P</i><0.05 <i>vs</i> OGD). <b>C</b>, The effect of Rapamycin on p65 translocation to the nuclei in the H9C2 cells after OGD tested by immunostaining assay. <b>D</b>, The effect of Rapamycin on the transcriptional activity of NFκB in the H9C2 cells after OGD (n = 6, *<i>P</i><0.05 <i>vs</i> CTL; #<i>P</i><0.05 <i>vs</i> OGD). <b>E and F</b>, inhibition of NFκB upregulated LC3 protein modification in the H9C2 cells 4 hours after oxygen glucose deprivation for by Western blot (E) and Immunostaining assay (F) (*<i>P</i><0.05 <i>vs</i> OGD, n = 6) OGD, oxygen glucose deprivation; CTL, normal control with serum and oxygen; C+Rapa, normal control with Rapamycin treatment; O+Rapa, OGD with Rapamycin treatment.</p