A Preliminary Study On The Early Myocardial Mitochondrial Damage And Its Mechanism In Mice With Restrictive Cardiomyopathy Caused By CTnIR193H Mutation

PART Ⅰ EARLY MYOCARDIAL MITOCHONDRIAL DAMAGE IN MICE WITH RESTRICTIVE CARDIOMYOPATHY CAUSED BY CTNIR193 H MUTATIONObjective: Myocardial mitochondrial damage is an important cause of various cardiovascular diseases,especially hypertrophic cardiomyopathy and dilated cardiomyopathy.It is unclear whether myocardial mitochondrial damage is also an important element in development of restrictive cardiomyopathy(RCM).One study reported that ATP production and mitochondrial metabolism disorder were associated with RCM,suggesting it might an important pathological process of cardiac pathology in RCM.Therefore this section of the study aimed to determine whether myocardial mitochondrial damage present at early stage in development of RCM transgenic(TG)mice due to myocardial troponin I(cTnI)R193H mutation.Materials and methods:(1)By crossing wild type(WT,C57BL/6)mice with cTnIR193 H transgenic(TG,with C57BL/6background)mice,we obtained two types of offspring:WT and transgenic mice,and 1-2,3-4 and 5-6 month-old mice were used for the present study with littermate WT mice as controls.(2)Cardiac function was measured by high-frequency ultrasound;(3)Using HE,Masson staining and transmission electron microscopy respectively detect myocardial tissue structure and myocardial interstitial fibrosis and mitochondrial strcuture;(4)Using multiscan spectrμm to detect complex I activity,ATP and ROS(reactive oxygen)level;(5)Using PET-CT to detect Left ventricular myocardial standare glucose uptake in mice.Results:(1)The ejection fraction(EF%),short axis shortening rate(FS%)and left Ventricular isovolμmetric systolic time(IVCT)and left ventricular isovolμmetric diastolic time(IVRT)were not statistically different in 1-2 and 3-4 month-old TG mice(P> 0.05),while IVRT was significantly prolonged(P <0.05)in 5-6 month-old TG mice with normal contractile function compared with age matched WT mice;(2)Transmission electron microscopic examination showed that significant changes in the ultra-structures were present in myocardial mitochondria with mild swelling in 1-2month-old TG mice with normal diastolic function,and severe swelling with distorted shapes,disorganized cristae and vacuolation in 5-6 month-old TG mice with diastolic dysfunction,compared with littermate wild-type(WT)mice.No myocardial fibrosis or myocardial hypertrophy was found in TG mice.(3)By measuring mitochondrial function,we found that complex I activities were progressively reduced with the growing up in TG mice(P <0.05),but ATP and ROS levels did not change significantly compared with age matched WT mice.(4)The level of standard glucose uptake(SUV)reduced in left ventricle in 5-6 month-old TG mice,while 1-2 and 3-4 month-old TG mice appeared to have a higher level of SUV when compared to littermate WT control mice.Conclusion: Early myocardial mitochondria damage is present in development of mice with RCM due to cTnIR193 H mutation.PART Ⅱ CTNIR193 H MUTATION MAY THROUGH REGULATE PGC-1Α RELATED PROTEIN LOW EXPRESSION RESULTING IN MYOCARDIAL MITOCHONDRIA DAMAGEObjective: The first part of the study found that the early mitochondrial damage in mice with RCM caused by cTnIR193 H mutant.However,whether cTnIR193 H was correlated with mitochondrial damage and how cTnIR193 H mutation causes mitochondrial damage? In the previous study,we found that cTnI can bind to mitochondrial-related proteins,and cTnI can enter the nucleus and regulate gene expression.Therefore,it is speculated that the mutant cTnI has similar functions.This section aims to determine whether cTnIR193 H mutation was correlated with mitochondrial damage,and explore the mechanism of mitochondrial damage by transfecting mutant cTnI in cardiomyocytes.Materials and Methods:(1)The primary cardiomyocytes were divided into over-expression mutant cTnIR193 H group(R193H)and negative control group(control);(2)Morphology of myocardial mitochondria was detected by laser confocal;(3)Structure of myocardial mitochondria measured by transmission electron microscope;(4)Mitochondrial function:using multiscan spectrμm to detect complex I activity,ATP and ROS(reactive oxygen)level;The oxygen consμmption rate(OCR)of myocardial mitochondria was measured by seahorse XFe24 analyzer.(5)To further determine the damage of mitochondrial structure and function,WB was used to detect the expression of mitochondria-related proteins.Results:(1)In vitro experiments showed that mitochondrial structure was abnormal with impaired function including decrease of complex I activity,ATP and ROS levels and oxygen consμmption rate(OCR)in R193 H cardiomyocytes compared with controls(P <0.05).(2)Western blot analysis confirmed that ND5,LRPPRC and PGC-1α were decreased in the R193 H over-expressing cardiomyocytes when compared to the controls(P<0.05),which supported this observation.Conclusion: cTnIR193 H mutation resulting in myocardial mitochondria damage may be by regulating PGC-1α related protein low expression.