Exploring The Effect Of Low-fat On Myocardial Energy Metabolism In Congenital Heart Disease Patients Based On IPSC-CMs

Objective:Adults with congenital heart disease(CHD)have a high risk of metabolic syndrome(MS),dyslipidemia is a common symptom of MS,and the difference between blood lipid levels and healthy people in CHD patients is still controversial,and the effect of dyslipidemia on CHD patients has not been reported.Therefore,this paper aims to explore the difference in lipid levels between CHD patients and healthy people through meta-analysis,and to explore the effect of low lipids on the energy metabolism of CMs in patients with CHD from the perspective of cardiomyocyte(CMs)energy metabolism by using the induced pluripotent stem cell-derived cardiomyocytes(iPSC-CMs)model,so as to provide useful guidance for the prognosis of CHD patients.Methods:(1)First,the literature on MS-related metabolite levels in CHD patients was collected by searching relevant databases,and the literature that met the requirements was extracted and the quality of the literature was evaluated.The Meta package of R software was used to compare the differences in MS-related metabolite levels between CHD patients and healthy controls under a random-effects model or a fixed-effect model,using the standardized mean difference(i.e.,Cohen’s d value)as the effect size.The symmetric distribution of the funnel plot and the Egger test assessed publication bias,and the P value and I~2 statistics of the Cochran Q test were used to assess the heterogeneity between studies,and the sources of inter-study heterogeneity were further explored by subgroup analysis in different geographical regions of the study.(2)Urine cells were isolated and cultured by centrifugation,and induced pluripotent stem cells(iPSCs)were further established by transfection of Sendai virus containing reprogramming transcription factors,and the pluripotency of iPSCs was identified by alkaline phosphatase staining,RT-PCR,cellular immunofluorescence,teratoma and other methods.iPSC-CMs were cultured in low-fat medium to simulate the low-fat environment of CMs in CHD patients,and the effects of low lipid on the expression of genes PPARα,PGC-1α,SREBP1a and SREBP1c related to mitochondrial genesis and fatty acid metabolism were detected by RT-PCR.Active staining of mitochondria of cells using Mito Tracker Red dye to investigate the effect of low lipids on cellular mitochondrial biogenesis.Results:(1)A total of 7257 eligible studys were collected in this paper,and 18studies were finally included in the meta-analysis after further screening.Results from meta analysis showed that TC(Cohen’d:-0.68,95%CI:-0.91 to-0.45,I~2=88%,P<0.01),HDL-C(Cohen’d:-0.63,95%CI:-0.89 to-0.37,I~2=89%,P<0.01)and LDL-C(Cohen’d:-0.32,95%CI:-0.54 to-0.10,I~2=87%,P<0.01)levels in CHD patients were significantly lower than control groups.Hb A1c(Cohen’d:0.93,95%CI:0.17 to1.70,I~2=91%,P<0.01)levels were higher in CHD compared with controls,and BMI(Cohen’d:-0.27,95%CI:-0.42 to-0.12,I~2=69%,P<0.01)were lower in CHD patients than control groups.Subgroup analysis of populations in different geographic regions showed that there were large differences in lipid levels between different subgroups.(2)iPSCs with stable passage and uniform morphology were constructed,and alkaline phosphatase staining experiments were performed,and the experimental results were positive,indicating that iPSCs had alkaline phosphatase activity.The RT-PCR experiment detected the expression of four pluripotent genes,CRIPTO,NANOG,OCT4 and SOX2,and found that the pluripotency genes of iPSCs were activated and highly expressed,and the expression was close to that of human embryonic stem cells(ESCs).Furthermore,the expression of two key transcription factors,NANOG and OCT4,was detected by immunofluorescence staining,and it was found that both transcription factors were positive expression,which proved that the cell had pluripotency close to human ESCs.The results of karyotype analysis showed that there were no abnormalities in the number and structure of chromosomes,indicating that there were no abnormal changes in karyotype during the culture of the cell line.The results of in vivo teratoma formation showed that the iPSCs constructed in this paper had differentiation totipotency.(3)The CMs induced by iPSCs in this paper stably expressed cardiac troponin T,indicating that the differentiation induced by iPSCs to CMs was successful.Compared with the control group treated with normal serum treatment,the expression of PGC-1α,PPARa,SREBP-1a and SREBP-1c associated with fatty acid synthesis and transport were significantly reduced and statistically significant in CMs(P<0.0001).Further mitochondrial activity staining of CMs found that low-fat treatment significantly reduced mitochondrial biogenesis in cells.Conclusions:(1)CHD patients had significantly lower TC,HDL-C and LDL-C levels and BMI than controls,and higher Hb A1c levels compared with controls,as well as large differences in lipid levels between populations in different geographical regions.This may be related to whether CHD patients underwent surgical intervention,nutritional status and genetic variation,and these data will be helpful for the health management of CHD patients.(2)iPSCs constructed by somatic cell reprogramming have similar pluripotency as ESCs and can be directed to induce differentiation into CMs,which can become a reliable platform for CHD-related research.(3)Low-lipid serum treatment reduced the expression of genes related to mitochondrial genesis and fatty acid metabolism in CMs,suggesting that lower lipid levels in CHD patients may affect mitochondrial biogenesis and fatty acid metabolism in their CMs,which in turn leads to changes in myocardial energy metabolism in CHD patients.