Compensatory changes in Ca(2+) and myocardial O(2) consumption in beta-tropomyosin transgenic hearts.

Transgenic mice overexpressing beta-tropomyosin have increased myofilament Ca(2+) sensitivity that we hypothesized would result in altered relationships among pressure and heart rates, intracellular Ca(2+), and myocardial O(2) consumption. In perfused hearts from transgenic mice there was a marked negative force-frequency response between 6 and 10 Hz with a 30 +/- 3% reduction in peak-positive first derivative of pressure development over time (dP/dt) compared with 14 +/- 2% in wild-type mice (P < 0.001). At 8 Hz systolic pressures were normal, though peak systolic intracellular Ca(2+) was significantly reduced in transgenic mice versus wild type (726 +/- 61 vs. 936 +/- 67 nM, P < 0.05) indicating an alteration in the pressure-Ca(2+) relationship. Over a wide range of positive and negative inotropic interventions there were normal developed pressures, though marked elevations in myocardial O(2) consumption (15-54%). Because pressures are normal and intracellular Ca(2+) decreased and myocardial O(2) consumption increased, this suggests that these abnormalities are at least in part compensatory mechanisms to the altered myofilament function.

A familial hypertrophic cardiomyopathy alpha-tropomyosin mutation causes severe cardiac hypertrophy and death in mice.

Tropomyosin, an essential component of the sarcomere, regulates muscle contraction through Ca(2+)-mediated activation. Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in numerous cardiac sarcomeric proteins, including myosin heavy and light chains, actin, troponin T and I, myosin binding protein C, and alpha-tropomyosin. This study developed transgenic mouse lines that encode an FHC mutation in alpha-tropomyosin; this mutation is an amino acid substitution at codon 180 (Glu180Gly) which occurs in a troponin T binding region. Non-transgenic and control mice expressing wild-type alpha-tropomyosin demonstrate no morphological or physiological changes. Expression of exogenous mutant tropomyosin leads to a concomitant decrease in endogenous alpha-tropomyosin without altering the expression of other contractile proteins. Histological analysis shows that initial pathological changes, which include ventricular concentric hypertrophy, fibrosis and atrial enlargement, are detected within 1 month. The disease-associated changes progressively increase and result in death between 4 and 5 months. Physiological analyses of the FHC mice using echocardiography, work-performing heart analyses, and force measurements of cardiac myofibers, demonstrate dramatic functional differences in diastolic performance and increased sensitivity to calcium. This report demonstrates that mutations in alpha-tropomyosin can be severely disruptive of sarcomeric function, which consequently triggers a dramatic hypertrophic response that culminates in lethality.

PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin.

In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% alpha-tropomyosin (alpha-Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+ sensitivity of force and the rate constant of force redevelopment (k(tr)). Experiments were also done using transgenic (TG) myocardium expressing approximately 60% beta-Tm to test the idea that the alpha-Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+ sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+ sensitivity of force in NTG and TG myocardium, had no effect on maximum k(tr) in either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.

Altered hemodynamics in transgenic mice harboring mutant tropomyosin linked to hypertrophic cardiomyopathy.

We used transgenic (TG) mice overexpressing mutant alpha-tropomyosin [alpha-Tm(Asp175Asn)], linked to familial hypertrophic cardiomyopathy (FHC), to test the hypothesis that this mutation impairs cardiac function by altering the sensitivity of myofilaments to Ca(2+). Left ventricular (LV) pressure was measured in anesthetized nontransgenic (NTG) and TG mice. In control conditions, LV relaxation was 6,970 +/- 297 mmHg/s in NTG and 5,624 +/- 392 mmHg/s in TG mice (P < 0.05). During beta-adrenergic stimulation, the rate of relaxation increased to 8,411 +/- 323 mmHg/s in NTG and to 6,080 +/- 413 mmHg/s in TG mice (P < 0.05). We measured the pCa-force relationship (pCa = -log [Ca(2+)]) in skinned fiber bundles from LV papillary muscles of NTG and TG hearts. In control conditions, the Ca(2+) concentration producing 50% maximal force (pCa(50)) was 5.77 +/- 0.02 in NTG and 5.84 +/- 0.01 in TG myofilament bundles (P < 0.05). After protein kinase A-dependent phosphorylation, the pCa(50) was 5.71 +/- 0.01 in NTG and 5.77 +/- 0. 02 in TG myofilament bundles (P < 0.05). Our results indicate that mutant alpha-Tm(Asp175Asn) increases myofilament Ca(2+)-sensitivity, which results in decreased relaxation rate and blunted response to beta-adrenergic stimulation.

Tropomyosin 3 increases striated muscle isoform diversity.

Tropomyosin (TM), a component of the thin filament of the sarcomere, is encoded by a four-member multigene family: alpha-TM, beta-TM, TPM 3, and TPM 4. The alpha-TM, beta-TM, and TPM 3 genes each utilize an alternative splicing mechanism to encode a striated muscle isoform. Although the alpha-TM and beta-TM striated muscle isoforms are well characterized, relatively little is known about the TPM 3 isoform. We cloned and sequenced the murine TPM 3 cDNA and found that it exhibits a 93% nucleotide homology and 99% amino acid homology to the human sequence. Results show that, unlike humans, TPM 3 is not expressed in any developmental stage of murine hearts. TPM 3 message is expressed in slow-twitch skeletal muscle but is not found in representative fast-twitch musculature. The soleus, a representative slow-twitch muscle, expresses transcript levels of 65% beta-TM, 15% alpha-TM, and 20% TPM 3, but the TPM 3 protein accounts for approximately 31% of the total striated tropomyosin in slow-twitch muscle. In fast-twitch muscle, alpha-TM comprises 71% of the total striated muscle TM protein, and beta-TM comprises 29%. The results demonstrate that a translational mechanism regulates the production of the TM proteins, with beta-TM message not being efficiently translated. The unique distribution pattern of TPM 3 adds to the diversity of the tropomyosin family and strongly suggests functional significance for the different striated muscle TM isoforms.

Alteration of cardiac myofibrillogenesis by liposome-mediated delivery of exogenous proteins and nucleic acids into whole embryonic hearts.

A precise organization of contractile proteins is essential for contraction of heart muscle. Without a necessary stoichiometry of proteins, beating is not possible. Disruption of this organization can be seen in diseases such as familial hypertrophic cardiomyopathy and also in acquired diseases. In addition, isoform diversity may affect contractile properties in such functional adaptations as cardiac hypertrophy. The Mexican axolotl provides an uncommon model in which to examine specific proteins involved with myofibril formation in the heart. Cardiac mutant embryos lack organized myofibrils and have altered expression of contractile proteins. In order to replicate the disruption of myofibril formation seen in mutant hearts, we have developed procedures for the introduction of contractile protein antibodies into normal hearts. Oligonucleotides specific to axolotl tropomyosin isoforms (ATmC-1 and ATmC-3), were also successfully introduced into the normal hearts. The antisense ATmC-3 oligonucleotide disrupted myofibril formation and beating, while the sense strands did not. A fluorescein-tagged sense oligonucleotide clearly showed that the oligonucleotide is introduced within the cells of the intact hearts. In contrast, ATmC-1 anti-sense oligonucleotide did not cause a disruption of the myofibrillar organization. Specifically, tropomyosin expression can be disrupted in normal hearts with a lack of organized myofibrils. In a broader approach, these procedures for whole hearts are important for studying myofibril formation in normal hearts at the DNA, RNA, and/or protein levels and can complement the studies of the cardiac mutant phenotype. All of these tools taken together present a powerful approach to the elucidation of myofibrillogenesis and show that embryonic heart cells can incorporate a wide variety of molecules with cationic liposomes.

Rescue of high expression beta-tropomyosin transgenic mice by 5-propyl-2-thiouracil. Regulating the alpha-myosin heavy chain promoter.

Tropomyosin is an essential component of the sarcomeric thin filament in striated muscle that participates in the regulation of muscle contraction through Ca(2+)-mediated activation. The two predominant tropomyosin isoforms expressed in striated muscle are alpha- and beta-tropomyosin, which exhibit an 86% amino acid identity between themselves. Previous studies by our laboratory utilized a transgenic mouse system to overexpress beta-tropomyosin in the heart to address the functional differences between these two tropomyosin isoforms. Interestingly, when a high percentage of beta-tropomyosin replaces alpha-tropomyosin in the hearts of transgenic mice, the mice die due to severe cardiac abnormalities. In this study, we have rescued these high expression beta-tropomyosin mice by turning off the alpha-myosin heavy chain promoter, which is driving the beta-tropomyosin transgene. This down-regulation of the alpha-myosin heavy chain promoter was accomplished by the administration of 5-propyl-2-thiouracil, which disrupts thyroid hormone synthesis and inhibits promoter activity through thyroid regulatory elements located in the 5'-flanking region of the promoter. Results show that as beta-tropomyosin expression is down-regulated, alpha-tropomyosin expression is increased. Also, alpha- and beta-myosin heavy chain expression is modified in response to the changes in thyroid hormone expression. Morphological analysis of these rescued mice show a moderate pathological phenotype, characterized by atrial myocytolysis; echocardiographic analyses demonstrate altered ventricular functions, such as peak filling rates and left ventricular fractional shortening. This is the first report demonstrating that transcriptional regulatory elements located within the alpha-myosin heavy chain promoter can be manipulated to rescue potentially lethal phenotypes, such as high expression beta-tropomyosin transgenic mice.

Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction.

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin.

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts.

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse alpha-tropomyosin cDNA under the control of a cardiac-specific alpha-myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well-organized sarcomeric myofibrils. Transfection of a beta-tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a beta-galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of alpha-tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres.

Beta-tropomyosin overexpression induces severe cardiac abnormalities.

Tropomyosin, a coiled-coil dimer, stabilizes actin filaments and is central to the control of calcium-regulated striated muscle contraction. Striated muscle-specific alpha-tropomyosin is the predominant isoform in cardiac muscle, with low levels of beta-tropomyosin restricted to fetal development in the mouse. To understand the functional role of various tropomyosin isoforms during myofilament activation and regulation in the intact sarcomere, we generated transgenic mice that overexpress striated muscle-specific beta-tropomyosin in the adult heart. Our earlier results succinctly demonstrate that overexpression of beta-tropomyosin in the hearts of transgenic mice decreases endogeneous alpha-tropomyosin levels while altering diastolic function of the myocardium. To explore further the significance of altering the alpha- to beta-tropomyosin isoform ratio in developing murine myocardium, we generated transgenic mice which express beta-tropomyosin at high levels in the heart. The data show that higher levels of beta-tropomyosin expression are lethal with death ensuing between 10-14 days postnatally. A detailed histological analysis demonstrates that the hearts of these mice exhibit several pathological abnormalities, including thrombus formation in the lumen of both atria and in the subendocardium of the left ventricle. Other changes include atrial enlargement and fibrosis, and diffuse myocytolysis, Physiological analyses using ventricular muscle strip preparations from these mice reveal that both myocardial contraction and relaxation parameters are severely impaired. Thus, these results firmly demonstrate an essential difference in tropomyosin isoform function in physiologically regulating cardiac performance.

Prevention of cardiac hypertrophy in mice by calcineurin inhibition.

Hypertrophic cardiomyopathy (HCM) is an inherited form of heart disease that affects 1 in 500 individuals. Here it is shown that calcineurin, a calcium-regulated phosphatase, plays a critical role in the pathogenesis of HCM. Administration of the calcineurin inhibitors cyclosporin and FK506 prevented disease in mice that were genetically predisposed to develop HCM as a result of aberrant expression of tropomodulin, myosin light chain-2, or fetal beta-tropomyosin in the heart. Cyclosporin had a similar effect in a rat model of pressure-overload hypertrophy. These results suggest that calcineurin inhibitors merit investigation as potential therapeutics for certain forms of human heart disease.

Molecular and physiological effects of alpha-tropomyosin ablation in the mouse.

Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of alpha-TM, the murine alpha-TM gene was disrupted by homologous recombination. Homozygous alpha-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for alpha-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle alpha-TM mRNA, with no compensatory increase in transcript levels by striated muscle beta-TM or TM-30 isoforms. Surprisingly, this reduction in alpha-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle alpha-TM protein are produced and integrated in the myofibril. Quantification of alpha-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of alpha-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous alpha-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression.

Tropomyosin structure and function new insights.

Cardiac muscle contraction is dependent upon a cooperative interaction between thick and thin filament sarcomeric proteins. Tropomyosin (TM), an essential thin filament protein, interacts with actin and the troponin complex to regulate contractile activity. During muscle contraction, an increase of calcium (Ca(2+)) in the myofilament space promotes binding of Ca(2+) to troponin C, which alters the conformational state of TM and facilitates acto-myosin interactions. By coupling classic genetic approaches with recent developments in transgenic animal model systems, new insights have been provided on the functional role of TM isoforms in both normal and disease states. The focus of this article is to review the current state of knowledge on TM structure and function, with a particular emphasis on myocardial expression in transgenic mouse model systems. (Trends Cardiovasc Med 1997;7:124-128). © 1997, Elsevier Science Inc.

Exchange of beta- for alpha-tropomyosin in hearts of transgenic mice induces changes in thin filament response to Ca2+, strong cross-bridge binding, and protein phosphorylation.

Despite its potential as a key determinant of the functional state of striated muscle, the impact of tropomyosin (Tm) isoform switching on mammalian myofilament activation and regulation in the intact lattice remains unclear. Using a transgenic approach to specifically exchange beta-Tm for the native alpha-Tm in mouse hearts, we have been able to uncover novel functions of Tm isoform switching in the heart. The myofilaments containing beta-Tm demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca2+ sensitivity of steady state force, and a decrease in the rightward shift of the Ca2+-force relation induced by cAMP-dependent phosphorylation. Our results are the first to demonstrate the specific effects of Tm isoform switching on mammalian thin filament activation in the intact lattice and suggest an important role for Tm in modulation of myofilament activity by phosphorylation of troponin.

Molecular and physiological effects of overexpressing striated muscle beta-tropomyosin in the adult murine heart.

Tropomyosins comprise a family of actin-binding proteins that are central to the control of calcium-regulated striated muscle contraction. To understand the functional role of tropomyosin isoform differences in cardiac muscle, we generated transgenic mice that overexpress striated muscle-specific beta-tropomyosin in the adult heart. Nine transgenic lines show a 150-fold increase in beta-tropomyosin mRNA expression in the heart, along with a 34-fold increase in the associated protein. This increase in beta-tropomyosin message and protein causes a concomitant decrease in the level of alpha-tropomyosin transcripts and their associated protein. There is a preferential formation of the alpha beta-heterodimer in the transgenic mouse myofibrils, and there are no detectable alterations in the expression of other contractile protein genes, including the endogenous beta-tropomyosin isoform. When expression from the beta-tropomyosin transgene is terminated, alpha-tropomyosin expression returns to normal levels. No structural changes were observed in these transgenic hearts nor in the associated sarcomeres. Interestingly, physiological analyses of these hearts using a work-performing model reveal a significant effect on diastolic function. As such, this study demonstrates that a coordinate regulatory mechanism exists between alpha- and beta-tropomyosin gene expression in the murine heart, which results in a functional correlation between alpha- and beta-tropomyosin isoform content and cardiac performance.

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

Induction of endogenous myosin light chain 1 and cardiac alpha-actin expression in L6E9 cells by MyoD1.

Rat L6E9 muscle cells commit to terminal differentiation by forming a large muscle syncitia complete with the expression of a large number of muscle-specific contractile protein genes. To determine whether these cells, which fail to synthesize MLC (myosin light chain) 1 and cardiac alpha-actin, exhibit a deficiency in the expression of muscle determination genes, we measured expression of MyoD1, myogenin, Myf-5, and MRF-4. Results show these cells do not synthesize MyoD1, yet express the other myogenic determination genes. Transient expression of exogenous MyoD1 in these cells is sufficient to activate endogenous MLC 1 and cardiac alpha-actin mRNA synthesis during muscle differentiation. Previously undetected myosin heavy chain (MHC) isoforms (beta-MHC and perinatal MHC) are also transcribed at low levels in L6E9 muscle cells, and in MyoD1-transfected L6E9 cells no change occurs in their expression. Furthermore, treatment with the demethylating agent 5-azacytidine activates expression of the endogenous MyoD1 gene in L6E9 cells and, subsequently, rescues deficiencies in their myogenic biochemical program. These results demonstrate that the endogenous MyoD1 gene in L6E9 cells is not defective and can be functionally activated. Also, the MyoD1 protein plays an essential role, which cannot be compensated by other known muscle determination proteins, in the induction of MLC 1 and cardiac alpha-actin expression.

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.

Reprogramming of myosin light chain 1/3 expression in muscle heterokaryons.

Fast myosin light chain (MLC) 1/3 is one of the few genes which regulates transcript production at both transcriptional and post-transcriptional levels, utilizing two functionally distinct promoters coupled with alternatively spliced exons. The transcriptional process controlling expression from this single gene locus is developmentally regulated, such that MLC 1 precedes MLC 3 during myogenesis. Results from our RNA analyses demonstrate that in differentiated rat L6E9 muscle, MLC 3 is the sole isoform expressed from the MLC 1/3 locus. However, we also show that by generating rat L6E9:mouse C2 muscle heterokaryons, MLC 1 expression from the L6E9 MLC locus can be induced. In addition to novel rat MLC 1 expression in the C2:L6E9 heterokaryons, we show that the synthesis profile of rat MLC 3 mRNA is also altered relative to L6E9 muscle cultured alone. Additional experiments demonstrate that the reprogramming of rat MLC 1 and 3 expression in the muscle heterokaryons requires that C2 and L6E9 nuclei be contained within a common cytoplasm. These results demonstrate that expression from the MLC 1/3 gene is "plastic," and is not under the control of a strict developmental program but, rather, can be modified by the environmental milieu.