| Background:Hypertrophic cardiomyopathy（HCM）can cause heart failure（HF）,malignant arrhythmia even sudden cardiac death（SCD）,and HCM is one of the main causes of adolescent SCD.It is mostly caused by genetic mutation encoding sarcomere structural proteins and is autosomal dominant inheritance.At present,the specific molecular mechanism of HCM caused by gene mutation is not fully clear and lack of specific treatment.To constructing animal models with mutation,exploring the pathogenesis and finding possible intervention targets through in vivo/in vitro experiments might provide a theoretical basis for the development of more effective treatments.ACTC1 gene encodes cardiacα-actin,which is the central cytoskeleton protein of sarcomere filaments and participates in cardiomyocyte contraction.ACTC1 gene is one of the main pathogenic genes of HCM,with 454 reported HCM-related mutations in Clin Var database.Previous studies mainly focused on exploring the relationship between ACTC1 gene mutation and clinical phenotype of HCM,with little studies investigated the specific molecular mechanism of HCM caused by ACTC1 gene mutation.In this study,a new ACTC1 gene mutation（p.Y308H）was screened for the first time in a HCM family,and the specific mechanism of this mutation remains to be elucidated.Objective:In this study,a gene knock in mouse model carrying Actc1 p.Y308H heterozygous mutation(Actc1+/Y308H)was constructed,and functional analysis and pathogenic mechanism of this mutation were carried out to search for possible intervention targets,and effective individual therapeutic drugs were screened.Methods:1.Whole exome sequencing was performed on the proband and family members,and the candidate mutations detected were verified by first-generation sequencing（Sanger sequencing）.2.Actc1+/Y308H mice carrying Actc1 p.Y308H mutation were constructed by homologous recombination technique.The control group was wild type(Actc1+/+)mice matched with sex and age in the same litter.3.The cardiac structural parameters of mice were detected by small animal ultrasound system and the level of cardiac function was calculated.4.Masson staining was performed to observe the myocardial fibrosis;wheat germ agglutinin（WGA）staining was performed to observe the cross-sectional area,;the dihydroethidium（DHE）staining was used to observe the level of oxidative stress.5.The morphological and structural changes of myocardial mitochondria in mice were observed by transmission electron microscope.6.The level of ATP in myocardial tissue of mice was detected.7.The differential expressed proteins in myocardium of Actc1+/Y308H and Actc1+/+mice were screened by TMT relative quantitative protein map.8.Overexpressed ACTC1 wild and mutation plasmids and adenoviruses,overexpressed FHL2 and UBC plasmids were constructed,and the neonatal rat cardiac myocytes and HEK293T cells was used as tool cells.9.The m RNA level was detected by real-time fluorescence quantitative PCR,protein expression was detected by Western blot,and protein interaction was detected by molecular docking,co-immunoprecipitation and immunofluorescence techniques.10.An adeno-associated virus 9 vector was used to overexpress FHL2,and AAV9-ad-Fhl2 was injected into tail vein of Actc1+/Y308H and Actc1+/+mice.11.Actc1+/+and Actc1+/Y308H mice received intragastric injection of a metformin solution.12.Statistical methods:all data were analyzed and plotted by Graph Pad Prism 8.0software.The measurement data are expressed as mean±standard deviation（mean±SD）or mean±standard error of mean（mean±SEM）.For two independent samples,if they obey normal distribution,unpaired t-test（uniform variance）or Welch’s method are used to correct t-test（uneven variance）,otherwise Mann-Whitney U test is used.The comparisons among groups were analyzed by ANOVA.P<0.05（*）is considered to have statistical difference,P<0.01（**）is considered to have significant statistical difference.Results:1.A novel ACTC1 p.Y308H mutation was identified by gene sequencing in a HCM familyWhole exome sequencing was performed on the pedigree and results were validated by targeted Sanger sequencing,a novel ACTC1 p.Y308H mutation was detected.Bioinformatics technology was used to predict the pathogenicity of the mutation,the results showed that the p.Y308 site is highly conserved and the mutation might cause the damage of ACTC1 protein function.2.Construction of Actc1+/Y308H mice carrying Actc1 p.Y308H mutation and functional analysis of the mutation in vivo.1)Compared with Actc1+/+mice,Actc1+/Y308H mice showed left ventricular diastolic anterior wall thickness（LVAW d）and left ventricular diastolic posterior wall thickness（LVPW d）（LVAW d:0.75±0.02 mm vs.0.86±0.03 mm;LVPW d:0.73±0.02 mm vs.0.90±0.03 mm;all P<0.05）,and decreased left ventricular ejection fraction（EF）and short axis shortening rate（FS）at 6 months of age（EF:61.21±1.06%vs.54.75±1.02%;FS:32.13±0.76%vs.27.67±0.66%;all P<0.001）.2)Compared with Actc1+/+mice,Actc1+/Y308H mice had an increased heart weight/body weight ratio（0.0049±0.0005 vs.0.0059±0.0005,P<0.01）,an increased degree of myocardial fibrosis（1.59±0.69 vs.6.00±1.35,P<0.01）,an increased cross-sectional area of individual cardiomyocytes（1.01±0.35 vs.1.32±0.45,P<0.001）,and increased expression of cardiac hypertrophy markers MYH7 and NPPA（MYH7:0.50±0.17 vs.1.34±0.23,P<0.001;NPPA:0.77±0.17 vs.0.99±0.15;all P<0.05）.3)The results of transmission electron microscopy showed that Actc1+/Y308H mice showed Mitochondrial structural damage was found in Actc1+/Y308H mice heart,which manifestating as loss of vacuolated ridge;the expressions of mitochondrial respiratory chain complex proteins ATP5A1,UQCRC2,MTCO1,SDHB and NDUFB8 decreased compared with Actc1+/+mice（ATP5A1:1.03±0.17 vs.0.55±0.16;UQCRC2:0.94±0.20 vs.069±0.15;MTCO1:1.05±0.19 vs.0.52±0.18;SDHB:0.89±0.19 vs.0.56±0.15;NDUFB8:1.58±0.20 vs.1.13±0.14;all P<0.05）.4)The results DHE staining showed that compared with Actc1+/+mice,oxidative stress level of Actc1+/Y308H mice increased（1.00±0.17 vs.4.40±0.32,P<0.001）and ATP level of myocardial tissue of Actc1+/Y308H mice decreased（1.00 vs.0.73±0.08,P<0.05）.5)Compared with Actc1+/+mice,the phosphorylation levels of Adenosine 5-monophosphate（AMP）-activated protein kinaseαsubunit（AMPKα）and Acetyl-Co A Carboxylase（ACC）in Actc1+/Y308H mice were decreased（AMPKα:0.92±0.28 vs.0.50±0.14;ACC:0.97±0.15 vs.0.53±0.19;all P<0.01）,which suggests that the AMPK/ACC-induced energy generation pathway was inhibited;the phosphorylation levels of mTOR,p70S6K and extracellular regulated protein kinases（ERK）were increased（mTOR:0.83±0.10 vs.1.18±0.22;p70S6K:0.88±0.17 vs.1.41±0.29;ERK:1.04±0.12 vs.1.61±0.23;P<0.01）,which suggests that the mTOR/p70S6K-induced and ERK-induced hypertrophy pathway was activated.3.Exploring the Molecular Mechanism of HCM caused by ACTC1 p.Y308H mutation1)Compared with Actc1+/+mice,the m RNA and protein levels of ACTC1in the heart of Actc1+/Y308H mice were not changed（all P>0.05）;2)The results of protein spectrum and western blot showed that the expression of FHL2was down-regulated in Actc1+/Y308H mice heart,and the GO functional enrichment analysis of FHL2 protein showed that the protein was bound to various sarcomere structures,such as M-band and Z-disc,and involved in various biological processes such as myocardial cell development.3)After specific overexpression of FHL2 in the heart of Actc1+/Y308H mice by AAV9-ad-Fhl2,Actc1+/Y308H mice showed an increased EF（52.59±1.06%vs.58.82±0.95%,P<0.05）,a reduction of cardiac fibrosis level（4.55±1.05%vs.1.36±0.69%,P<0.01）,reduced cardiomyocyte cross-sectional area（1.48±0.46 vs.1.24±0.43,P<0.001）and decreased expression of MYH7 and NPPA protein level（MYH7:1.33±0.33 vs.0.69±0.25;NPPA:1.37±0.24 vs.0.98±0.15,all P<0.05）,suggesting an improvement of hypertrophic phenotype.4)After specific overexpression of FHL2 in the heart of Actc1+/Y308H mice by AAV9-ad-Fhl2,Actc1+/Y308H mice showed an reduction of mitochondrial structural damage and increased expression levels of respiratory chain complex proteins（ATP5A1:0.51±0.13 vs.0.89±0.20;UQCRC2:0.55±0.06 vs.0.89±0.19;MTCO1:0.49±0.19 vs.0.86±0.23;SDHB:0.60±0.16 vs.0.94±0.19;NDUFB8:0.99±0.23 vs.1.70±0.47,all P<0.05）,suggesting an improvement of mitochondrial fuction.5)The protein expression levels of signaling pathways was dected after specific overexpression of FHL2 in the heart of Actc1+/Y308H mice by AAV9-ad-Fhl2,the phosphorylation level of AMPKαand ACC was increased after FHL2 overexpressed in Actc1+/Y308H mice（AMPKα:0.54±0.26 vs.0.99±0.23;ACC:0.30±0.15 vs.0.54±0.19;all P<0.05）,suggesting an elevated activity of AMPK/ACC-induced energy generation pathway;the phosphorylation level of mTOR,p70S6K and ERK was decreased after FHL2 overexpressed in Actc1+/Y308H mice（mTOR:1.29±0.30 vs.0.82±0.20;p70S6K:1.50±0.30 vs.0.84±0.30;ERK:1.30±0.12 vs.0.92±0.14;P<0.05）,suggesting a reduced activity of mTOR/p70S6K-indued and ERK-induced hypertrophy pathway.6)Compared with Actc1+/+mice,the m RNA level of FHL2 in Actc1+/Y308H mice had no change（P>0.05）.Under the action of cycloheximide（CHX）,compared with the degradation rate of FHL2 after overexpression of wild-type ACTC1,the degradation rate of FHL2 after overexpression of mutated ACTC1 was accelerated.Compared with the co-transfection of wild-type ACTC1,FHL2 and ubiquitin（UBC）plasmid group,the immunoprecipitation assay showed that the binding of FHL2 to ubiquitin was increased in the mutated ACTC1,FHL2 and ubiquitin（UBC）plasmid group.7)Molecular docking,co-immunoprecipitation and immunofluorescence experiments showed that there was interaction between ACTC1 and FHL2,and ACTC1 p.Y308H mutation could reduce the binding of ACTC1 and FHL2（0.66±0.09 vs.0.50±0.13,P<0.05）.4.Pharmacological interventions1)Afted metformin intragastric administration,the Actc1+/Y308H mice showed increased LVEF（53.12±1.12%vs.59.73±0.75%,P<0.05）,reduced cardiac fibrosis levels（5.11±0.16 vs.1.34±0.39,P<0.01）,reduced cardiomyocyte cross-sectional area（1.38±0.46 vs.1.10±0.33,P<0.001）and decreased expression of MYH7 and NPPA protein level（MYH7:1.65±0.24 vs.0.58±0.21;NPPA:1.38±0.26 vs.0.83±0.16,all P<0.05）,suggesting that metformin could lead to an improvement of hypertrophic phenotype of Actc1+/Y308H mice.2)The FHL2 protein expression level was not altered after metformin intragastric administration Actc1+/Y308H mice（P>0.05）,suggesting that metformin may not improve cardiac function and hypertrophy phenotype of Actc1+/Y308H mice through FHL2pathway.3)After metformin intragastric administration,Actc1+/Y308H mice showed an reduction of mitochondrial structural damage and increased expression levels of respiratory chain complex proteins（ATP5A1:0.57±0.12 vs.0.83±0.17;UQCRC2:0.44±0.11 vs.0.75±0.20;MTCO1:0.58±0.14 vs.0.92±0.22;SDHB:0.62±0.10 vs.0.92±0.19;NDUFB8:1.21±0.13 vs.1.65±0.17,all P<0.05）,suggesting an improvement of mitochondrial fuction.4)The protein expression levels of signaling pathways was dected after metformin intragastric administration,the phosphorylation level of AMPKαand ACC was increased in Actc1+/Y308H mice（AMPKα:0.46±0.15 vs.0.95±0.25;ACC:0.29±0.05vs.0.62±0.19;all P<0.05）,suggesting an elevated activity of AMPK/ACC-induced energy generation pathway;the phosphorylation level of mTOR,p70S6K and ERK was decreased in Actc1+/Y308H mice（mTOR:1.14±0.23 vs.0.73±0.21;p70S6K:1.36±0.26 vs.0.81±0.19;ERK:1.22±0.30 vs.0.53±0.25;all P<0.05）,suggesting a reduced activity of mTOR/p70S6K-indued and ERK-induced hypertrophy pathway.Conclusion:1.Actc1+/Y308Hmouse model was constructed for the first time and the clinical phenotype of the patients was successfully reproduced,it was confirmed that the ACTC1 p.Y308H mutation can cause HCM.2.It was found for the first time that ACTC1 p.Y308H mutation can cause obvious HCM phenotypes such as myocardial hypertrophy,fibrosis and decreased cardiac function,and can lead to AMPK-mediated mitochondrial damage,impaired energy metabolism and activation of mTOR/p70S6K and ERK-mediated hypertrophy pathway.3.It is found for the first time that ACTC1 p.Y308H mutation reduces the binding of ACTC1 to FHL2,and then increases the degradation of FHL2 through ubiquitination pathway,which may be an important molecular basis for the pathogenesis of the mutation,and FHL2 may be an effective target for intervention.4.It was found for the first time that metformin can rescue the myocardial damage and energetic abnormalities caused by ACTC1 p.Y308H mutation. |
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