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PubMed1992

Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes.

Costameres, the vinculin-rich, sub-membranous transverse ribs found in many skeletal and cardiac muscle cells (Pardo, J. V., J. D. Siciliano, and S. W. Craig. 1983. Proc. Natl. Acad. Sci. USA. 80:363-367.) are thought to anchor the Z-lines of the myofibrils to the sarcolemma. In addition, it has been postulated that costameres provide mechanical linkage between the cells' internal contractile machinery and the extracellular matrix, but direct evidence for this supposition has been lacking. By combining the flexible silicone rubber substratum technique (Harris, A. K., P. Wild, and D. Stopak. 1980. Science (Wash. DC). 208:177-179.) with the microinjection of fluorescently labeled vinculin and alpha-actinin, we have been able to correlate the distribution of costameres in adult rat cardiac myocytes with the pattern of forces these cells exert on the flexible substratum. In addition, we used interference reflection microscopy to identify areas of the cells which are in close contact to the underlying substratum. Our results indicate that, in older cell cultures, costameres can transmit forces to the extracellular environment. We base this conclusion on the following observations: (a) adult rat heart cells, cultured on the silicone rubber substratum for 8 or more days, produce pleat-like wrinkles during contraction, which diminish or disappear during relaxation; (b) the pleat-like wrinkles form between adjacent alpha-actinin-positive Z-lines; (c) the presence of pleat-like wrinkles is always associated with a periodic, "costameric" distribution of vinculin in the areas where the pleats form; and (d) a banded or periodic pattern of dark gray or close contacts (as determined by interference reflection microscopy) has been observed in many cells which have been in culture for eight or more days, and these close contacts contain vinculin. A surprising finding is that vinculin can be found in a costameric pattern in cells which are contracting, but not producing pleat-like wrinkles in the substratum. This suggests that additional proteins or posttranslational modifications of known costamere proteins are necessary to form a continuous linkage between the myofibrils and the extracellular matrix. These results confirm the hypothesis that costameres mechanically link the myofibrils to the extracellular matrix. We put forth the hypothesis that costameres are composite structures, made up of many protein components; some of these components function primarily to anchor myofibrils to the sarcolemma, while others form transmembrane linkages to the extracellular matrix.

Actins↗
PubMed2001

The Caenorhabditis elegans unc-78 gene encodes a homologue of actin-interacting protein 1 required for organized assembly of muscle actin filaments.

Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491--502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.

Actins↗
PubMed2001

Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle.

Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

Actins↗
PubMed1968

The effect of colchicine on myogenesis in vivo in Rana pipiens and Rhodnius prolixus (Hemiptera).

The effect of colchicine on myogenesis in vivo has been studied in the regenerating tadpole tail of the frog, Rana pipiens, and in the abdominal molting muscles of a blood-sucking bug, Rhodnius prolixus Stål. Colchicine is shown to disrupt microtubules in the differentiating muscle cells of both these organisms. The disruption of microtubules is correlated with a loss of longitudinal anisometry in the myoblasts and myotubes of the regeneration blastema in the tadpole tail. Before colchicine treatment, the myotubes contain longitudinally oriented myofibrils. After colchicine treatment, rounded, multinucleate myosacs containing randomly oriented myofibrils are present. It is suggested that the primary function of microtubules in myogenesis in the Rana pipiens tadpole is the maintenance of cell shape. The abdominal molting muscles of Rhodnius undergo repeated phases of differentiation and dedifferentiation of the sarcoplasm. However, the longitudinal anisometry of the muscle fibers is maintained in all phases by the attachments of the ends of the fibers to the exoskeleton, and microtubule disruption does not alter cell shape. The orientation of the developing myofibrils is also unaltered, indicating that the microtubules do not directly align or support the myofibrils in this system.

Animals↗
PubMed1972

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle.

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca(2+) and 5 nM Mg(2+) for 9 hr at 37 degrees C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca(2+) and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37 degrees C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca(2+) and 5 mM Mg(2+) in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca(2+) at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0-7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca(2+) levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca(2+) in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.

Animals↗
PubMed1978

Assays of the metabolic viability of single giant mitochondria. Experiments with intact and impaled mitochondria.

Single giant mitochondria isolated from mice fed cuprizone were assayed for their metabolic viability. Two tests were devised. One test optically detected the accumulation of calcium phosphate within the mitochondria under massive loading conditions (including the presence of succinate and ATP). The accumulation corresponds to a test of energy coupling from either electron transport or the hydrolysis of ATP since it is blocked by either antimycin A or oligomycin. The other assay tested for the production of ATP from ADP and Pi, using myofibrils. Myofibrils prepared from glycerinated rabbit psoas muscle contract only in the presence of ATP and not in the presence of ADP. Myofibrillar contraction is unaffected by the presence of antimycin A or oligomycin. However, myofibrils in the presence of mitochondria that are phosphorylating ADP to ATP do contract. This contraction is blocked by antimycin A and/or oligomycin. Hence, the ATP which causes myofibrillar contraction is produced by oxidative phosphorylation. At low mitochondrial concentration, only the myofibrils in close proximity with mitochondria contract in the presence of ADP. Therefore the assay can be used to test the viability of individual mitochondria. Individual giant mitochondria were found to be viable, using both of these assays. Comparable results were obtained in mitochondria impaled with microelectrodes. The potentials and resistances were unaffected by concomitant calcium phosphate accumulation or oxidative phosphorylation.

Adenosine Diphosphate↗
PubMed1981

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells.

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross-striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M-line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'-bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.

Animals↗
PubMed1981

Connectin, an elastic protein of muscle. Identification of "titin" with connectin.

When whole muscle fibers or myofibrils of rabbit and chicken skeletal muscles are directly solubilized in hot SDS solution, a very high molecular protein called titin can be isolated by gel filtration (Wang et al. 1979). Connectin, an elastic protein of muscle (Maruyama et al. 1977), can be isolated by a similar method from thoroughly extracted muscle residues. Studies of electrophoretic mobility on 2-3% polyacrylamide gel electrophoresis, amino acid composition, and localization in myofibrils determined by the indirect immunofluorescence technique showed that titin and connectin are identical. Connectin was found to be unstable in SDS solution on storage for a few days at room temperature; the doublet band of connectin on SDS gel electrophoresis became diffuse and eventually disappeared. Connectin was concentrated around the A-I junction region of a myofibril, although it was present in an entire sarcomere except for the Z lines. On removal of myosin, the A-I junction was still fluorescent, when treated with fluorescent antibody against connectin. In the KI-extracted myofibril, materials accumulated on both sides of the Z lines were strongly stained, and there were fluorescent filaments between the neighboring Z lines, but the Z lines were not stained at all.

Animals↗
PubMed1984

Two-dimensional electrophoresis of troponin complex with nonequilibrium pH gradient-sodium dodecyl sulfate polyacrylamide slab gel.

A two-dimensional electrophoresis procedure for the separation and analysis of troponin subunits is described in which the protein solution supplemented with 50 mM each of both glutamic and aspartic acids is subjected to nonequilibrium pH gradient electrophoresis in the first dimension. Complete dissolution and gelation of the sample with agarose are essential for analysis of constituent proteins of cardiac myofibrils. Electrophoresis in the first dimension gel is carried out for a relatively short time, 2-3 h. In combination with sodium dodecyl sulfate slab gel electrophoresis (second dimension), three subunits, troponin T, troponin I, and troponin C, of dog cardiac troponin-tropomyosin complex and myofibrils can be simultaneously analyzed quantitatively on a slab gel. The contents of troponin and tropomyosin of cardiac myofibrils were 275 +/- 34 pmol/mg of myofibrillar protein. The molar ratio of troponin T, troponin I, troponin C, and tropomyosin was close to 1 : 1 : 1 : 1 in troponin-tropomyosin complex and myofibrils.

Animals↗
PubMed2000

Denaturation of myofibrillar proteins from chickens as affected by pH, temperature, and adenosine triphosphate concentration.

The susceptibility to denaturation of myofibrillar protein from chicken muscles was investigated and compared with denaturation of myofibrillar protein from pork. Immediately postmortem, the Pectoralis profundus (white muscle) and the Pubo-ishio femorale (red muscle) of six Arbor Acres chickens were collected. The Semimembranosus (white muscle) and Psoas major (red muscle) of three Yorkshire x Landrace and three Yorkshire x Landrace x Duroc pigs were collected at 45 min postmortem. Protein denaturation was prevented by keeping the muscles at 0 to 2 C in a buffer (pH 7.2) containing ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) (to sequester Ca ions). After purification, myofibrils were incubated at 25 or 40 C, pH 5.4 or 6.5, with 0, 0.68, or 3.4 mM adenosine triphosphate (ATP). Protein solubility, an indicator of denaturation, was assessed after 0, 10, 20, and 60 min incubation. Protein solubility of chicken pectoralis myofibrils was not affected by any of the conditions. In the other myofibrils, pH 5.4 caused significantly (P < 0.05) more protein denaturation than pH 6.5, and incubation at 40 C resulted in significantly more protein denaturation than incubation at 25 C. The presence of ATP (tested at pH 6.5) affected denaturation; higher ATP concentrations resulted in increased loss of solubility. We concluded that chicken red myofibrillar proteins are equally susceptible to denaturation as are pork red and white myofibrils. Chicken pectoralis (white) muscle fibers are least susceptible to denaturation. The results of this study indicate that factors other than protein denaturation are responsible for the low water-holding capacity of pale, soft, exudative chicken breast muscle.

Adenosine Triphosphate↗
PubMed1998

Changes in force and cytosolic Ca2+ concentration after length changes in isolated rat ventricular trabeculae.

1. Changes in cytosolic [Ca2+] ([Ca2+]i) were measured in isolated rat trabeculae that had been micro-injected with fura-2 salt, in order to investigate the mechanism by which twitch force changes following an alteration of muscle length. 2. A step increase in length of the muscle produced a rapid potentiation of twitch force but not of the Ca2+ transient. The rapid rise of force was unaffected by inhibiting the sarcoplasmic reticulum (SR) with ryanodine and cyclopiazonic acid. 3. The force-[Ca2+]i relationship of the myofibrils in situ, determined from twitches and tetanic contractions in SR-inhibited muscles, showed that the rapid rise of force was due primarily to an increase in myofibrillar Ca2+ sensitivity, with a contribution from an increase in the maximum force production of the myofibrils. 4. After stretch of the muscle there was a further, slow increase of twitch force which was due entirely to a slow increase of the Ca2+ transient, since there was no change in the myofibrillar force-[Ca2+]i relationship. SR inhibition slowed down, but did not alter the magnitude of, the slow force response. 5. During the slow rise of force there was no slow increase of diastolic [Ca2+]i, whether or not the SR was inhibited. The same was true in unstimulated muscles. 6. We conclude that the rapid increase in twitch force after muscle stretch is due to the length-dependent properties of the myofibrils. The slow force increase is not explained by length dependence of the myofibrils or the SR, or by a rise in diastolic [Ca2+]i. Evidence from tetani suggests the slow force responses result from increased Ca2+ loading of the cell during the action potential.

Animals↗
PubMed1972

Sodium dependence of the inward spread of activation in isolated twitch muscle fibres of the frog.

1. The excitatory process travelling along the T-system may be either electrotonic or regenerative. If Na(+) dependent action potential is present in the tubular membranes, high frequency of stimulation might cause a Na(+) depletion in the tubules sufficient to abolish this process.2. We tested this hypothesis by recording tension in isolated muscle fibres stimulated tetanically (up to 60 shocks/sec). In low [Na(+)] solutions, output tension was initially similar to that in normal Ringer, but then fell smoothly to a substantially lower value.3. The activity of individual myofibrils was recorded directly with ciné-micrographs during isotonic contractions while the fibres were stimulated at high frequencies. In low [Na(+)](o) wavy myofibrils appeared in the centre of the fibre and spread towards the periphery, indicating failure of activation. Wavy myofibrils never appeared in normal Ringer.4. Intracellular action potentials recorded during the tetanic stimulation indicated that the inactivated myofibrils present in low [Na(+)] solutions cannot be explained by the changes in size and duration of the action potential.5. Our results strongly suggest the existence of a regenerative Na(+) conductance in the tubular membrane during the inward spread of an excitatory process.

Action Potentials↗
PubMed1989

Binding of adenylosuccinate synthetase to contractile proteins of muscle.

The muscle isozyme of adenylosuccinate synthetase (AdSS), an enzyme of the purine nucleotide cycle, has previously been shown to bind to purified F-actin in buffers of low ionic strength and pH (Ogawa et al. Eur. J. Biochem. 85: 331-338, 1978). We have extended these observations by measuring the association of both crude and purified AdSS with the contractile proteins of muscle in buffers of physiological ionic strength and pH. Under these conditions, the enzyme binds to F-actin, actin-tropomyosin complexes, reconstructed thin filaments, and myofibrils but not to myosin. The apparent dissociation constant of 1.2 microM and binding maximum of 2.6 nmol enzyme/mg myofibrils indicate that binding of AdSS to myofibrils can be physiologically significant. The results suggest that AdSS in muscle may be associated with the thin filament of myofibrils.

Actins↗
PubMed1997

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading.

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Adaptation, Physiological↗
PubMed2000

Decreased expression of the cardiac LIM domain protein MLP in chronic human heart failure.

BACKGROUND: The cardiac LIM domain protein MLP, a member of the cysteine-rich protein family, is an essential regulator of cardiac muscle development. Mice with a disruption of the MLP gene resemble the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans. We investigated whether altered MLP expression is significant for the pathogenesis of human heart failure. METHODS AND RESULTS: Immunohistochemistry and in situ hybridization confirmed the expression of MLP protein and mRNA in human cardiomyocytes. Western blot analysis revealed that the MLP peptide was present in the contractile protein fraction but not in the cytosolic or membrane fraction and that the binding of MLP to myofibrils required functional zinc finger domains. MLP immunoreactivity was decreased approximately 50% (P<0.05) in the left ventricular myocardium of patients with chronic heart failure due to dilated or ischemic cardiomyopathy compared with non-failing donor hearts. MLP mRNA expression, as assessed by Northern blot experiments, was not significantly different between failing and non-failing control hearts, which suggests that decreased MLP synthesis or increased MLP protein turnover, rather than a decreased number of RNA transcripts, may play a role. CONCLUSIONS: Because MLP may promote myofibril assembly, the down-regulation of this adapter protein might play an essential role in myofibril derangement or impaired myofibril rearrangement in the failing human myocardium.

Adult↗
PubMed1987

Disruption of myofibrillar energy use: dual mechanisms that may contribute to postischemic dysfunction in stunned myocardium.

The abnormalities in regional function produced by myocardial ischemia persist after the ischemic episode resolves. Since a close functional coupling exists between myofibrillar creatine kinase and myosin ATPase, a disruption of this coupling could adversely influence myocardial function and might provide a mechanism for the myocardial dysfunction observed. The purpose of the present study was to determine if an alteration in the activity of creatine kinase associated with the myofibril occurs in the postischemic period. Anesthetized open-chest dogs (n = 6) underwent coronary occlusion for 15 minutes, followed by reperfusion for 15 minutes. In reperfused myocardium, adenine nucleotide content was decreased (72 +/- 10% of nonischemic myocardium, p less than 0.05), documenting the presence of previous ischemia. The creatine phosphate content of reperfused myocardium returned to normal, indicating resumption of myocardial energy production. The creatine kinase activity of purified myofibrils isolated from reperfused myocardium was decreased by 17 +/- 7% compared to that of nonischemic myofibrils (p less than 0.03). In addition, the free adenosine diphosphate concentration in reperfused myocardium was calculated to be 96 microM and was less than the Km of adenosine diphosphate determined for myofibrillar creatine kinase (105 microM). The results suggest two putative mechanisms for disruption of energy use in postischemic myocardium: decreased creatine kinase activity associated with the myofibril, and limitation of substrate necessary for maximal creatine kinase activity.

Adenosine Diphosphate↗
PubMed1994

Tropomodulin in rat cardiac muscle. Localization of protein is independent of messenger RNA distribution during myofibrillar development.

Tropomodulin is a 40.6-kD protein that colocalizes with actin filament pointed ends in skeletal muscle. We report the sequence of two partial-length complementary DNA (cDNA) clones of rat cardiac tropomodulin that cover 90% of the coding region. The cDNA sequence is 90% conserved between human and rat, with the predicted amino acid sequence similarity even higher at 95%. Anti-tropomodulin antibodies label a single polypeptide with an apparent mobility of 43,000 in Western blot analysis of rat cardiac muscle. Immunofluorescence experiments using this anti-tropomodulin antibody result in labeling that is coincident with thin filament ends, as demonstrated by double localization with alpha-actinin antibody. Tropomodulin protein is organized into a sarcomeric staining pattern with the earliest appearance of myofibrils in rat cardiocytes. The localization of tropomodulin protein at or near thin filament ends led us to examine the distribution of tropomodulin messenger RNA (mRNA) during myofibrillar development in vitro. Fluorescent in situ hybridization experiments using tropomodulin cDNA probe in cardiocytes that have been cultured for 3 to 5 days show a distribution of large mRNA patches. The cytoplasmic location of tropomodulin mRNA at this time, which bears no relation to the developed myofibrils, suggests that tropomodulin protein is targeted to thin filament ends rather than using localized translational machinery. However, the distribution of tropomodulin mRNA in cultured cardiocytes changes over the next 2 weeks from large perinuclear patches to small concentrations arranged along myofibrils throughout the cell. The reorganization of tropomodulin mRNA throughout the cardiocyte appears to be distinct from the pattern of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same time period. Increasing intracellular density of myofibrils within developing cardiocytes may lead to redistribution of selected mRNAs for localized translation.

Amino Acid Sequence↗
PubMed1997

Localization of cardiac (alpha)-myosin heavy chain mRNA is regulated by its 3' untranslated region via mechanical activity and translational block.

We have altered the spontaneous contractile activity of neonatal cardiac myocytes in culture to investigate the re-lationship between mechanical forces, myofibril assembly, and the localization and translation of (alpha)-myosin heavy chain mRNA. Immunofluorescence and in situ hybridization techniques revealed that contracting myocytes display well aligned myofibrils and a diffuse distribution of (alpha)-myosin heavy chain mRNA. Inhibition of contractile activity with the calcium channel blocker verapamil (10 microM) resulted in myofibril disassembly and a perinuclear mRNA distribution within six hours. There was a significant decrease (P<0. 05) of mRNA levels, 5 to 15 micron away from the nucleus following 6 hours of verapamil treatment compared with control cells. Inhibition of protein synthesis with cycloheximide (10 microM) also resulted in perinuclear mRNA localization despite having little effect on contractile activity or myofibril assembly. To determine if the 3' untranslated region of (alpha)-myosin heavy chain mRNA was sufficient for localizing the entire message, a chimeric construct composed of beta-galactosidase coding region followed by (alpha)-myosin heavy chain 3' untranslated region sequences was made as a reporter plasmid and transfected into cultured myocytes. A perinuclear accumulation of ss-galactosidase was exhibited in many of the contractile arrested cells (48.3+/-2.4%, n=7). In contrast, significantly fewer (P<0.05) contracting control (29.1+/-3.3%, n=7) and strongly contracting, isoproterenol-treated cells (27.2+/-6.1%, n=3) exhibited a perinuclear localization of protein. The distribution of the reporter protein was not affected by the contractile state in cells transfected with a constitutively translated 3'UTR. We propose that mechanical activity of neonatal cardiac myocytes regulates the intracellular localization of alpha-myosin heavy chain mRNA via the 3' untranslated region mediated by an initial block in translation.

Animals↗