Modulating Autophagy To Influence The Cardiac Function In A Rat Model Of Dilated Cardiomyopathy

Part I To set up and evaluate the model of dilated cardiomyopathyObjective:To set up a rat model of dilated cardiomyopathy.Methods:We immunized Lewis rat with porcine cardiac myosin to set up a rat model of auto-immune myocarditis which can simulate dilated cardiomyopathy in its chronic phase. Ten weeks after immunization, Echo-cardiogram was adopted to evaluate cardiac function. HE stain and Masson’s trichrome stain was adopted to evaluate pathological change. Cell structure and autophagosome were observed by transmission electron microscopy. Cardiomyoctye apoptosis was observed by TUNEL.Results:Compared to the control group(EF=81.3±3.8%), the average eject fraction was decreased in dilated cardiomyopathy group (EF=56.1±3.3%). Tissue fibrosis could be found in the dilated cardiomyopathy group. Under transmission electron microscopy, sarcomeric disarray could be observed and swollen mitochondria were accumulated in dilated cardiomyopathy group. Vacuolized mitochondria and double membrane autophagosome could also be found. Apoptosis could be found in dilated cardiomyopathy group, but not in the control group.Conclusions:We successfully set up the model of dilated cardiomyopathy. Autophagysome can easily be found in dilated cardiomyopathy. Apoptosis could also be found in dilated cardiomyopathy. Part II Activating autophagy in the model of dilated cardiomyopathyObjectives:It is found that autophagy is activated in human dilated cardiomyopathy. It is still unknown whether up-regulating autophagy can improve the cardiac function of the failing heart. Methods:We immunized rats with porcine cardiac myosin to set up a model of dilated cardiomyopathy. Rapamycin, a kind of mTOR inhibitor upregulating autophagy, was given to rats intraperitoneally eight weeks after the immunization by low dose (1mg/kg.d), mid dose (2mg/kg.d) and high dose (4mg/kg.d) separately for2weeks. Cardiac function was evaluated by echocardiography, expression of phospho-p70s6k, phospho-4EBP-1and LC3BⅠ/Ⅱ were tested by western blotting, autophagic vacuoles were observed by transmission electron microscopy. Cardiomyocyte apoptosis was observed by TUNEL.Results:We found that compared to the control group (EF=81.3±3.8%), the average ejection fraction decreased in both the dilated cardiomyopathy group (EF=56.1±3.3%) and the high dose rapamycin group (EF=55.9±3.6%), but recovered in the low/mid dose rapamycin groups (EF=64.9±4.6%, EF=69.4±4.4%). The phosphorylation of p70s6k and4E-BP1decreased and the expression of LC3BI/II increased in all the groups of rapamycin. Autophagic vacuoles were easily found in the groups of rapamycin. Apoptosis could be found in all the groups except the control group. However, the body weight reduced significantly in the rapamycin groups. Furthermore, the mortality increased in the high dose rapamycin group.Conclusions:Rapamycin could improve the cardiac function of dilated cardiomyopathy, but the effect of rapamycin turned out to be biphasic and the effective range appeared narrow. Apoptosis was increased in all of the rapamycin groups. Part Ⅲ Inhibiting autophagy in the model of dilated cardiomyopathyObjective:It is still unknown whether the accumulation of autophagosome adds to the deterioration of cardiac function in dilated cardiomyopathy. We tried to improve the cardiac function by inhibiting autophagy in a rat model of dilated cardiomyopathy.Methods:We immunized rats with porcine cardiac myosin to set up a model of dilated cardiomyopathy. Eight weeks after immunization, rats were given 3-Methyladenine(3-MA)1.5mg/kg.d intraperitoneally for2weeks. Cardiac function was evaluated by echocardiography, expression of Beclin-1and LC3B91ⅠⅡ were tested by western blotting, autophagic vacuoles were observed by transmission electron microscopy. Cardiomyoctye apoptosis was observed by TUNEL.Results:Compared to the control group (EF=81.3±3.8%), the average ejection fraction decreased both in the DCM group (EF=56.1±3.3%)and the3-MA group (EF=58.6±5.4%). Beclin-1and LC3BI/II were suppressed in the group of3-MA. Autophagic vacuoles were seldom found in the group of3-MA. However,3-MA failed to reverse the pathologic process of dilated cardiomyopathy. Apoptosis was found both in3-MA group and dilated cardiomyopathy group.Conclusions:Inhibiting autophagy in dilated cardiomyopathy could neither reverse fibrosis nor improve cardiac function. Apoptosis was also increased in the3-MA group. Part IV Evaluating cardiomyocyte apoptosis when modulating autophagyObjective:To further evaluate cardiomyocyte apoptosis in dilated cardiomyopathy when autophagy was modulated.Methods:Apoptosis level was found to be elevated in all the other groups except the control group. We further evaluated several apoptosis related protein including anti-apoptosis BCL2、pro-apoptosis BAX and the terminal effective molecular caspase3by Western.Results:Except control group, apoptosis could be found in all the other groups by TUNEL. Among these groups, the expression of anti-apoptosis BCL2was elevated, especially in rapamycin4mg group. Except control group, the expression of pro-apoptosis BAX was also increased in all the other groups, especially in rapamycin4mg group. The expression of the terminal effective molecular caspase3was also increased especially in rapamycin4mg group, which meant apoptosis was significantly increased in rapamycin4mg group. The expression of caspase3did not differ significantly in3-MA group and rapamycin2mg group, which meant the apoptosis level in3-MA group was similar to that in rapamycin2mg group.Conclusions:Apoptosis was significantly elevated in high dose rapamycin group, which could partly explain that the cardiac function was deteriorated in rapamycin4mg group.Apoptosis level was similar in3-MA group and rapamycin2mg group. However, the cardiac function did not get improved in the3-MA group but got improved in rapamycin2mg group. The up-regulation of autophagy should be considered as the reason.