BACKGROUNDHypertrophic cardiomyopathy (HCM) is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain (beta-MHC) are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype.

METHODSThe exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed.

RESULTSThe mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13,619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated.

CONCLUSIONThe Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Adolescent ; Adult ; Cardiomyopathy, Hypertrophic, Familial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Myosin Heavy Chains ; genetics ; Ventricular Myosins ; genetics

Alterations of cardiovascular function associated with various thyroid states have been studied. In hyperthyroidism left ventricular contractility and relaxation velocity were increased, whereas these parameters were decreased in hypothyroidism. The mechanisms for these changes have been suggested to include alterations in the expression and/or activity levels of various proteins; alpha-myosin heavy chain, beta-myosin heavy chain, beta-receptors, the guanine nucleotide-binding regulatory protein, and the sarcolemmal Ca2+-ATPase. All these cellular alterations may be associated with changes in the intracellular Ca2+ concentration. The most important regulator of intracellular Ca2+ concentration is the sarcoplasmic reticulum (SR), which serves as a Ca2+ sink during relaxation and as a Ca2+ source during contraction. The Ca2+-ATPase and phospholamban are the most important proteins in the SR membrane for muscle relaxation. The dephosphorylated phospholamban inhibits the SR Ca2+-ATPase through a direct interaction, and phosphorylation of phospholamban relieves the inhibition. In the present study, quantitative changes of Ca2+-ATPase and phospholamban expression and the functional consequences of these changes in various thyroid states were investigated. The effects of thyroid hormones on (1) SR Ca2+ uptake, (2) phosphorylation levels of phospholamban, (3) SR Ca2+-ATPase and phospholamban protein levels, (4) phospholamban mRNA levels were examined. Our findings indicate that hyperthyroidism is associated with increases in Ca2+-ATPase and decreases in phospholamban levels whereas opposite changes in these proteins occur in hypothyroidism.
Ethanol ; Guanine ; Hyperthyroidism ; Hypothyroidism ; Membranes ; Muscle Relaxation ; Phosphorylation ; Relaxation ; RNA, Messenger ; Sarcoplasmic Reticulum* ; Thyroid Gland* ; Thyroid Hormones ; Ventricular Myosins

OBJECTIVETo explore the genetic basis and phenotypic correlation with disease severity in a large cohort of Chinese patients with hypertrophic cardiomyopathy (HCM).

METHODSA total of 179 unrelated Chinese HCM patients admitted to our department from 2002 to 2011 were enrolled in this study. Direct gene sequencing of β-myosin heavy chain (MYH7), myosin binding protein-C ( MYBPC3), and cardiac troponin T (TNNT2) were performed and clinical data were obtained in these patients.

RESULTSA total of 34 mutations were identified in 40 patients (22.3%), 79.4% (27/34) mutations occurred only once and a possible hot spot, A26 in MYH7, was found. Distribution of mutations was 52.9% (18/34) (MYBPC3), 35.3% (12/34) ( MYH7) and 11.8% (4/34) (TNNT2) respectively. Double mutations were identified in 2.2% (4/179) patients. Genotype-positive patients were associated with an earlier symptom onset, severer left ventricular hypertrophy, a higher incidence of syncope, and were more likely to have positive family history of HCM or sudden cardiac death (SCD) , and were more likely to progress into heart failure (24.2% vs. 5.0%, P = 0.002) and at a higher risk of SCD (9.1% vs. 0, P = 0.009) during the 6.5-year follow-up. No statistical difference in any clinical parameters and outcomes was found between patients carrying MYBPC3 and MYH7 mutations. Double mutations were associated with malignant clinical progression in this cohort. Different phenotype severity could be seen in HCM patients with same genotype (e. g. MYH7-1736T, TNNT2-R92W).

CONCLUSIONMYBPC3 is the most predominant gene mutation in this HCM cohort. The presence of a sarcomere mutation in patients with HCM is associated with poor clinical outcome, although no specific genes or mutations can exactly predict the severity of clinical phenotypes.

Asian Continental Ancestry Group ; Cardiomyopathy, Hypertrophic ; Carrier Proteins ; Death, Sudden, Cardiac ; Disease Progression ; Genotype ; Humans ; Hypertrophy, Left Ventricular ; Mutation ; Phenotype ; Sarcomeres ; Troponin T ; Ventricular Myosins

BACKGROUND: Single or multiple episodes of brief period of ischemia and reperfusion(ischemic preconditioning, IP) have been shown to limit infarct size after a subsequent longer period of ischemia. A considerable number of possible mechanisms has been proposed, however, controversies still remain. Accordingly, we evaluated the effect of four cycles of 5 minutes ischemia and 5 minutes reperfusion(IP) followed by subsequent 30 minutes ischemia(ISCH) and 60 minutes reperfusion using isolated Langendorff-Perfused rabbit hearts. Methods and RESULTS: After a 50-minute recovery phase, parameters of the left ventricular function(LVF) including left ventricular developed pressure(LVDP), contractility and the heart rate were recorded, and ultrastructure was examined. Myosin ATPase activity was determined by measurement of inorganic phosphorus and isozymes of the myosin heavy chain were examined by polyacrylamide gel electrophoresis containing pyrophosphate buffer. The ISCH hearts showed severe to irreversible change of the cardiac myocytes homogenously in contrast to the IP hearts in which changes were not homogenous and irreversible injury was only focal. However, parameters of the LVF were not significantly different between the IP and the ISHC hearts during reperfusion. Myosin ATPase activities were also not significantly different(0.67+/-0.123 micromol/mg protein/h in the IP hearts, 0.56+/-0.172 micromol/mg protein/h in the ISCH hearts, and 0.76+/-0.239 micromol/mg protein/h in the control hearts). Band patterns of the myofibrillar proteins, separated by sodium ddodecyl sulfate-polyacrylamide gel electrophoresis, revealed no differences between the IP, ISCH and the control hearts. Myosin heavy chains in the IP and the ISCH hearts were separated into 3 isozymes, V1,V2and V3in pyrophosphate gel electrophoresis in contrast that the control hearts revealed two isozymes, V1and V2. However, there were no differences in the protein composition and electrophoretic motility between the IP and the ISCH hearts. CONCLUSION: These results indicate that IP could not attenuate the changes in LVF, myosin ATPase activity and myosin isozymes on reperfusion, however, it could attenuate the ultrastructural changes of the cardiac myocytes.
Adenosine Triphosphatases* ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Heart Rate ; Heart* ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Myocytes, Cardiac ; Myosin Heavy Chains ; Myosins* ; Phosphorus ; Reperfusion ; Sodium ; Ventricular Function, Left*

BACKGROUND AND OBJECTIVES: We investigated the effects of different concentrations of serum, 5-azacytidine, and culture time on the cardiomyogenic differentiation of P19 embryonal carcinoma stem cells in the course of developing an efficient protocol for generating the cardiomyogenic lineage. MATERIALS AND METHODS: P19 cells were plated at a density of 1x10(6) cells on 10-cm bacterial dishes for 96 hours in the presence of 1% dimethyl sulfoxide to form embryoid bodies. The embryoid bodies were cultured in medium with 2% or 10% fetal bovine serum for an additional 10 or 15 consecutive days in the presence of 0, 1, or 3 microM 5-azacytidine. RESULTS: Quantitative real-time polymerase chain reaction (PCR) analysis showed that the messenger ribonucleic acid (mRNA) expression of cardiac muscle-specific genes, such as GATA4, alpha-actin, alpha-myosin heavy chain, and cardiac troponin T, were significantly higher in the 15-day culture groups than in the 10-day culture groups. Furthermore, the cardiac muscle-specific genes were expressed more in the high-serum groups compared to the low-serum groups regardless of the culture time. Cardiomyogenic differentiation of the P19 cells was most effective in 1 microM 5-azacytidine regardless of the serum concentrations. In addition, the stimulation effects of 5-azacytidine on cardiomyogenic differentiation were more significant under low-serum culture conditions compared to high-serum culture conditions. Cardiomyogenic differentiation of P19 cells was further confirmed by immunostaining with cardiac muscle-specific antibodies. CONCLUSION:Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.
Actins ; Antibodies ; Azacitidine ; Carcinoma, Embryonal ; Cell Differentiation ; Dimethyl Sulfoxide ; Embryoid Bodies ; Embryonal Carcinoma Stem Cells ; Myocytes, Cardiac ; Real-Time Polymerase Chain Reaction ; RNA ; Safrole ; Troponin T ; Ventricular Myosins

OBJECTIVETo investigate the negative regulation of microRNA-1 (miR-1) on L-type calcium channel beta2 subunit (Cavbeta 2) during cardiomyocyte hypertrophy and its mechanism.

METHODSCardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (HJ2000). The targets of miR-1 were predicted by online database microCosm. The 3' untranslated region sequence of Cavbeta 2 was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression of atrial natriuretic peptide (ANP), beta-myosin heavy chain (beta-MHC), miR-1 and the Cavbeta 2 mRNA were detected by qRT-PCR. The protein expression of Cavbeta 2 was detected by Western blot. The level of miR-1 was up-regulated by miR-1 mimic transfection and the expression level of Cavbeta 2 was down-regulated by RNAi, then effects of which on cardiomyocyte hypertrophy were investigated.

RESULTS(1) The expression of miR-1 was significantly reduced in cardiomyocyte hypertrophy. Upregulating the miR-1 level could suppress the increase of cell surface area, the expression of ANP and beta-MHC mRNA (P < 0.05). (2) Cavbeta 2 was the one of potential targets of miR-1 by prediction using online database microCosm. The luciferase activities of HEK293 cells with the plasmid containing miR-1 and wide type Cavbeta 3' UTR sequence was significantly decreased when compared with that of control group (P < 0.01). Up-regulation of the miR-1 level could suppress the protein expression of Cavbeta 2. (3) The expression of Cavbeta 2 was significantly increased in cardiomyocyte hypertrophy induced by ISO. Downregulation of Cavbeta by RNAi could markedly inhibit the increase of cell surface area, the expression of ANP and beta-MHC mRNA.

CONCLUSIONCavbeta2 is one of potential targets of miR-1 by bioinformatics prediction. The experiment data confirms that Cavbeta2 is truly the target of miR-1. MiR-1 can negatively regulate the expression of Cavbeta 2, resulting in the decrease of intracellular Ca2+ content and the attenuation of cardiomyocyte hypertrophy.

Animals ; Atrial Natriuretic Factor ; metabolism ; Calcium Channels, L-Type ; genetics ; Cardiomegaly ; genetics ; Gene Expression Regulation ; HEK293 Cells ; Humans ; MicroRNAs ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection ; Ventricular Myosins ; metabolism

OBJECTIVETo identify the casual mutation of a Chinese pedigree with hypertrophic cardiomyopathy (HCM), and to analyze the genotype-phenotype relationship.

METHODSThe coding exons of 26 reported disease genes were sequenced by targeted resequencing in the proband and the identified mutation were detected with bi-directional Sanger sequencing in all family members and 307 healthy controls. The genotype-phenotype correlation was analyzed in the family.

RESULTSA missense mutation (c.2191C > T, p. Pro731Ser) in the 20th exon of MYH7 gene was identified. This mutation was absent in 307 healthy controls and predicted to be pathogenic by PolyPhen-HCM. Totally 13 family members carried this mutation, including 10 patients with HCM and 3 asymptomatic mutation carriers. The proband manifested severe congestive heart failure and 8 patients expressed various clinical manifestations of heart failure, including dyspnea, palpitations, chest pain, amaurosis or syncope. Five patients were diagnosed as HCM at the age of 16 or younger. One family member suffered sudden cardiac death.

CONCLUSIONSThe Pro731Ser of MYH7 gene mutation is a causal and malignant mutation linked with familiar HCM.

Adolescent ; Asian Continental Ancestry Group ; Base Sequence ; Cardiomyopathy, Hypertrophic ; ethnology ; genetics ; Death, Sudden, Cardiac ; Exons ; Humans ; Mutation, Missense ; Myosin Heavy Chains ; genetics ; Pedigree ; Phenotype ; Research Design ; Ventricular Myosins

BACKGROUND AND OBJECTIVES: Most studies in cardiac regeneration have explored bone marrow mesenchymal stem cells (BM-MSC) with variable therapeutic effects. Amniotic fluid MSC (AF-MSC) having extended self-renewal and multi-potent properties may be superior to bone marrow MSC (BM-MSC). However, a comparison of their cardiomyogenic potency has not been studied yet.METHODS: The 5-azacytidine (5-aza) treated AF-MSC and BM-MSC were evaluated for the expression of GATA-4, Nkx2.5 and ISL-1 transcripts and proteins by quantitative RT-PCR and Western blotting, respectively as well as for the expression of cardiomyogenic differentiation markers cardiac troponin-T (cTNT), beta myosin heavy chain (βMHC) and alpha sarcomeric actinin (ASA) by immunocytochemistry.RESULTS: The AF-MSC as compared to BM-MSC had significantly higher expression of GATA-4 (183.06±29.85 vs. 9.80±0.05; p<0.01), Nkx2.5 (8.3±1.4 vs. 1.82±0.32; p<0.05), and ISL-1 (39.59±4.05 vs. 4.36±0.39; p<0.01) genes as well as GATA-4 (2.01±0.5 vs. 0.6±0.1; p<0.05), NKx2.5 (1.9±0.14 vs. 0.8±0.2; p<0.01) and ISL-1 (1.7±0.3 vs. 0.9±0.1; p<0.05) proteins. The AF-MSC also had significantly elevated expression of cTNT (5.0×10⁴±0.6×10⁴ vs. 3.5 ×10⁴±0.8×10⁴; p<0.01), β-MHC (15.7×10⁴±0.9×10⁴ vs. 8.2×10⁴±0.6×10⁴; p<0.01) and ASA (18.6×10⁴±4.9×10⁴ vs. 13.1×10⁴±3.0×10⁴; p<0.05) than BM-MSC.CONCLUSIONS: Our data suggest that AF-MSC have greater cardiomyogenic potency than BM-MSC, and thus may be a better source of MSC for therapeutic applications in cardiac regenerative medicine.
Actinin ; Amniotic Fluid ; Antigens, Differentiation ; Azacitidine ; Blotting, Western ; Bone Marrow ; Female ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; Regeneration ; Regenerative Medicine ; Therapeutic Uses ; Troponin T ; Ventricular Myosins

BACKGROUND: Through a genome-wide search using the genetic markers(RFLP genetic markers), the familial hypertrophic cardiomyopathy(FHCM) with an autosomal dominant mode of inheritance has been firstly detected to be genetically linked to chromosome 14q1. The subsequent studies have shown that the point mutations at the exons encoding for the head and head /rod junction of the cardiac beta myosin heavy chain(beta-MHC) are the most frequent type of mutation in the FHCM families genetically implicated with a linkage to beta-MHC, whereas the alpha/beta-MHC hybrid gene and a large deletion at the 3' region of beta-MHC gene were also rarely detected. With the other families genetically implicated with the chromosomes 1,11,15,16 and 18, FHCM also manifests locus heterogeneity, a phenomenon in which abnormalities at different genes are involved in different families. In addition, a korean FHCM family with 403Arg-->Gln mutation of beta-MHC gene has been previously found by an american research group. METHODS: For clinical diagnosis, echocardiography and electrocardiography were performed on the individual members of a korean FHCM family. The microsatellite markers(MYO-I,MYO-II) located in the beta-MHC gene region were amplified by PCR(polymerase chain reaction) and the polymorphism was analyzed for the possible linkage to the phenotypic expression of FHCM. Independently, the same PCR products of the exons 13 and 23 were digested with the specific restriction enzymes for the presence of the most frequently reported point mutations of beta-MHC gene (403 and 908 amino acid mutations). Single strand conformation polymorphism(SSCP) of the exon 13 and 23 of the beta-MHC gene was also analyzed of the mobility shift expected if any point mutation is present at these two exons. RESULTS: The inheritance pattern of HCM(hypertrophic cardiomyopathy) in the family is considered as autosomal dominant. In this family(KU 101), one of the microsatellite markers(MYO-II) indicated the possible cosegregation between the allele was also present in the 32-year-old brother of the proband, who reveals no clinical signs of the disease. The other microsatellite genetic marker(MYO-I) was uninformative, without giving the discriminating power to verify the linkage to beta-MHC gene. In the analysis for two common mutations of beta-MHC gene by PCR-RFLP and PCR-SSCP, no evidence was found for 403 and 908 amino acid mutations and any point mutation in the exons 13 and 23. CONCLUSIONS: Based on the linkage analysis using microsatellite genetic markers, there was a possibility that the disease could be linked to an abnormality in the beta-MHC gene of the chromosome 14q1.
Adult ; Alleles ; Cardiomyopathy, Hypertrophic, Familial* ; Diagnosis ; Echocardiography ; Electrocardiography ; Exons ; Genetic Markers ; Head ; Humans ; Inheritance Patterns ; Mass Screening* ; Microsatellite Repeats ; Point Mutation ; Polymerase Chain Reaction ; Population Characteristics ; Siblings ; Ventricular Myosins* ; Wills

BACKGROUND: Through a genome-wide search using the genetic markers(RFLP genetic markers), the familial hypertrophic cardiomyopathy(FHCM) with an autosomal dominant mode of inheritance has been firstly detected to be genetically linked to chromosome 14q1. The subsequent studies have shown that the point mutations at the exons encoding for the head and head /rod junction of the cardiac beta myosin heavy chain(beta-MHC) are the most frequent type of mutation in the FHCM families genetically implicated with a linkage to beta-MHC, whereas the alpha/beta-MHC hybrid gene and a large deletion at the 3' region of beta-MHC gene were also rarely detected. With the other families genetically implicated with the chromosomes 1,11,15,16 and 18, FHCM also manifests locus heterogeneity, a phenomenon in which abnormalities at different genes are involved in different families. In addition, a korean FHCM family with 403Arg-->Gln mutation of beta-MHC gene has been previously found by an american research group. METHODS: For clinical diagnosis, echocardiography and electrocardiography were performed on the individual members of a korean FHCM family. The microsatellite markers(MYO-I,MYO-II) located in the beta-MHC gene region were amplified by PCR(polymerase chain reaction) and the polymorphism was analyzed for the possible linkage to the phenotypic expression of FHCM. Independently, the same PCR products of the exons 13 and 23 were digested with the specific restriction enzymes for the presence of the most frequently reported point mutations of beta-MHC gene (403 and 908 amino acid mutations). Single strand conformation polymorphism(SSCP) of the exon 13 and 23 of the beta-MHC gene was also analyzed of the mobility shift expected if any point mutation is present at these two exons. RESULTS: The inheritance pattern of HCM(hypertrophic cardiomyopathy) in the family is considered as autosomal dominant. In this family(KU 101), one of the microsatellite markers(MYO-II) indicated the possible cosegregation between the allele was also present in the 32-year-old brother of the proband, who reveals no clinical signs of the disease. The other microsatellite genetic marker(MYO-I) was uninformative, without giving the discriminating power to verify the linkage to beta-MHC gene. In the analysis for two common mutations of beta-MHC gene by PCR-RFLP and PCR-SSCP, no evidence was found for 403 and 908 amino acid mutations and any point mutation in the exons 13 and 23. CONCLUSIONS: Based on the linkage analysis using microsatellite genetic markers, there was a possibility that the disease could be linked to an abnormality in the beta-MHC gene of the chromosome 14q1.
Adult ; Alleles ; Cardiomyopathy, Hypertrophic, Familial* ; Diagnosis ; Echocardiography ; Electrocardiography ; Exons ; Genetic Markers ; Head ; Humans ; Inheritance Patterns ; Mass Screening* ; Microsatellite Repeats ; Point Mutation ; Polymerase Chain Reaction ; Population Characteristics ; Siblings ; Ventricular Myosins* ; Wills