Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells

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.

Efectos de claritromicina sobre el Intervalo QT corregido, en pacientes añosos hospitalizados / Comparative Pharmacodynamic analysis of QT interval prolongation. Induced by Clarithromycin in elderly hospitalized patients

Se estableció los efectos de la claritromicina sobre el intervalo QT corregido (iQTc) en pacientes gerontes, que requirieron hospitalización por infección respiratoria, mediante registro electrocardiográfico al inicio y al final del tratamiento. Se observó en 61% de los casos un aumento del iQTC (0,04 seg en promedio). No obstante las comorbilidades asociadas, ningún caso evolucionó a taquicardia ventricular polimórfica

The effects of clarithromycin on the corrected QT interval (iQTc) in elderly patients, who required hospitalization due to respiratory infection, were established by electrocardiographic recording at the beginning and at the end of the treatment. An increase in iQTC was observed in all cases (0.04 sec on average). Despite the associated comorbidities, no case evolved to polymorphic ventricular tachycardia

Mutation and clinical relevance in a large cohort of unrelated Chinese patients with hypertrophic cardiomyopathy / 中华心血管病杂志

<p><b>OBJECTIVE</b>To explore the genetic basis and phenotypic correlation with disease severity in a large cohort of Chinese patients with hypertrophic cardiomyopathy (HCM).</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>A 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).</p><p><b>CONCLUSION</b>MYBPC3 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.</p>

Pro731Ser mutation in the β-myosin heavy chain and hypertrophic cardiomyopathy in a Chinese pedigree / 中华心血管病杂志

<p><b>OBJECTIVE</b>To identify the casual mutation of a Chinese pedigree with hypertrophic cardiomyopathy (HCM), and to analyze the genotype-phenotype relationship.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSIONS</b>The Pro731Ser of MYH7 gene mutation is a causal and malignant mutation linked with familiar HCM.</p>

Effects of microRNA-1 on negatively regulating L-type calcium channel beta2 subunit gene expression during cardiac hypertrophy / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the negative regulation of microRNA-1 (miR-1) on L-type calcium channel beta2 subunit (Cavbeta 2) during cardiomyocyte hypertrophy and its mechanism.</p><p><b>METHODS</b>Cardiomyocyte 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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>Cavbeta2 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.</p>

Tension cost correlates with mechanical and biochemical parameters in different myocardial contractility conditions

OBJECTIVES: Tension cost, the ratio of myosin ATPase activity to tension, reflects the economy of tension development in the myocardium. To evaluate the mechanical advantage represented by the tension cost, we studied papillary muscle contractility and the activity of myosin ATPase in the left ventricles in normal and pathophysiological conditions. METHODS: Experimental protocols were performed using rat left ventricles from: (1) streptozotocin-induced diabetic and control Wistar rats; (2) N-nitro-L-arginine methyl ester (L-NAME) hypertensive and untreated Wistar rats; (3) deoxycorticosterone acetate (DOCA) salt-treated, nephrectomized and salt- and DOCA-treated rats; (4) spontaneous hypertensive rats (SHR) and Wistar Kyoto (WKY) rats; (5) rats with myocardial infarction and shamoperated rats. The isometric force, tetanic tension, and the activity of myosin ATPase were measured. RESULTS: The results obtained from infarcted, diabetic, and deoxycorticosterone acetate-salt-treated rats showed reductions in twitch and tetanic tension compared to the control and sham-operated groups. Twitch and tetanic tension increased in the N-nitro-L-arginine methyl ester-treated rats compared with the Wistar rats. Myosin ATPase activity was depressed in the infarcted, diabetic, and deoxycorticosterone acetate salt-treated rats compared with control and sham-operated rats and was increased in N-nitro-L-arginine methyl ester-treated rats. These parameters did not differ between SHR and WKY rats. In the studied conditions (e.g., post-myocardial infarction, deoxycorticosterone acetate salt-induced hypertension, chronic N-nitro-L-arginine methyl ester treatment, and streptozotocin-induced diabetes), a positive correlation between force or plateau tetanic tension and myosin ATPase activity was observed. CONCLUSION: Our results suggest that the myocardium adapts to force generation by increasing or reducing the tension cost to maintain myocardial contractility with a better mechanical advantage.

Enhanced Cardiomyogenic Differentiation of P19 Embryonal Carcinoma Stem Cells

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.

Novel Val606Met mutation in beta myosin heavy chain gene in Chinese pedigrees with familiar hypertrophic cardiomyopathy / 中华心血管病杂志

<p><b>OBJECTIVE</b>To screen the disease-causing gene mutation in Chinese patients with familiar hypertrophic cardiomyopathy (HCM) and to analyse the correlation between the genotype and phenotype.</p><p><b>METHODS</b>Eight Chinese pedigrees with HCM and 80 age-matched normal control subjects were studied. The exons in the functional regions of the beta myosin heavy chain gene (beta-MHC) were amplified with PCR and the products were sequenced. The relation between the genotype and phenotype was analyzed.</p><p><b>RESULT</b>Val606Met mutation was identified in exon 16 in one family and Val606Met mutation was identified in 4 out of 8 family members in this pedigree and 3 out of 4 Val606Met carriers suffered from HCM. No similar mutation was identified in controls.</p><p><b>CONCLUSION</b>The Val606Met mutation located at the actin-binding region of the cardiac beta-MHC gene is involved in the pathogenesis of HCM in this Chinese pedigree.</p>

Mutation of Arg723Gly in beta-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hypertrophic 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.</p>

Pesquisa de marcadores para os genes da cadeia pesada da beta-miosina cardíaca e da proteína C de ligacão à miosina em familiares de pacientes com cardiomiopatia hipertrófica / Research of markers for the genes of the heavy chain of cardiac beta-myosin and myosin binding protein C in relatives of patients with hypertrophic cardiomyopathy

OBJETIVO: Estudar os marcadores moleculares para os genes da cadeia pesada da beta-miosina cardíaca e da proteína-C de ligacão à miosina em familiares de portadores de cardiomiopatia hipertrófica. MÉTODOS: Foram estudadas 12 famílias que realizaram anamnese, exame físico, eletrocardiograma, ecocardiograma e coleta de sangue para o estudo genético através da reacão em cadeia da polimerasse. RESULTADOS: Dos 227 familiares 25 por cento eram acometidos, sendo 51 por cento do sexo masculino com idade média de 35n19 (2 a 95) anos. A análise genética mostrou ligacão com o gene da b-miosina cardíaca em uma família e, em outra, ligacão com o gene da proteína C de ligacão à miosina. Em cinco famílias foram excluídas ligacões com os dois genes; em duas, a ligacão com o gene da proteína C de ligacão à miosina, porém para o gene da b-miosina os resultados foram inconclusivos; em duas famílias os resultados foram inconclusivos para os dois genes e em uma foi excluída ligacão para o gene da b-miosina mas ficou inconclusivo para o gene da proteína C de ligacão à miosina. CONCLUSAO: Em nosso meio, talvez predominem outros genes que não aqueles descritos na literatura, ou que existam outras diferencas genéticas relacionadas com a origem de nossa populacão e/ou fatores ambientais.

Long-term effects of TCV116 on cardiac function changes after myocardial infarction / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the long-term effects of TCV116 (candesartan cilexetil) on cardiac function changes after myocardial infarction.</p><p><b>METHODS</b>Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in rats. One week after the surgical performance,the surviving rats were randomly assigned to the following treatment groups: (1) MI rats with no therapy; (2) MI rats treated with TCV116 2 mg/kg per day; (3) Sham-operated control and (4) Sham-operated rats treated with TCV116 2 mg/kg per day. At 22 weeks, left ventricular function and cardiac histomorphometric parameters were measured, mRNA expression of cardiac genes such as beta myosin heavy chain, B-type natriuretic peptide, transforming growth factor beta1, collagen I and III quantified, and survival rates calculated.</p><p><b>RESULTS</b>Treatment with TCV116 significantly improved LV function, suppressed mRNA expression of cardiac genes,and extended the survival period compared with MI rats with no therapy (P<0.05).</p><p><b>CONCLUSION</b>Treatment with long-term angiotensin II type 1 receptor blocker may improve LV function and prolong the survival of rats after MI.</p>

Thyroid hormone-induced alterations of Ca2+-ATPase and phospholamban protein expression in cardiac sarcoplasmic reticulum

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.

Screening of the Cardiac Beta Myosin Heavy Chain Gene for the Linkage to Familial Hypertrophic Cardiomyopathy in a Korean Family

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.

Screening of the Cardiac Beta Myosin Heavy Chain Gene for the Linkage to Familial Hypertrophic Cardiomyopathy in a Korean Family

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.