[The molecular basis of cardiac contractility].
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Cardiomyopathy, Hypertrophic↗
Biomedical subjects
V Mahdavi
Publications and source records attributed to V Mahdavi.
Rapid and reversible changes in myosin heavy chain expression in response to increased neuromuscular activity of rat fast-twitch muscle.
Chronic 10 Hz stimulation of rat fast-twitch muscle induced rapid and reversible changes in the tissue levels of fast myosin heavy chain (HC) mRNA isoforms. These changes consisted of a rapid decrease in HCIIb mRNA and a progressive increase in HCIIa mRNA. After 15 days, the HCIIb mRNA normally amounting to approximately 80%, had decreased to less than 5% of the sum of the two HC mRNA isoforms. HCIIb mRNA was again detectable one day after cessation of stimulation and progressively increased at the expense of HCIIa mRNA with ongoing recovery. These results point to a down-regulation of the HCIIb gene by the applied stimulus pattern which, conversely, enhances the expression of the HCIIa gene.
Animals↗
Molecular basis of cardiac performance. Plasticity of the myocardium generated through protein isoform switches.
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Animals↗
Thyroid hormone receptor alpha isoforms generated by alternative splicing differentially activate myosin HC gene transcription.
Thyroid hormones are thought to modulate gene expression positively or negatively through interactions with chromatin-associated receptors. Recently, the c-erb A proto-oncogene products have been shown to be nuclear thyroid hormone (T3) receptors (TR) by sequence similarity with other steroid receptors and by their ability to bind thyroid hormone. But it has not been shown that these receptors directly activate transcription of the responsive genes in vivo. In addition, the rat TR alpha gene encodes several messenger RNA (mRNA) species, generated by differential processing of its transcripts (ref. 22). For these reasons we investigated the ability of two major isoforms of the rat TR alpha gene products to activate transcription of a sarcomeric myosin heavy chain (mHC) gene, because expression of all members of this gene family is responsive to T3. We show here that the rTR alpha 1 receptor is a thyroid hormone-dependent transcriptional factor, which upon binding the T3 responsive element of the alpha-mHC gene, activates expression of this gene in vivo. The rTR alpha 2 isoform, which is identical to rTR alpha 1 except for its carboxyl terminal portion, is generated by alternative splicing of the rTR alpha gene transcript. This peptide, when produced in vitro and in vivo failed to bind T3 or other hormones or to trans-activate alpha-mHC gene expression. Thus, alternative splicing can produce marked differences in the functional properties of a transcriptional factor.
Acetyltransferases↗
Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload.
Hypertrophy, an increase in cell size without cell division, is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle cells. Cardiac myosins undergo an adult-to-fetal isoform transition in various models of hypertrophy. Using gene-specific cDNA probes, we show here that in the adult myocardium the mRNAs encoding the fetal (skeletal muscle type) isoforms of alpha-actin and sarcomeric tropomyosin are re-expressed within 2 days in response to pressure overload. In addition, atrial natriuretic factor mRNA, so far believed to be expressed primarily in the atria, was readily detectable in the ventricles of neonates and was induced to markedly high levels in pressure-overloaded adult ventricles. In contrast, cardiac hypertrophy produced by thyroid hormone excess was not associated with induction of the atrial natriuretic factor gene or fetal contractile protein isogenes. Furthermore, the c-fos and c-myc protooncogenes and a major heat shock protein gene (hsp70) are induced in the ventricular myocardium within 1 hr after imposition of pressure overload. These results suggest that induction of cellular protooncogenes and heat shock (stress) protein genes is an early response to pressure overload, whereas reinduction of the genes normally expressed only in perinatal life, such as fetal isoforms of contractile proteins and atrial natriuretic factor, is a later event. These two types of responses might represent the general pattern of growth induction to work overload by terminally differentiated cells that have lost the ability to undergo DNA replication.
Aging↗
Multiple positive and negative 5' regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene.
To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins.
Animals↗
Developmental and hormonal regulation of sarcomeric myosin heavy chain gene family.
Sarcomeric myosin heavy chain (MHC), the main component of the sarcomere, contains the ATPase activity that generates the contractile force of cardiac and skeletal muscles. The different MHC isoforms are encoded by a closely related multigene family. Most members (seven) of this gene family have been isolated and characterized in the rat, including the alpha- and beta-cardiac, skeletal embryonic, neonatal, fast IIA, fast IIB, and extraocular specific MHC. The slow type I skeletal MHC is encoded by the same gene that codes for the cardiac beta-MHC. Each MHC gene studied displays a pattern of expression that is tissue and developmental stage specific, both in cardiac and skeletal muscles. Furthermore, more than one MHC gene is expressed in each muscle while each gene is expressed in more than one tissue. The expression of each MHC gene in cardiac and skeletal muscles is modulated by thyroid hormone. Surprisingly, however, the same MHC gene can be regulated by the hormone in a significantly different manner, even in opposite directions, depending on the muscle in which it is expressed. Moreover, the skeletal embryonic and neonatal MHC genes, so far considered specific to these 2 developmental stages, are normally expressed in certain adult muscles and can be reinduced by hypothyroidism in specific muscles. This complex pattern of expression and regulation of the MHC gene family in cardiac and skeletal muscle sheds new light on the mechanisms involved in determining the biochemical basis of the contractile state. It also indicates that the cardiac contractile system needs to be examined in a broader context, including skeletal muscles, in order to understand fully its developmental and physiologic regulation.
Animals↗
Myosin heavy chain messenger RNA and protein isoform transitions during cardiac hypertrophy. Interaction between hemodynamic and thyroid hormone-induced signals.
Expression of the cardiac myosin isozymes is regulated during development, by hormonal stimuli and hemodynamic load. In this study, the levels of expression of the two isoforms (alpha and beta) of myosin heavy chain (MHC) during cardiac hypertrophy were investigated at the messenger RNA (mRNA) and protein levels. In normal control and sham-operated rats, the alpha-MHC mRNA predominated in the ventricular myocardium. In response to aortic coarctation, there was a rapid induction of the beta-MHC mRNA followed by the appearance of comparable levels of the beta-MHC protein in parallel to an increase in the left ventricular weight. Administration of thyroxine to coarctated animals caused a rapid deinduction of beta-MHC and induction of alpha-MHC, both at the mRNA and protein levels, despite progression of left ventricular hypertrophy. These results suggest that the MHC isozyme transition during hemodynamic overload is mainly regulated by pretranslational mechanisms, and that a complex interplay exists between hemodynamic and hormonal stimuli in MHC gene expression.
Animals↗
All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner.
In mammals different isoforms of myosin heavy chain are encoded by the members of a multigene family. The expression of each gene of this family is regulated in a tissue- and developmental stage-specific manner as well as by hormonal and various pathological stimuli. In this study the molecular basis of isoform switches induced in myosin heavy chain by thyroid hormone was investigated. The expression of the myosin heavy chain gene family was analyzed in seven different muscles of adult rats subjected to hypo- or hyperthyroidism with complementary DNA probes specific for six different myosin heavy chain genes. The results demonstrate that all six genes are responsive to thyroid hormone. More interestingly, the same myosin heavy chain gene can be regulated by thyroid hormone in highly different modes, even in opposite directions, depending on the tissue in which it is expressed. Furthermore, the skeletal embryonic and neonatal myosin heavy chain genes, so far considered specific to these two developmental stages, can be reinduced by hypothyroidism in specific adult muscles.
Animals↗
Sarcomeric myosin heavy chain gene family: organization and pattern of expression.
The sarcomeric myosin heavy chains (MHCs), which exhibit different levels of ATPase activity, are encoded by a closely related multigene family from which seven members have been identified and characterized in the rat. The MHC genes appear to map to a single chromosome, and at least two of them, alpha- and beta-cardiac, are closely linked in the genome. Each of these genes is approximately 25 kilobases long, and their coding sequences are interrupted by 40 introns. Each MHC gene displays a pattern of expression that is tissue-specific and developmentally regulated, with more than one MHC gene expressed in each muscle and developmental stage. Moreover, with the exception of the extra-ocular muscle MHC gene that has a very specific pattern of expression, the other genes are all expressed in more than one tissue. The expression of all MHC genes can be modulated by thyroid hormone. Surprisingly, however, the same myosin heavy chain gene can be regulated by thyroid hormone in highly different modes, even in opposite directions, depending on the tissue in which it is expressed. Furthermore, the skeletal embryonic and neonatal myosin heavy chain genes, so far considered specific to these two developmental stages, can be re-induced by hypothyroidism in specific adult muscles.
Animals↗
Intron positions are conserved in the 5' end region of myosin heavy-chain genes.
We have determined the 5' end sequence of the rat embryonic skeletal muscle myosin heavy-chain (MHC) gene comprising the first three amino-terminal coding exons. Comparison with the corresponding regions of the rat ventricular alpha and the nematode Caenorhabditis elegans unc-54 MHC genes (Mahdavi, V., Chambers, A.P., and Nadal-Ginard, B. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2626-2630; Karn, J., Brenner, S., and Barnett, L. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4253-4257) shows that the degree of amino acid sequence conservation increases from the first to the third exon. Intron positions between these exons are maintained in all three genes studied, whereas size and sequence of corresponding introns are highly divergent. In contrast to the rat MHC genes where the coding region is highly split throughout its entire length, only the 5' end region is frequently interrupted by introns in the nematode gene indicating the potential importance of these introns in gene structure and expression. The occurrence of "preferential" intron positions in the MHC genes suggests the existence of a highly split ancestral MHC gene from which different evolutionary lineages removed and/or added specific sets of introns.
Amino Acid Sequence↗
Intricate combinatorial patterns of exon splicing generate multiple regulated troponin T isoforms from a single gene.
Mechanisms of alternative RNA splicing, important in the generation of protein diversity, are common but incompletely understood. Among the contractile proteins, troponin T exists in several isoforms, shown to be derived in part from a novel pattern of differential RNA splicing in the 3' region of the rat skeletal fast troponin T gene. In fact, this gene has a previously unsuspected capacity to encode multiple isoforms. The isolation of four distinct but related cDNAs from this gene, which share discontinuous subsegments of sequence identity in their 5' regions, and the determination of the genomic sequence, demonstrate that small exons with characteristic split codon structure are differentially spliced in intricate combinatorial patterns to generate a minimum of 10, and potentially 64, distinct troponin T mRNAs, encoding different isoforms, in a developmentally regulated and tissue-specific manner. At least two of these mRNAs are spliced from structurally identical primary transcripts, necessitating control by trans-acting factors.
Amino Acid Sequence↗
Expression of the cardiac ventricular alpha- and beta-myosin heavy chain genes is developmentally and hormonally regulated.
The cardiac ventricular myosin phenotype is developmentally and hormonally regulated. The genes coding for the two myosin heavy chains ( MHCs ), alpha and beta, have been recently isolated and characterized. In this study, we establish the precise temporal expression of these MHC genes in correlation with the myosin phenotype both during cardiac development and in response to different thyroid hormone levels and also document their expression in other muscle tissues. The close correlation observed between the relative abundance of the alpha- and beta-MHC mRNAs and corresponding isozymes demonstrates that the MHC phenotype is produced by the expression of the alpha- and beta-MHC genes and is regulated by changes in the level of their respective mRNAs. The opposite effect of thyroid hormone on the expression of the alpha- and beta-MHC genes in the ventricular myocardium indicates that these genes are regulated in an antithetic fashion. Finally, the MHC mRNAs encoded by the alpha- and beta-MHC genes are also present in the atrial myocardium and in the soleus, respectively.
Animals↗
Cardiac alpha- and beta-myosin heavy chain genes are organized in tandem.
Two ventricular myosin heavy chains (MHCs), alpha and beta, which exhibit different levels of ATPase activity, are differentially expressed during development, in response to thyroid hormone and in several pathological conditions. We have isolated and analyzed the structure of the genes coding for alpha- and beta-MHC mRNAs in the rat. Detailed analysis of eight overlapping MHC genomic clones shows that the alpha- and beta-MHC genes are organized in tandem and span 50 kilobases of the chromosome. The beta-MHC gene, predominantly expressed in late fetal life, is located 4 kilobases upstream from the alpha-MHC gene, predominantly expressed in the adult. These two genes are very closely related at the nucleotide sequence level, suggesting that they have arisen by duplication of a common ancestor, yet their expression in the ventricular myocardium has been shown to be regulated in an antithetic fashion by thyroid hormone.
Animals↗
Cardiac myosin heavy chain isozymic transitions during development and under pathological conditions are regulated at the level of mRNA availability.
We have shown that the level of two ventricular MHC mRNAs, fetal and adult, can account for the observed isozymic transitions of the myosin protein during normal development and under pathological conditions. Moreover, these MHC mRNAs are encoded by two genes that are linked in the genome, less than 5 kilobases apart and are organized according to their developmental expression. The fact that the two ventricular MHC genes are very closely related at the nucleotide sequence level, and yet respond in opposite direction to the same stimulus, makes these genes an excellent model in which to study the possible mechanisms involved in the gene switching.
Animals↗
Molecular characterization of two myosin heavy chain genes expressed in the adult heart.
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Amino Acid Sequence↗
Proteolytic enzymes in normal and transformed cells.
Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62,000 was increased 5--8-fold on transformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.
Animals↗
Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.
Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.
Animals↗