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Recent clinical studies have documented the efficacy of trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA) as a new biologically targeted therapy for erbB-2 receptor-positive forms of breast cancer. During the course of a large-scale clinical trial, a subset of patients reported the onset of symptoms and signs of cardiac failure that appeared to be aggravated by concomitant exposure to anthracyclines. The mechanisms responsible for this cardiac toxicity are unclear. However, new insights into the pathways that lead to other forms of heart failure have identified a pivotal role for myocyte survival pathways in preventing the onset of cardiomyopathy and associated heart failure in genetically engineered animal models of the disease. This mini-review highlights these recent findings and suggests the possibility that the loss of erbB-2 receptor-dependent myocyte survival pathways may create a susceptibility for the onset of heart failure in response to the cardiotoxicity of anthracycline treatment. The possibility exists that the divergent susceptibility for the onset of cardiotoxicity among patients who have received trastuzumab might ultimately reflect an inherent genetic susceptibility to the diverse mechanisms that initiate, promote, and suppress the complex pathways to heart failure.

The assembly of contractile proteins into organized sarcomeric units is one of the most distinctive features of cardiac myocyte hypertrophy. In a well characterized in vitro model system using cultured neonatal rat ventricular myocytes, a subset of G protein-coupled receptor agonists has been shown to induce actin-myosin filament organization. Pretreatment of myocytes with C3 exoenzyme ADP-ribosylated Rho and inhibited the characteristic alpha1-adrenergic receptor agonist-induced myofibrillar organization, suggesting involvement of the Rho GTPase in cardiac myofibrillogenesis. We used adenoviral mediated gene transfer to examine the effects of activated Rho and inhibitory mutants of one of its effectors, Rho kinase, in myocytes. Rho immunoreactivity was increased in the particulate fraction of myocytes infected with a recombinant adenovirus expressing constitutively activated Rho. Rho-infected cells demonstrated a striking increase in the assembly and organization of sarcomeric units and in the expression of the atrial natriuretic factor protein. These Rho-induced responses were markedly inhibited by co-infection with adenoviruses expressing putative dominant negative forms of Rho kinase. A parallel pathway involving Ras-induced myofibrillar organization and atrial natriuretic factor expression was only minimally affected. alpha1-Adrenergic receptor agonist-induced myofibrillogenesis was inhibited by some but not all of the Rho kinase mutants. Our data demonstrate that activated Rho has profound effects on myofibrillar organization in cardiac myocytes and suggest that Rho kinase mediates Rho-induced hypertrophic responses.

Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development (Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase.

G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy. These responses have been previously shown to be dependent on Gq and Ras. Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fos serum response element. Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic. We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinum C3 transferase, to examine the possible requirement for Rho in alpha1-adrenergic receptor-mediated hypertrophy. Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by alpha1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Galphaq. These effects were reproduced with the myosin light chain-2 reporter gene. Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression. Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA. Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras. Our results provide direct evidence that Rho is an effector of Galphaq signaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.

Cardiotrophin-1 (CT-1) was recently isolated by expression cloning based on its ability to induce an increase in cell size in neonatal rat ventricular cardiomyocytes. Sequence similarity data suggested that CT-1 is a novel member of a family of structurally related cytokines sharing the receptor component gp130. The present study documents that gp130 is required for CT-1 signaling in cardiomyocytes, by demonstrating that a monoclonal anti-gp130 antibody completely inhibits c-fos induction by CT-1. Similarly, a leukemia inhibitory factor receptor subunit beta (LIFRbeta) antagonist effectively blocks the CT-1 induction of c-fos, indicating a requirement for LIFRbeta in the hypertrophic response, as well. Upon stimulation with CT-1, both gpl30 and the LIFRbeta are tyrosine-phosphorylated, providing further evidence that CT-1 signals through the gp130/LIFRbeta heterodimer in cardiomyocytes. CT-1 induces a hypertrophic response in cardiomyocytes that is distinct from the phenotype seen after alpha-adrenergic stimulation, both with regard to cell morphology and gene expression pattern. Stimulation with CT-1 results in an increase in cardiac cell size that is characterized by an increase in cell length but no significant change in cell width. Confocal laser microscopy of CT-1 stimulated cells reveals the assembly of sarcomeric units in series rather than in parallel, as seen after alpha-adrenergic stimulation. CT-1 induces a distinct pattern of immediate early genes, and up-regulates the atrial natriuretic factor (ANF) gene, but does not affect skeletal alpha-actin or myosin light chain-2v expression. As evidenced by nuclear run-on transcription assays, both CT-1 and alpha-adrenergic stimulation lead to an increase in ANF gene transcription. Transient transfection analyses document that, in contrast to alpha-adrenergic stimulation, the CT-1 responsive cis-regulatory elements are located outside of the proximal 3 kilobase pairs of the ANF 5'-flanking region. These studies indicate that CT-1 can activate a distinct form of myocardial cell hypertrophy, characterized by the promotion of sarcomere assembly in series, via gpl30/LIFRbeta-dependent signaling pathways.

To study the molecular mechanisms that control patterning of the heart tube during early cardiogenesis, we have used the ventricular myosin regulatory light chain (MLC-2v), which is expressed in the ventricular segment of the primitive heart tube, as a genetic marker for ventricular specification in rodents. To assess whether the atrial isoform, MLC-2a, could also serve as a chamber-specific marker, we cloned an atrial MLC-2 cDNA (554 base pairs) which displayed homology to the human MLC-2a cDNA at both the nucleotide (87%) and amino acid (95%) levels. Northern blot, reverse transcriptase-linked polymerase chain reaction, RNase protection, and Western blot analysis revealed atrial restricted expression in the adult mouse heart, very low levels in aorta, and no detectable expression in ventricle, skeletal muscle, uterus, or liver. In situ hybridization studies during mouse embryogenesis revealed cardiac specific expression throughout days 8-16 postcoitum, with atrial restricted expression from day 12 and qualitatively greater atrial expression than ventricular from day 9. Thus, preferential pattern of expression in the atria occurs prior to septation. The MLC-2a gene was differentially regulated when compared with MLC-2v expression during embryonic stem cell cardiogenesis in vitro with MLC-2a transcript levels detectable from day 6 in suspension cultures as compared with day 9 for MLC-2v. The region-specific expression of the MLC-2a and MLC-2v genes in their respective chambers during early cardiogenesis provides genetic markers for chamber specification (atrial and ventricular) in both the in vitro and in vivo context.

alpha 1-Adrenergic agonists activate a hypertrophic response in cultured neonatal ventricular myocytes, which include an increase in cell size, organization of contractile proteins into sarcomeric units, and the induction of the atrial natriuretic factor (ANF) gene. Previous findings have supported a role for ras in this signaling pathway. Utilizing microinjection techniques to delivery affinity-purified neutralizing antibodies to G alpha q,11 into cultured ventricular myocytes, the current studies demonstrate a functional requirement for the heterotrimeric G protein, Gq, in the alpha 1-adrenergic induction of the ANF gene, changes in cell size, organization of myofilaments, and phosphoinositide hydrolysis. Expression of a constitutively active mutant of G alpha q leads to the expression of ANF protein in these cells. Taken together, these data suggest that G q-dependent pathways are necessary and sufficient to activate defined features of the hypertrophic response. In attempts to further delineate the relative roles of ras and Gq in this pathway, we found that G alpha q is required for alpha 1-adrenergic phosphoinositide hydrolysis, though ras does not appear to be necessary for this response. In addition, we coexpressed an inhibitory ras mutant, along with the constitutively active G alpha q. Expression of ANF protein stimulated by the G alpha q mutant was not inhibited. Thus, both ras- and Gq-dependent pathways are necessary to fully transduce defined features of alpha 1-adrenergic-stimulated hypertrophy of neonatal cardiac ventricular myocytes, but activated Gq may be able to induce ANF expression independent of inhibitory ras.

Over the last few years many of the difficulties encountered in applying molecular biology techniques to cardiac muscle have been addressed. Although continuous cardiac cell lines are still, as yet, unavailable, progress in embryonic stem cell systems and in our fundamental understanding of the critical steps that lead to cessation of cardiac myocyte cell division suggests that one may soon emerge. The cis-acting promoter elements and transacting factors responsible for cardiac specificity and inducible expression of cardiac genes are being characterized at a rapid pace. Transgenic models of cardiac disease are being generated and new insights are being made towards the goal of gene therapy in the heart. As the repertoire of techniques for transferring DNA into heart cells continues to expand, so the prospect for efficient and long-term expression of exogenous DNA in heart cells improves. The recent rapid rate of progress promises that ideas once fanciful may ultimately be within our reach.

alpha 1-Adrenergic agonists induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stimulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which alpha 1-adrenergic receptor subtype mediates the acquisition of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log Ki = -8.7) and (+)-niguldipine (log Ki = -10.6). The alpha-adrenergic receptor-induced increase in transcriptional activation of an ANF luciferase reporter gene is inhibited over the same range of concentrations of 5-methylurapidil (log Ki = -8.2) and (+)-niguldipine (log Ki = -11.2) that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies alpha-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurapidil (log Ki = -8.0) and (+)-niguldipine (log Ki = -10.6). In contrast, treatment with the alpha 1B selective antagonist chlorethylclonidine at a concentration of 10 microM had no effect on the adrenergically mediated induction of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alpha 1A-adrenergic receptors mediate not only the early effects of alpha 1-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using alpha 1-adrenergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, e.g. GTP binding proteins, should provide a powerful means of assessing their role in the induction of the molecular and morphologic parameters of myocardial cell hypertrophy.

We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By direct needle microinjection of activated Ras protein into primary neonatal rat ventricular cardiac myocytes, we find that, unlike many other cell types, Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c-Fos and atrial natriuretic factor (ANF) genes. Expression of both these genes is associated with the hypertrophic response in ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hypertrophic effect of an alpha-adrenergic agonist.

To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.

We evaluated the contributions of calcium loading and impaired energy production to metabolic and ultrastructural manifestations of cell injury in a cultured neonatal rat ventriculocyte model. Direct calcium loading was produced by incubation in K(+)-free medium to inhibit the Na+,K(+)-ATPase and promote Na(+)-Ca2+ exchange, and inhibition of energy metabolism was produced by incubation with 30 microM iodoacetic acid (IAA). Measurements were made of total cell calcium, [3H] arachidonic acid (AA) release (an index of membrane phospholipid degradation), ATP, and ultrastructural features of cell damage. Inhibition of the Na(+),K(+) pump resulted in the rapid onset of cellular calcium loading, increased [3H]AA release, and moderate ATP reduction. After return to control medium for 24 hours, myocytes previously exposed to K(+)-free medium for 1 hour showed recovery of ATP level and little additional [3H]AA release. However, after 2 to 3 hours of calcium loading, the ATP level remained moderately depressed, residual [3H]AA release was greater, and a mixed population of relatively normal and severely damaged myocytes was observed by electron microscopy. IAA treatment for 1 hour resulted in moderate ATP reduction without calcium accumulation or [3H]AA release, whereas IAA treatment for 3 hours resulted in marked ATP reduction associated with calcium accumulation and [3H]AA release. Reversal experiments showed substantial recovery of ATP level after 1 hour of IAA exposure, and marked ATP depression and [3H]AA release associated with widespread irreversible injury after 3 hours. Thus, the data indicate that increased calcium accumulation itself can initiate accelerated membrane phospholipid degradation, but that progression to irreversible injury is influenced by other factors, including the magnitude of ATP depression associated with calcium loading.

The present study was designed to determine if alpha- and beta-adrenergic stimulation of neonatal rat myocardial cells might induce common and/or distinct members of the immediate early gene program and to assess whether their induction might correlate with the differential effects of these adrenergic agonists on cardiac gene expression, sarcomere assembly, and several features of myocardial cell hypertrophy. Alpha- and beta-adrenergic stimulation of neonatal rat myocardial cells both produce an increase in the assembly of an individual contractile protein (myosin light chain-2) into organized sarcomeric units and also rapidly induce mRNAs for the immediate early genes c-fos and c-jun, thereby suggesting a potential role for these protooncogenes in sarcomerogenesis. alpha-Adrenergic stimulation results in the co-induction of mRNAs encoding a zinc finger protein gene (Egr-1). However, beta-adrenergic stimulation does not produce a significant increase in the levels of Egr-1 mRNA, providing the first evidence in any cell system that c-fos and Egr-1 expression can regulated separately. Studies with norepinephrine in combination with various adrenergic receptor antagonists revealed that the induction of Egr-1 is primarily an alpha 1-mediated, pertussis toxin-insensitive response. These studies provide the first evidence for the induction of immediate early genes following adrenergic stimulation of myocardial cells and demonstrate alpha- and beta-adrenergic stimulation can rapidly activate the expression of common and distinct subsets of these transcriptional regulators. Since alpha- and beta-adrenergic agonists have differential effects on cardiac gene expression and on the acquisition of several features of myocardial cell hypertrophy, the induction of Egr-1 provides a potential mechanism for the induction of genes that are exclusively induced during alpha-adrenergic-mediated myocardial cell hypertrophy.

Early in neonatal development, differentiated myocardial cells lose their ability to proliferate, and further enlargement of the heart occurs through hypertrophy of existing cardiac muscle cells. To study the process of myocardial growth and hypertrophy we have recently utilized a neonatal rat myocardial cell model (Lee, H. R., Henderson, S. A., Reynolds, R., Dunnmon, P., Yuan, D., and Chien, K. R. (1988) J. Biol. Chem. 263, 7352-7538). The present study was designed to determine if the expression of SV40 large T antigen would be capable of restoring the proliferative capacity of terminally differentiated neonatal rat myocardial cells. Utilizing a replication-defective recombinant human adenovirus which contains an SV40 early T antigen insert, maximal expression of T antigen was achieved at 24-48 h postinfection, with over 85-90% of the cells displaying positive T antigen staining. Furthermore, the expression of the T antigen-induced proliferation of the myocardial cells without the loss of expression of certain differentiated properties, including myosin light chain expression and assembly into organized myofibrils, spontaneous contractile activity, and a chronotropic response to adrenergic agonists. These results demonstrate the utility of recombinant human adenoviruses to achieve high efficiency transient expression of foreign genes in differentiated myocardial cells and suggest that the expression of T antigen may provide a suitable model to study the biochemical events which are required to maintain the proliferative capacity of myocardial cells.

We have isolated and characterized a cDNA recombinant plasmid (pRLC429) specific for the rat heart myosin light chain 2 (MLC2). The cDNA insert consists of 446 base pairs, including a 72-base pair segment of the 3'-untranslated region. Additional 5'-sequence, not present in plasmid pRLC429, was obtained by primer extension of the cDNA. The extended cDNA sequence combined with the plasmid pRLC429 sequence provided the codon information for the entire MLC2 polypeptide and partial sequences for the 3'- and 5'-noncoding regions of MLC2 mRNA. The predicted amino acid sequence for rat heart MLC2 showed a high homology with the sequences available for the chicken (83%) and human heart (80%) MLC2s. However, the homology between rat heart MLC2 and its counterpart in rat skeletal muscle is relatively low (67%). On the basis of the nuclease S1 protection assay with uniformly labeled single-stranded pRLC429 DNA, subcloned into M13mp18 phage vector, we conclude that the rat atrial muscle also contains MLC2 of the ventricular type. In an attempt to ascertain whether structural variants of MLC2 are expressed in hypertrophic heart muscle, we examined the RNAs from spontaneously hypertensive rat where there is a natural progression of hypertrophy associated with an increase in blood pressure. The RNA isolated from 7-, 13-, and 18-week-old spontaneously hypertensive rat hearts protected the same length DNA against S1 nuclease as was observed with RNAs from the age-matched normal rat hearts, suggesting that there is a single MLC2 gene transcript expressed in both the normal and hypertrophic heart muscle cells.

Alterations in myocardial membrane phospholipids may play an important role in the pathogenesis of ischaemic myocardial cell injury. Studies in canine myocardium, perfused rat heart, and cultured myocardial cells have demonstrated that the accumulation of free arachidonic acid correlates with the development of irreversible cell injury. Accumulation of other phospholipid hydrolysis products, including amphiphilic compounds such as lysophosphatidylcholine, has also been reported. The biochemical mechanisms which are responsible for phospholipid hydrolysis and arachidonic acid accumulation during ischaemia are unknown. This manuscript provides a synopsis of previous work in this field and suggests new directions for the field of myocardial phospholipid metabolism.

Several independent studies have demonstrated that there is a degradation of membrane phospholipids during myocardial ischemia. At present, most of the data support the initial activation of a phospholipase A pathway of phospholipid degradation. The extent of total phospholipid degradation is in the nanomole per gram wet weight quantity, as opposed to ischemic liver, in which the extent of phospholipid depletion approaches the micromole per gram wet weight level. However, in vitro studies suggest that calcium permeability properties and other myocardial cell membrane functions are sensitive to nanomole levels of phospholipid degradation. Clearly, further work is necessary in intact cell and heart preparations to correlate the degradation of phospholipid with the development of irreversible membrane injury during ATP depletion and hypoxia.

Previous studies have demonstrated that alpha-adrenergic stimulation of cultured, neonatal rat myocardial cells results in an increase in intracellular volume and protein content of cultured neonatal rat myocardial cells. Utilizing this model of cardiac hypertrophy, we have examined the effects of alpha-adrenergic stimulation on the accumulation of sarcomeres and the expression of a rat cardiac myofibrillar gene, myosin light chain-2 (MLC-2). Following alpha-adrenergic stimulation, cultured myocardial cells displayed a severalfold increase in the number of sarcomeric units, as assessed by electron microscopy, an increase in cellular MLC-2 content, and a 2-3 fold increase in the steady state levels of MLC-2 mRNA. This effect of alpha-adrenergic stimulation was accompanied by a 2-3-fold increase in total transcriptional activity, which was dependent on the concentration and duration of exposure to the agonist, and displayed alpha 1-adrenergic receptor specificity. The transcriptional response was not immediate, with a lag period of at least 1 h, and a maximal effect required continuous occupancy of the receptor. The increase in steady state levels of MLC-2 mRNA is regulated, in part, at the level of transcription of the cardiac MLC-2 gene. These results suggest that alpha 1-adrenergic stimulation may be important in the growth of the neonatal heart through the activation of total transcriptional activity. In addition, increases in the levels of myofibrillar proteins during myocardial cell growth and hypertrophy, may be mediated in part by the stimulation of transcription of myofibrillar genes.

The present study characterized the structure, organization, and expression of the rat cardiac myosin light chain (MLC) -2 gene. The rat cardiac MLC-2 gene has seven exons which display complete conservation with the exon structure of the rat fast twitch skeletal MLC-2 gene. A 250-base pair (bp) sequence of the 5'-flanking region contains CArG motifs and additional cis elements, each greater than 10 bp in length, which were conserved in sequence and relative position with the chick cardiac MLC-2 gene. A series of MLC-2/luciferase fusion genes consisting of nested 5' deletions of the MLC-2 5'-flanking region were constructed and transfected into primary neonatal rat myocardial cells and a non-myocardial cell line (CV-1), demonstrating that this 250 bp of the MLC-2 5'-flanking region was sufficient to confer cardiac specific expression on a luciferase reporter gene. This study suggests the presence of important proximal regulatory sequences in the MLC-2 5'-flanking region which are capable of directing the cardiac specific expression of the rat cardiac myosin light chain-2 gene.