Molecular Mechanism Of Mitochondrial Transmembrane Protein 11(TMEM11) Regulating Cardiomyocyte Proliferation And Cardiac Repair

Objective Cardiovascular disease is one of the main causes of human death,among which heart failure is the main cause of death.The pathology of heart failure lies in the massive loss of myocardial functional cells caused by myocardial infarction.For many years,impairment of mitochondrial function,the source of cardiac energy,has been recognized as a key molecular mechanism of heart failure.With the deepening of research,it is recognized that mitochondrial function is not limited to providing energy,but also participates in important biochemical and regulatory processes,including redox,biosynthesis,reactive oxygen species signaling,cell growth and death,and ion homeostasis,protein folding,and inflammation,etc.It can be seen that mitochondria are not only involved in heart failure as an energy source,but whether they are involved in the biochemical regulation process of cardiomyocytes and the molecular mechanism need to be further studied.Induction of myocardial proliferation holds promise for the clinical treatment of myocardial infarction and heart failure,but the regenerative capacity of mature cardiomyocytes is weak.As the most important organelle in cardiomyocytes,mitochondria have a key regulatory role in cardiomyocyte proliferation.Previous results found that the expression level of mitochondrial transmembrane protein11(TMEM11)increased with the development of mouse hearts,indicating that TMEM11 has a potential role in cardiomyocyte proliferation.To this end,we focus on TMEM11,aiming to elucidate its function and molecular mechanism in cardiomyocyte proliferation and cardiac repair,and provide a new theoretical basis for treating cardiovascular diseases.Experimental method1.Real-time fluorescence quantitative PCR(RT-q PCR),Western blot,immunofluorescence,nuclear-cytoplasmic separation and mitochondrial separation were used to detect the expression,and distribution of TMEM11 in mouse hearts.2.The adenovirus vector Ad V-sh TMEM11 was constructed to knock down the expression of TMEM11 in primary mouse neonatal cardiomyocytes.RT-q PCR and Western Blot were used to detect the effect of adenovirus knockdown of TMEM11,and immunofluorescence was used to detect the proliferation of cardiomyocytes after knockdown of TMEM11.3.PCR and Western Blot were used to identify the knockout effect of TMEM11 knockout(TMEM11 Ko)mice.Heart body weight ratio,H/E staining,and immunofluorescence were used to detect the effect of TMEM11 Ko mice on the proliferation of mature cardiomyocytes.4.PCR and Western Blot were used to identify the overexpression effect of TMEM11transgenic(TMEM11 Tg)mice,to construct a neonatal mouse model of myocardial infarction in TMEM11 Tg mice,and to detect its effects on myocardial cell proliferation and cardiac injury repair by mouse echocardiography analysis and immunofluorescence.5.In order to observe the effects of TMEM11 Ko mice on cardiomyocyte proliferation and cardiac injury repair,a 60-day TMEM11 Ko adult mouse model of myocardial infarction was constructed,and the effects of TMEM11 deletion on cardiomyocyte proliferation and cardiac injury repair were detected by mouse echocardiography,Masson staining,and immunofluorescence.Experimental results1.RT-q PCR and Western Blot detected the expression level of TMEM11 in each developmental stage,and the results showed that the expression level of TMEM11 m RNA was up-regulated with the development of the mouse heart.Western Blot results showed that compared with other tissues,TMEM11 was expressed relatively high in the heart.The results of nuclear-cytoplasmic separation and immunofluorescence showed that TMEM11 was expressed in the nucleus and cytoplasm in addition to the mitochondria.In conclusion,TMEM11 is highly expressed in mouse cardiomyocytes,suggesting that it may be involved in regulating cardiomyocyte proliferation.2.Western Blot and RT-q PCR results showed that adenovirus Ad V-sh TMEM11 inhibited the expression of TMEM11,and immunofluorescence results showed that knockdown of TMEM11 could effectively promote the proliferation of cardiomyocytes.These results indicated that the downregulation of TMEM11 promoted the proliferation of cardiomyocytes.3.PCR and Western Blot results proved that TMEM11 Ko mice were successfully constructed.Immunofluorescence results showed that TMEM11 Ko mice effectively promoted the proliferation of cardiomyocytes.The results of heart weight ratio and H/E staining showed that there was no significant difference in heart weight ratio and heart shape between TMEM11 Ko and wild-type mice,indicating that TMEM11 Ko mice did not affect mouse cardiac function.Taken together,these data demonstrate that TMEM11 Ko stimulates cell cycle reentry and cardiomyocyte proliferation in postnatal and adult hearts.4.PCR and Western Blot results showed that TMEM11 Tg mice were successfully constructed,and immunofluorescence results showed that the proliferation of cardiomyocytes in neonatal TMEM11 Tg mice after myocardial infarction was inhibited;The results of echocardiographic analysis in mice showed that compared with wild-type,the cardiac function impairment of TMEM11 Tg mice after myocardial infarction surgery was more serious.Therefore,TMEM11 overexpression inhibits cardiomyocyte proliferation and cardiac injury repair.5.To explore the effect of TMEM11 deletion on cardiomyocyte proliferation and repair of cardiac injury by constructing a TMEM11 Ko mouse model of myocardial infarction for 60 days.The results of mouse echocardiography,Masson staining and immunofluorescence showed that compared with wild-type mice,myocardial cell proliferation and cardiac repair ability of TMEM11 Ko mice at 60 days after myocardial infarction were significantly enhanced,and their cardiac function was significantly improved.Conclusion TMEM11 has a high expression level in the heart and is involved in the regulation of cardiomyocyte proliferation.TMEM11 deletion significantly improves cardiac function after myocardial infarction injury,reduces myocardial infarct size,and promotes myocardial cell proliferation.Overexpression of TMEM11 inhibits the proliferation of neonatal mouse cardiomyocytes,suggesting that TMEM11 has therapeutic potential for cardiac repair and regeneration,and is expected to be applied in the clinical treatment of cardiovascular diseases.