| Background Mitochondrial cardiomyopathy is a type of disease in which myocardial morphology and dysfunction are caused by mitochondrial respiratory chain gene defects in myocardial tissue.There are many major clinical phenotypes involved in mitochondrial cardiomyopathy,including hypertrophic cardiomyopathy,dilated cardiomyopathy(DCM),restrictive cardiomyopathy,and left ventricular noncompaction cardiomyopathy etc.Iron-sulfur clusters are one of the key components of the energy production process in the mitochondrial respiratory chain.And ISCA1(Iron-sulfur cluster assembly 1)is an important assembly factor for iron-sulfur cluster which is essential for the maturation of Fe4S4 protein.It has been found that ISCA1 abnormality affects the mitochondrial respiratory chain electron transport process in vitro.The current disease reports related to Isca1 are mainly focused on encephalopathy and myopathy.We speculate that Isca1 also plays an important role in heart which is another highly energy-consuming organ of the body.Therefore,we established Isca1 traditional knockout(Iscal-/-)rat model and Isca1 conditional knockout(Iscalflox/flox/MHC-Cre)rat model to explore the effect of Isca1 on the development of the rat and morphology and function of the heart,and to verify the role of Isca1 in heart development and pathogenesis of heart failure,to provide theoretical basis and clues for the prevention and treatment of cardiovascular diseases such as heart failure.Methods 1)Establishing Isca1 traditional knockout rat and Isca1 conditional knockout rat by CRISPR-Cas9 technology and breeding.2)Localization and expression of ISCA1 in tissues of rat:RNA level of Isca1 in tissues of WT rats was detected by PCR;the expression of ISCA1 was observed by the self-contained fluorescent of Iscal traditional knockout rat model;the localization of ISCA I in cardiomyocytes of WT rats was detected by immunofluorescence;expression of ISCA 1 in the myocardial tissue of cardiomyopathy patients and cardiomyopathy mouse model was determined by Western blot.3)Genotyping identification by PCR and survival rate analysis of Isca1 traditional knockout rats and Isca1 conditional knockout rats.4)Embryonic development analysis of Isca1 traditional knockout rats:2.5-day-old and 8.5-day-old embryos preparation and morphological observation of Isca1 traditional knockout rats by microphotographing;genotyping of the embryos was detected by PCR.5)Heart morphology and function analysis of Isca1 conditional knockout rats:record heart shape and heart weight/body weight ratio of 6.5-day-old Isca1 conditional knockout rats;heart structure and function of 0.5-day-old,2.5-day-old,6.5-day-old Isca1 conditional knockout rats were analyzed by magnetic resonance imaging;heart structure and function of embryo 17.5-day-old,embryo 20.5-day-old,0.5-day-old,2.5-day-old,6.5-day-old Isca1 conditional knockout rats were analyzed by echocardiography;pathological analysis of 6.5 day-old Isca1 conditional knockout rats were conducted by H&E staining and transmission electron microscopy.6)Energy metabolism analysis:NDUFA9 and AC02 levels of embryo 8.5-day-old Isca1 traditional knockout rats were determined by Western blot.expression of mitochondrial respiratory chain-associated protein of Isca1 conditional knockout rats was determined by Western blot;mitochondrial respiratory chain-related enzyme activity was detected by colorimetry;the ATP content of myocardial tissue was determined by colorimetry.Results 1.Iscal traditional knockout rat establishment,phenotype observation and mechanism analysis.1)Iscal traditional knockout rat model was established using CRISPR-Cas9 technology and selected based on the sequencing results.2)At RNA level,Isca1 expressed in heart,liver,spleen,lung,kidney,muscle,adipose and brain in different degrees,and highly expressed in spleen,brain and heart,and lower expressed in liver;immunofluorescence localization revealed that ISCA1 expressed systemically in rats and highly expressed in heart,muscle,liver,kidney and testis.3)we cannot obtain homozygous offspring of Isca1 traditional knockout rat,and the Iscal traditional knockout rat embryo developed normally at the 4-cell stage(embryo 2.5-day-old),but it showed abnormal development or developmental arrest at embryo 8.5-day-old;NDUFA9 protein level was down-regulated and AC02 protein level was up-regulated significantly in Iscal traditional knockout embryos(P<0.05).2.Iscal conditional knockout rat establishment,phenotype observation and mechanism analysis.1)The Isca1 knockout efficiency in the myocardium of Iscal conditional knockout rat was higher than 90%;ISCA1 is mainly localized to myocardial mitochondria;ISCA1 level increased in the myocardial tissue of cardiomyopathy patients and cardiomyopathy mouse model.2)Iscal conditional knockout rats all died from 7 to 10 days;Iscal conditional knockout rats were in dying state at 6.5 day with weak breathing,the heart showed significantly hypertrophy and increased heart weight/body weight ratio(P<0.05);MRI and echocardiography showed shape of heart increased,ventricular dilatation,heart function decreased and severe heart failure in 6.5-day-old Iscal conditional knockout rats;we observed at five time points including embryo 17.5-day-old and 20.5-day-old,0.5-day-old,2.5-day-old and 6.5-day-old after birth,we found the pathological phenotype of Isca1 conditional knockout rats is in progressive development,and embryo 17.5-day-old Iscal conditional knockout rats showed no differences compared with the same litter control rats.3)At the time of 6.5-day-old,the levels of mitochondrial complex Ⅰ subtype proteins NDUFA9 and NDUFS3,and complex Ⅱ subtype protein SDHB in the myocardium of Isca1 conditional knockout rat decreased significantly(P<0.01).The levels of AC02,complex Ⅲ subtype protein UQCRC2 and complex IV subtype protein COX Ⅳ have no significant difference(P>0.05).The enzyme activity assay showed that the mitochondrial complex Ⅰ,complex Ⅱ and complex Ⅳ activity decreased significantly(P<0.01).Conclusion ISCA1was expressed in the whole body of rats,and was highly expressed in high-energy-consuming organs such as heart and muscle.Iscal knockout led to abnormal development at embryo 8.5-day-old.NDUFA9 and AC02 are key proteins of mitochondrial respiratory chain.NDUFA9 expression are down-regulated,but AC02 up-regulated,suggesting that mitochondrial energy metabolism is abnormal;Isca1 conditional knockout caused ventricular dilatation,heart function decrease and leading to heart failure 6-8 days after birth until death.Abnormal expression and activity of mitochondrial respiratory chain complex-related molecules in myocardial tissue,indicating that ISCA1 plays an important role in development especially in heart development.We speculated that ISCA1 plays an important role in mitochondrial energy metabolism.This provides new ideas and directions for the identification of biological function of ISCA1 and the prevention and treatment of cardiovascular diseases. |
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