β-MHC Expression Research Articles

Characterization of naturally occurring myosin heavy chain antisense mRNA in rat heart

Analysis of mRNA by Northern blot and reverse transcription–polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for β-myosin heavy chain (MHC) and α-MHC in the neonatal rat heart: antisense MHC mRNA expression of α-MHC and β-MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the β-MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and β-MHC consisted of approximately 2,700 bp, suggesting a higher probability of α-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of α-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of α-MHC was 52.0 ± 2.6% on the sense mRNA but only 36.3 ± 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart. J. Cell. Biochem. 70:110–120, 1998. © 1998 Wiley-Liss, Inc.

Abstract

Familial hypertrophic cardiomyopathy (FHC) is a genetically and phenotypically heterogeneous disease with an autosomal dominant Mendelian inheritance. Mutations in four different genes coding for sarcomeric proteins have been identified to cause the disease. In 1990, a mutation in the β-myosin heavy chain (βMHC) gene was identified to cause familial hypertrophic cardiomyopathy. Soon after, more than 30 missense mutations and one deletion in the βMHC gene were identified to be responsible for FHC. This was followed by mutations in troponin T, α tropomyosin and cardiac myosin binding protein-C genes. In patients with familial hypertrophic cardiomyopathy due to βMHC mutations, the mutant βMHC mRNA and protein have been isolated from cardiac and skeletal muscle of these individuals. Functional studies have shown that the mutant βMHC protein has impaired actin-myosin interaction and expression of the mutant βMHC in feline cardiocytes disrupts the assembly of the sarcomere. Similarly, mutations in the other three genes lead to mutant proteins which interfere with the normal structure and function of the sarcomere, resulting in an identical phenotype of sarcomere disarray, the hallmark of FHC.

Molecular genetics of familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is a genetically and phenotypically heterogeneous disease with an autosomal dominant Mendelian inheritance. Mutations in four different genes coding for sarcomeric proteins have been identified to cause the disease. In 1990, a mutation in the β-myosin heavy chain (βMHC) gene was identified to cause familial hypertrophic cardiomyopathy. Soon after, more than 30 missense mutations and one deletion in the βMHC gene were identified to be responsible for FHC. This was followed by mutations in troponin T, α tropomyosin and cardiac myosin binding protein-C genes. In patients with familial hypertrophic cardiomyopathy due to βMHC mutations, the mutant βMHC mRNA and protein have been isolated from cardiac and skeletal muscle of these individuals. Functional studies have shown that the mutant βMHC protein has impaired actin-myosin interaction and expression of the mutant βMHC in feline cardiocytes disrupts the assembly of the sarcomere. Similarly, mutations in the other three genes lead to mutant proteins which interfere with the normal structure and function of the sarcomere, resulting in an identical phenotype of sarcomere disarray, the hallmark of FHC.

The effect of endotoxin on skeletal muscle protein gene expression in the rat

Sepsis is associated with net breakdown of skeletal muscle protein, mediated partly by reduced rates of muscle protein synthesis. This study investigated the role of altered gene expression for specific muscle proteins in mediating reduced protein synthesis in a rat model of acute severe sepsis. Adult rats were given a single sublethal intraperitoneal dose of endotoxin (bacterial lipopolysaccharide). Protein, RNA and DNA contents of muscle were meaured and changes in expression of mRNA in tibialis anterior and extensor digitorum longus muscles were detected by quantification of Northern blots at 6, 24, 48 and 72 hr after endotoxin and in animals starved for 24 hr. Results showed that at 24 hr after endotoxin there was loss of about 14% of muscle protein content. No reduction in mRNA was found at any time point for β-myosin heavy chain (MHC), fast-MHC, α-actin, skeletal muscle troponin or carbonic anhydrase III (CA III); rather, at 48 hr there was increased expression of β-MHC (224 ± 123% control) and CA III (202 ± 56%). Blocking TNF-α by pre-treatment with a monoclonal antibody did not appear to influence this. Total RNA content of muscle was reduced to 67% of the control values 24 hr after LPS, although this was no different to pair-fed animals starved for 24 hr. It is concluded that reduced protein synthesis in skeletal muscle in early acute sepsis is not primarily associated with reduced muscle protein gene expression.

Inhibition by angiotensin II type 1 receptor antagonist of cardiac phenotypic modulation after myocardial infarction

The purpose of this study was to examine the cardiac phenotype and remodeling after myocardial infarction and the effect of the angiotensin II type 1 (AT1) receptor antagonist (TCV-116) on the gene expression. Myocardial infarction in rats was produced by ligation of the coronary artery. TCV-116 (10 mg/kg/day) was administered orally to rats from 1 day after myocardial infarction. At 1, 2 and 3 weeks after myocardial infarction, blood pressure and heart rate were measured, and the heart was removed. The left ventricle was measured for infarct size and weight, and then the total RNA from the non-ischemic left ventricle was extracted. mRNAs in the non-ischemic left ventricle were measured by Northern blot analysis. The weight of the non-ischemic left ventricle was significantly increased 3 weeks after infarction. This was completely prevented by TCV 116 treatment. mRNA levels for β-myosin heavy chain (β-MHC), atrial natriuretic polypeptide (ANP), collagen types I and III and transforming growth factor-β1 (TGF-β1) in the non-ischemic left ventricle were increased by a factor of 3.0, 6.7, 7.9, 4.0 and 1.4 ( P<0.01), respectively, 1 week after infarction. There was no increase in a-skeletal actin mRNA at 1 and 2 weeks, but it was increased by a factor of 2.9 ( P<0.05) at 3 weeks. On the other hand, there was no change in α-MHC mRNA during the 3 weeks. TCV-116 significantly suppressed the increased gene expression of β-MHC and α-skeletal actin in the non-ischemic myocardium at all time points, and also suppressed the expression of ANP at 2 and 3 weeks. However, TCV-116 failed to inhibit the expression of collagen I and III mRNAS at 1 and 3 weeks. These results show that myocardial infarction causes a rapid shift of myocytes to fetal phenotype and a rapid activation of collagen genes in the non-ischemic myocardium. AT 1 receptor may be responsible for the phenotypic modulation of myocotes following myocardial infarction.

Thyroid Hormone Influences Beta Myosin Heavy Chain (βMHC) Expression

3,5,3′-Triiodo-L-thyronine (T 3) regulation of β-MHC expression was studied in rat fetal heart cells using deletion mutants of both the rat and human promoter regions fused to a CAT expression vector. T 3 inhibited the expression of human and rat β-MHC constructs with an IC 50 of about 1nM, which was similar to the EC 50 for βMHC-mRNA observed in cardiomyocytes. Deletion analysis of βMHC promoter constructs suggested that a T 3 response element (TRE) is located near the start of transcription. Possibly, T 3-receptor binding at this site interferes with formation of the transcriptional initiation complex.