Expression Of Myosin Light Chain Research Articles

Myogenin and oxidative enzyme gene expression levels are elevated in rat soleus muscles after endurance training.

The intent of this study was to determine whether endurance exercise training regulates increases in metabolic enzymes, which parallel modulations of myogenin and MyoD in skeletal muscle of rats. Adult Sprague-Dawley rats were endurance trained (TR) 5 days weekly for 8 wk on a motorized treadmill. They were killed 48 h after their last bout of exercise. Sedentary control (Con) rats were killed at the same time as TR animals. Myogenin, MyoD, citrate synthase (CS), cytochrome-c oxidase (COX) subunits II and VI, lactate dehydrogenase (LDH), and myosin light chain mRNA contents were determined in soleus muscles by using RT-PCR. Myogenin mRNA content was also estimated by using dot-blot hybridization. Protein expression levels of myogenin and MyoD were measured by Western blots. CS enzymatic activity was also measured. RT-PCR measurements showed that the mRNA contents of myogenin, CS, COX II, COX VI, and LDH were 25, 20, 17, 16, and 18% greater, respectively, in TR animals compared with Con animals (P < 0.05). The ratio of myogenin to MyoD mRNA content estimated by RT-PCR in TR animals was 28% higher than that in Con animals (P < 0.05). Myosin light chain expression was similar in Con and TR muscles. Results from dot-blot hybridization to a riboprobe further confirmed the increase in myogenin mRNA level in TR group. Western blot analysis indicated a 24% greater level of myogenin protein in TR animals compared with Con animals (P < 0.01). The soleus muscles from TR animals had a 25% greater CS enzymatic activity than the Con animals (P < 0.01). Moreover, myogenin mRNA and protein contents were positively correlated to CS activity and mRNA contents of CS, COX II, and COX VI (P < 0.05). These data are consistent with the hypothesis that myogenin is in the pathway for exercise-induced changes in mitochondrial enzymes.

Reciprocal Inhibition between MyoD and STAT3 in the Regulation of Growth and Differentiation of Myoblasts

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.

A dual role of the GTPase Rac in cardiac differentiation of stem cells.

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.

Microarray expression analysis of effects of exercise training: increase in atrial MLC-1 in rat ventricles.

Previous studies have shown that endurance exercise training increases myocardial contractility. We have previously described training-induced alterations in myocardial contractile function at the cellular level, including an increase in the Ca(2+) sensitivity of tension. To determine the molecular mechanism(s) of these changes, oligonucleotide microarrays were used to analyze the gene expression profile in ventricles from endurance-trained rats. We used an 11-wk treadmill training protocol that we have previously shown results in increased contractility in cardiac myocytes. After the training, the hearts were removed and RNA was isolated from the ventricles of nine trained and nine control rats. With the use of an Affymetrix Rat Genome U34A Array, we detected altered expression of 27 genes. Several genes previously found to have increased expression in hypertrophied myocardium, such as atrial natriuretic factor and skeletal alpha-actin, were decreased with training in this study. From the standpoint of altered contractile performance, the most significant finding was an increase in the expression of atrial myosin light chain 1 (aMLC-1) in the trained ventricular tissue. We confirmed microarray results for aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca(2+) sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may be responsible, at least in part, for previously observed training-induced enhancement of contractile function.

Targeted Deletion of the MLC1f/3f Downstream Enhancer Results in Precocious MLC Expression and Mesoderm Ablation

The expression of skeletal muscle contractile proteins is tightly regulated during embryonic development. In the mouse, the myosin light chain (MLC) 1f/3f gene locus is not activated until E9.5, exclusively in skeletal muscle precursor cells. A potent enhancer downstream of the MLC1f/3f locus confers correct temporal and spatial activation of linked reporter gene in transgenic mouse embryos. To examine roles of the MLC downstream enhancer (MLCE) in its native context of the MLC1f/3f gene locus, we eliminated a 1.5-kb DNA segment containing the enhancer from the mouse genome by targeted deletion, leaving no exogenous sequences at the deletion site. Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by E7.5, and were resorbed almost completely at E8.5. In situ hybridization and RT-PCR analyses of affected mutant embryos at E7.5 revealed ectopic MLC transcripts, whose products would be predicted to interfere with a variety of nonmuscle cell functions determining differentiation of mesoderm. These results suggest that the MLC downstream enhancer and its flanking sequences include negative regulatory elements which block precocious activation of MLC expression in mesodermal precursors during a critical window of development, as well as positive elements which subsequently permit tissue-restricted MLC transcription in differentiating skeletal muscles.

Impact of HMG CoA reductase inhibition on small GTPases in the heart.

Members of the Rho GTPase family, Rac1 and RhoA have been suggested to be mediators of cardiac hypertrophy in mice. Rho proteins are posttranslationally isoprenylated. In addition to cholesterol-lowering, statins inhibit the isoprenylation of small G proteins. Therefore, it was tested if these drugs inhibit Rac1 and RhoA activity in cardiomyocytes and, thereby, prevent angiotensin II-mediated expression of atrial natriuretic factor (ANF) and myosin light chain (MLC)-2 in the heart. Western and Northern analysis of rat neonatal cardiomyocytes and H9C2 cells showed inhibition of basal and angiotensin-stimulated Rac1 expression, membrane-translocation and activity after statin treatment. Similarly, basal and stimulated RhoA membrane expression was inhibited. Statins concentration- and time-dependently downregulated basal as well as angiotensin-induced expression of ANF by 86+/-2.3% and 89+/-1.7%, as well as MLC-2 by 75+/-4.1% and 84+/-6%, respectively. Direct inhibition of Rac GTPase by overexpression of the dominant negative mutant RacN17 or by Clostridium sordellii lethal toxin in rat H9C2 cells inhibited ANF expression by 70+/-4.9% and 78+/-10%, respectively. Inhibition of RhoA by Clostridium botulinum C3 transferase or the dominant negative mutant RhoN19 reduced ANF mRNA by 19+/-11% and 23+/-8%, respectively. To test these findings in vivo, spontaneously hypertensive rats were treated with atorvastatin, leading to a decrease in cardiac Rac1 and RhoA activity as determined by [35S]-GTP gamma S-binding assays by 61+/-16% and 72+/-24%, and downregulation of MLC-2 as well as ANF mRNA expression by 31+/-16% and 80+/-24%, respectively. (1) Statins downregulate the activity of small G proteins in cardiomyocytes in culture as well as in vivo. (2) Inhibition of Rac1 and RhoA by statins reduces myocardial expression of ANF and MLC-2. (3) Targeting myocardial Rho GTPases by statins may be a novel treatment strategy to prevent cardiac hypertrophy.

220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns

To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.

Analysis of the energetic state of heart cells after adenovirus-mediated expression of hALC-1.

Expression of the human atrial myosin light chain 1 (hALC-1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC-1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC-1 cDNA (AdCMV.hALC-1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC-1 upon infection with AdCMV.hALC-1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC-1 localization by double-labeling immunofluorescence of AdCMV.hALC-1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co-localization of hALC-1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC-1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC-1 is not associated with enhanced oxygen consumption.

Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibres.

Isoforms of myosin heavy chain (MHC) and myosin light chain (MLC) influence contractile kinetics of skeletal muscle. We previously showed that the four major skeletal muscle fibre types in Rana pipiens (type 1, type 2, type 3 and tonic; amphibian nomenclature) contain four unique MHC isoforms. In the present study we defined the MLCs expressed in each of these R. pipiens fibre types. The MLC composition of single MHC-typed fibres was determined from western blots using a panel of monoclonal MLC antibodies. A total of seven MLCs were identified, including four types of MLC1, two of MLC2 and a single MLC3. Twitch fibre types (types 1, 2 and 3) expressed MLC1(f) and MLC2(f), while tonic fibres contained a unique set of isoforms, MLC1(Ta), MLC1(Tb) and MLC2(T). MLC3 was expressed primarily in type 1, type 1-2 and type 2 fibres. Surprisingly, some frogs displayed a striking pattern of MLC expression where a unique isoform of MLC1 (MLC1(x)) was coexpressed along with the normal MLC1 isoform(s) in all fibre types. MLC1(x) was either expressed in all fibres of a given frog or was completely absent. The intraspecific polymorphism in MLC1 expression is likely to have a genetic basis, but is unlikely to be caused by allelic variation. The ratio of MLC3/MLC1 increased in direct proportion to the percentage of type 1 MHC, but was only weakly correlated. The variability in MLC3/MLC1 within a fibre type was extremely large. Both the MHC isoform and MLC3/MLC1 ratio varied significantly between 1 mm segments along the length of fibres. For all segments combined, MLC3/MLC1 increased with the percentage of type 1 MHC, but the correlation between segments was weaker than between fibres.

Plasticity of skeletal myosin in endurance-trained rats (I). A quantitative study.

The aim of this study was to quantify modifications in the expression of skeletal myosin light chain (MLC) and myosin heavy chain (MHC) isoforms of five muscles, according to their fiber composition and function, following endurance training in rats. Rodents were assigned randomly to one of two groups: caged sedentary controls (C) or endurance-trained rats (T). In T rats, three out of the four fast and mixed muscles studied exhibited a significant increase in the expression of MLC1s, 1f and 2s and a significant decrease in MLC2f and 3f, the exception being the plantaris muscle. In two out of the four muscles we observed a significant increase in MHCI and IIa, the exception being both gastrocnemii, where the expression of MHCI did not change. In the soleus of T rats, the expression of MLC1s, 2f and 3f decreased significantly, while that of MLC2s increased significantly, compared with those of C rats. The expression of MHCIIa in T rats decreased significantly compared with that of C rats, while the expression of MHCI increased significantly. In all muscles studied, a significant slowing of myosin isoforms was observed after endurance training.

Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells.

These experiments were performed to test the hypotheses that myosin light chain 17 (MLC(17)) a and b isoform expression varies between individual vascular smooth muscle (SM) cells and that their expression correlates with cell unloaded shortening velocity. Single SM cells isolated from rabbit aorta and carotid arteries were used to measure unloaded shortening velocity and subsequently were analyzed via RT-PCR for MLC(17) a and b mRNA ratio. The MLC(17b/a) mRNA and protein ratios from adjacent tissue sections correlate very well (R(2) = 0.68), allowing use of the mRNA ratio to predict the protein ratio. The rabbit MLC(17) isoform protein sequence was found to be similar to, but unique from, the swine, mouse, and chicken sequences. Isolated single SM cells from the aorta and carotid have resting lengths of 70-280 microm and shorten to 33-88 microm after contraction. Isolated cell maximum unloaded shortening velocity is highly variable (0.5-7.5 microm/s) but becomes more uniform when normalized to initial cell length (0.01-0.05 cell lengths/s). Carotid cells activated in the presence of okadaic acid (1 microm) have mean maximal unloaded shortening velocities not significantly different from carotid cells activated without okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting cell length before activation is significantly correlated with final cell length after unloaded shortening. Neither initial cell length, final cell length, total cell length change, nor maximum unloaded shortening velocity (absolute or normalized) was significantly correlated with single-cell MLC(17b/a) mRNA ratio. These studies were performed in isolated single SM cells where unloaded shortening velocity and MLC(17b/a) mRNA ratios were measured in the same cell. In this preparation, the three-dimensional organization and milieu of the cell is kept intact, but without the intercellular heterogeneity concerns of multicellular preparations. These results suggest the MLC(17b/a) ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloaded shortening velocity in single SM cells.

Skeletal muscle gene transfer: regeneration-associated deregulation of fast troponin I fiber type specificity.

Direct gene transfer into skeletal muscle in vivo presents a convenient experimental approach for studies of adult muscle gene regulatory mechanisms, including fast vs. slow fiber type specificity. Previous studies have reported preferential expression of fast myosin heavy chain and slow myosin light chain and troponin I (TnIslow) gene constructs in muscles enriched in the appropriate fiber type. We now report a troponin I fast (TnIfast) direct gene transfer study. We injected into the mouse soleus muscle plasmid DNA or recombinant adenovirus carrying a TnIfast/ beta-galactosidase (beta-gal) reporter construct that had previously been shown to be expressed specifically in fast fibers in transgenic mice. Surprisingly, microscopic histochemical analysis 1 and 4 wk postinjection showed similar TnIfast/beta-gal expression in fast and slow fibers. A low but significant level of muscle fiber segmental regeneration was evident in muscles 1 wk postinjection, and TnIfast/beta-gal expression was preferentially targeted to regenerating fiber segments. This finding can explain why TnIfast constructs are deregulated with regard to fiber type specificity, whereas the myosin constructs previously studied are not. The involvement of regenerating fiber segments in transduction by plasmid DNA and recombinant adenoviruses injected into intact normal adult muscle is an unanticipated factor that should be taken into account in the planning and interpretation of direct gene transfer experiments.

Myosin isoforms in anuran skeletal muscle: their influence on contractile properties and in vivo muscle function.

Functional studies on isolated single anuran skeletal muscle cells represent classic experiments from which much of our understanding of muscle contraction mechanisms have been derived. Because of their superb mechanical stability when isolated, single anuran fibers provide a uniquely powerful model system that can be exploited to understand the relationship between myosin heavy chain (MHC) and myosin light chain (MLC) composition and muscle fiber function. In this review, we summarize historic and recent studies of MHC and MLC expression patterns in the fiber types of anuran species. We extend the traditional classification scheme, using data from recent reports in which frog MHCs have been cloned, to reveal the molecular basis of frog muscle fiber types. The influence of MHC and MLC isoforms on contractile kinetics of single intact fibers is reviewed. In addition, we discuss more subtle questions such as variability of myosin coexpression along a single cell, and its potential influence on contractile function. The frog jump is used as a model system to elucidate principles of muscular system design, including the role of MHC isoforms on in vivo muscle function. Sequence information is used from cloned frog MHCs to understand the role of specific regions of the myosin motor domain in regulating contractile function and the evolutionary origins of fast and slow amphibian MHCs. Finally, we offer promising future possibilities that combine molecular methods (such as recombinant gene transfer) with single cell contractile measurements to address questions regarding myosin structure/function and gene regulation.

Expression of atrial myosin light chains but not alpha-myosin heavy chains is correlated in vivo with increased ventricular function in patients with hypertrophic obstructive cardiomyopathy.

The adult rodent heart adapts to increased work load by reexpression of its fetal genes, for example, beta-myosin heavy chain (MHC), in order to improve contractile function. However, the human ventricle regulates contractility by expression of atrial essential myosin light chain (ALC-1) rather than beta-MHC. We evaluated the impact of both mechanisms in patients with hypertrophic cardiomyopathy. MHC isoform expression was quantified at the mRNA and protein levels by reverse transcriptase polymerase chain reaction and immunoblotting, respectively. Although alpha-MHC mRNA was detected in control and hypertrophied human ventricular tissue, alpha-MHC protein was not observed. Similarly, we investigated the expression of ALC-1 by two-dimensional polyacrylamide gel electrophoresis and the clinical and hemodynamic parameters of the patients with hypertrophic cardiomyopathy. We found a significant positive correlation between ALC-1 protein expression and dP/dtmax in the hypertrophied human ventricle in vivo. Correlations between dP/dtmax and expression of protein for the ryanodine receptor and L-type Ca2+ channel were excluded. Our data suggest that reexpression of ALC-1 improves the contractile state of the adult human heart. We propose that two evolutionarily divergent compensatory mechanisms for increased work demand exist in the mammalian heart: MHC regulation in rodents and essential MLC regulation, of cardiac contractility, in humans.

Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart.

The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium.

Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart

The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium. Anat Rec 254:135–146, 1999. © 1999 Wiley-Liss, Inc.

Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle.

A zebrafish myosin light chain 2 cDNA clone was isolated and characterized. Sequence analysis of the clone revealed a high homology with the mammalian and avian genes encoding the fast skeletal muscle isoform, MLC2f. In situ hybridization and Northern blot hybridization analyses indicated that the zebrafish MLC2f mRNA is expressed exclusively in the fast skeletal muscle. Ontogenetically, the MLC2f mRNA appears around 16 hours postfertilization (hpf) in the first few well-formed anterior somites. At later stages, the MLC2f mRNA can also be detected in fin buds, eye muscles, and jaw muscles. To develop a useful model system for analyzing muscle gene regulation, the promoter of the zebrafish MLC2f gene was isolated and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The MLC2f/CAT chimeric constructs were analyzed by direct injection into the zebrafish skeletal muscle, and significant CAT activity was observed; in contrast, little or no CAT activity was generated from a similarly injected prolactin gene promoter/CAT gene construct. Within the 1 kb of the MLC2f promoter region, several MEF2-binding sites and E-boxes were identified, suggesting that MLC2f can be regulated by muscle transcription factors MEF2 and myogenic bHLH proteins. A 5' deletion analysis indicated that the proximal 79 nucleotides from the transcription start site, which contains a single MEF2-binding site, is sufficient to drive a high level of CAT activity in injected muscle. Internal deletion of the MEF2 element in the -79-bp construct caused an 80% decrease in CAT activity, whereas internal deletion of the same MEF2 element in a -1044-bp construct had no effect on induced CAT activity. These observations suggest that an MEF2 element is important to activate the MLC2f gene in muscle cells, and the effect of loss of the proximal MEF2 element can be compensated for by the presence of the upstream MEF2 elements. This study also demonstrated that direct injection of DNA into skeletal muscle is a valid and valuable approach to analyze muscle gene promoters in the zebrafish.

Expression and function of atrial myosin light chain 1 in the porcine right ventricle of normal and pulmonary hypertensive animals.

We investigated the expression of atrial myosin light chain 1 (ALC-1) and myosin cycling kinetics in the normal and hypertrophied right ventricle of the neonatal porcine heart. Right ventricular hypertrophy was induced by exposing piglets immediately after birth to hypobaric hypoxia for 3 days. Control piglets were kept under normal conditions for the same time. ALC-1 expression in the hypertrophied right ventricle was 16.9%. No ALC-1 expression could be observed in the right ventricle of control pigs. Force-velocity of chemically skinned right ventricular fibers was analyzed in order to examine the functional role of ALC-1 expression on myosin cross-bridge kinetics. Force generation per cross-section at maximal Ca2+ activation (pCa 4.5) was significantly higher in the hypertrophied group. Maximal shortening velocity (Vmax) of skinned fibers increased statistically significant from 0.69 muscle length per second (ML/s) in the control to 1.2 ML/s in the hypertrophied right ventricle. We conclude that the expression of ALC-1 in the hypertrophied ventricle of neonatal pigs increased cross-bridge cycling kinetics and contractility.

Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

Changes in Essential Myosin Light Chain Isoform Expression Provide a Molecular Basis for Isometric Force Regulation in the Failing Human Heart

We investigated the effects of the expression of myosin light chain (MLC) isoforms on the Ca2+ sensitivity of isometric force production of demembranated (skinned) fibers of papillary muscle from the left ventricle of three groups: patients with ischemic cardiomyopathy, patients with dilated cardiomyopathy (NYHA IV) and normal human hearts. Expression and phosphorylation of the phosphorylatable MLC isoforms (MLC-2) was equal within all three groups. However, 72% of the patients investigated in this study expressed the atrial essential MLC (ALC-1) in addition to the essential ventricular MLC (VLC-1) ranging between 2.4% and 10.3%. Using fibers from failing hearts, we observed a significant positive correlation between ALC-1 and Ca2+ sensitivity in that the higher the ALC-1 expression the higher the Ca2(+)-sensitivity: pCa50 (Ca2+ required for half-maximal force production) was 5.87 without ALC-1 and 6.08 with 10.3% ALC-1. Fibers from a normal heart (no ALC-1) revealed a pCa50 of 5.85. Isoform and phosphorylation patterns of tropomyosin and troponin I remained unchanged in the patients and normal hearts. Our results suggest that Ca2+ responsiveness and force development of the human heart is regulated by the expression of different MLC-1 isoforms.