Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticity-regulated PI3K/AKT pathway.

Matrix elasticity regulates proliferation, apoptosis, and differentiation of many cell types across various tissues. In particular, stiffened matrix in fibrotic lesions has been shown to promote pathogenic myofibroblast activation. To better understand the underlying pathways by which fibroblasts mechano-sense matrix elasticity, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves on poly(ethylene glycol)-based hydrogels with physiologically relevant and tunable elasticities. We show that soft hydrogels preserve the quiescent fibroblast phenotype of VICs much better than stiff plastic plates. We demonstrate that the PI3K/AKT pathway is significantly up-regulated when VICs are cultured on stiff gels or tissue culture polystyrene compared with freshly isolated VICs. In contrast, myofibroblasts de-activate and pAKT/AKT decreases as early as 2 h after reducing the substrate modulus. When PI3K or AKT is inhibited on stiff substrates, myofibroblast activation is blocked. When constitutively active PI3K is overexpressed, the myofibroblast phenotype is promoted even on soft substrates. These data suggest that valvular fibroblasts are sensing the changes in matrix elasticity through the PI3K/AKT pathway. This mechanism may be used by other mechano-sensitive cells in response to substrate modulus, and this pathway may be a worthwhile target for treating matrix stiffness-associated diseases. Furthermore, hydrogels can be designed to recapitulate important mechanical cues in native tissues to preserve aspects of the native phenotype of primary cells for understanding basic cellular responses to biophysical and biochemical signals, and for tissue-engineering applications.

A Conserved Mechanism of Cardiac Hypertrophy Regression through FoxO1.

The heart is a highly plastic organ that responds to diverse stimuli to modify form and function. The molecular mechanisms of adaptive physiological cardiac hypertrophy are well-established; however, the regulation of hypertrophy regression is poorly understood. To identify molecular features of regression, we studied Burmese pythons which experience reversible cardiac hypertrophy following large, infrequent meals. Using multi-omics screens followed by targeted analyses, we found forkhead box protein O1 (FoxO1) transcription factor signaling, and downstream autophagy activity, were downregulated during hypertrophy, but re-activated with regression. To determine whether these events were mechanistically related to regression, we established an in vitro platform of cardiomyocyte hypertrophy and regression from treatment with fed python plasma. FoxO1 inhibition prevented regression in this system, while FoxO1 activation reversed fed python plasma-induced hypertrophy in an autophagy-dependent manner. We next examined whether FoxO1 was implicated in mammalian models of reversible hypertrophy from exercise and pregnancy and found that in both cases FoxO1 was activated during regression. In these models, as in pythons, activation of FoxO1 was associated with increased expression FoxO1 target genes involved in autophagy. Taken together, our findings suggest FoxO1-dependent autophagy is a conserved mechanism for regression of physiological cardiac hypertrophy across species.

Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies.

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain (ß-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human ß-MHC Subfragment 1 (S1) in in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical function, including unchanged ATPase and enhanced actin velocity but had opposing effects on the super-relaxed (SRX) state of myosin. HCM-I467V S1 showed a small reduction in the SRX state, shifting myosin to a more actin-available state that may lead to the "gain-of-function" phenotype commonly described in HCM. In contrast, LVNC-I467T significantly increased the population of myosin in the ultra-slow SRX state. Interestingly, molecular dynamics simulations reveal that I467T allosterically disrupts interactions between ADP and the nucleotide-binding pocket, which may result in an increased ADP release rate. This predicted change in ADP release rate may define the enhanced actin velocity measured in LVNC-I467T, but also describe the uncoupled mechanochemical function for this mutation where the enhanced ADP release rate may be sufficient to offset the increased SRX population of myosin. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

A mediator of Rho-dependent invasion moonlights as a methionine salvage enzyme.

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Intrathecal injection of naked plasmid DNA provides long-term expression of secreted proteins.

Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (approximately vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF.

Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression.

BACKGROUND: One method for the delivery of therapeutic proteins to the spinal cord is to inject nonviral gene vectors including plasmid DNA into the cerebrospinal fluid (CSF) that surrounds the spinal cord (intrathecal space). This approach has produced therapeutic benefits in animal models of disease and several months of protein expression; however, there is little information available on the immune response to these treatments in the intrathecal space, the relevance of plasmid CpG sequences to any plasmid-induced immune response, or the effect of this immune response on transgene expression. METHODS: In the present study, coding or noncoding plasmids were delivered to the intrathecal space of the lumbar spinal region in rats. Lumbosacral CSF was then collected at various time points afterwards for monitoring of cytokines and transgene expression. RESULTS: This work demonstrates, for the first time, increased tumor necrosis factor-alpha and interleukin-1 in response to intrathecal plasmid vector injection and provides evidence indicating that this response is largely absent in a CpG-depleted vector. Transgene expression in the CSF is not significantly affected by this immune response. Expression after intrathecal plasmid injection is variable across rats but correlates with the amount of tissue associated plasmid and is increased by disrupting normal CSF flow. CONCLUSIONS: The data obtained in the present study indicate that plasmid immunogenicity may affect intrathecal plasmid gene therapy safety but not transgene expression in the CSF. Furthermore, the development of methods to prevent loss of plasmid via CSF flow out of the central nervous system through the injection hole and/or natural outflow routes may increase intrathecal plasmid gene delivery efficacy.

A randomized library approach to identifying functional lox site domains for the Cre recombinase.

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.

The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression.

Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.

A genetic screen identifies novel non-compatible loxP sites.

The ability of the Cre/lox system to make precise genomic modifications is a tremendous accomplishment. However, recombination between cis-linked heterospecific lox sites limits the use of Cre- mediated exchange of DNA to systems where genetic selection can be applied. To circumvent this problem we carried out a genetic screen designed to identify novel mutant spacer-containing lox sites displaying enhanced incompatibility with the canonical loxP site. One of the mutant sites recovered appears to be completely stable in HEK293 cells constitutively expressing Cre recombinase and supports recombinase-mediated cassette exchange (RMCE) in bacteria and mammalian cell culture. By preventing undesirable recombination, these novel lox sites could improve the efficiency of in vivo gene transfer.

Estrogen receptor profiling and activity in cardiac myocytes.

Estrogen signaling appears critical in the heart. However a mechanistic understanding of the role of estrogen in the cardiac myocyte is lacking. Moreover, there are multiple cell types in the heart and multiple estrogen receptor (ER) isoforms. Therefore, we studied expression, localization, transcriptional and signaling activity of ERs in isolated cardiac myocytes. We found only ERα RNA (but no ERß RNA) in cardiac myocytes using two independent methods. The vast majority of full-length ERα protein (ERα66) localizes to cardiac myocyte nuclei where it is competent to activate transcription. Alternate isoforms of ERα encoded by the same genomic locus (ERα46 and ERα36) have differential transcriptional activity in cardiac myocytes but also primarily localize to nuclei. In contrast to other reports, no ERα isoform is competent to activate MAPK or PI3K signaling in cardiac myocytes. Together these data support a role for ERα at the level of transcription in cardiac myocytes.

Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10.

Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.

Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats.

Paclitaxel is a commonly used cancer chemotherapy drug that frequently causes painful peripheral neuropathies. The mechanisms underlying this dose-limiting side effect are poorly understood. Growing evidence supports that proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), released by activated spinal glial cells and within the dorsal root ganglia (DRG) are critical in enhancing pain in various animal models of neuropathic pain. Whether these cytokines are involved in paclitaxel-induced neuropathy is unknown. Here, using a rat neuropathic pain model induced by repeated systemic paclitaxel injections, we examined whether paclitaxel upregulates proinflammatory cytokine gene expression, and whether these changes and paclitaxel-induced mechanical allodynia can be attenuated by intrathecal IL-1 receptor antagonist (IL-1ra) or intrathecal delivery of plasmid DNA encoding the anti-inflammatory cytokine, interleukin-10 (IL-10). The data show that paclitaxel treatment induces mRNA expression of IL-1, TNF, and immune cell markers in lumbar DRG. Intrathecal IL-1ra reversed paclitaxel-induced allodynia and intrathecal IL-10 gene therapy both prevented, and progressively reversed, this allodynic state. Moreover, IL-10 gene therapy resulted in increased IL-10 mRNA levels in lumbar DRG and meninges, measured 2 weeks after initiation of therapy, whereas paclitaxel-induced expression of IL-1, TNF, and CD11b mRNA in lumbar DRG was markedly decreased. Taken together, these data support that paclitaxel-induced neuropathic pain is mediated by proinflammatory cytokines, possibly released by activated immune cells in the DRG. We propose that targeting the production of proinflammatory cytokines by intrathecal IL-10 gene therapy may be a promising therapeutic strategy for the relief of paclitaxel-induced neuropathic pain.

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling.

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.

Intrathecal polymer-based interleukin-10 gene delivery for neuropathic pain.

Research on communication between glia and neurons has increased in the past decade. The onset of neuropathic pain, a major clinical problem that is not resolved by available therapeutics, involves activation of spinal cord glia through the release of proinflammatory cytokines in acute animal models of neuropathic pain. Here, we demonstrate for the first time that the spinal action of the proinflammatory cytokine, interleukin 1 (IL-1) is involved in maintaining persistent (2 months) allodynia induced by chronic-constriction injury (CCI). The anti-inflammatory cytokine IL-10 can suppress proinflammatory cytokines and spinal cord glial amplification of pain. Given that IL-1 is a key mediator of neuropathic pain, developing a clinically viable means of long-term delivery of IL-10 to the spinal cord is desirable. High doses of intrathecal IL-10-gene therapy using naked plasmid DNA (free pDNA-IL-10) is effective, but the dose required limits its potential clinical utility. Here we show that intrathecal gene therapy for neuropathic pain is improved sufficiently using two, distinct synthetic polymers, poly(lactic-co-glycolic) and polyethylenimine, that substantially lower doses of pDNA-IL-10 are effective. In conclusion, synthetic polymers used as i.t. gene-delivery systems are well-tolerated and improve the long-duration efficacy of pDNA-IL-10 gene therapy.

Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain.

Neuropathic pain is a major clinical problem unresolved by available therapeutics. Spinal cord glia play a pivotal role in neuropathic pain, via the release of proinflammatory cytokines. Anti-inflammatory cytokines, like interleukin-10 (IL-10), suppress proinflammatory cytokines. Thus, IL-10 may provide a means for controlling glial amplification of pain. We recently documented that intrathecal IL-10 protein resolves neuropathic pain, albeit briefly (approximately 2-3 h), given its short half-life. Intrathecal gene therapy using viruses encoding IL-10 can also resolve neuropathic pain, but for only approximately 2 weeks. Here, we report a novel approach that dramatically increases the efficacy of intrathecal IL-10 gene therapy. Repeated intrathecal delivery of plasmid DNA vectors encoding IL-10 (pDNA-IL-10) abolished neuropathic pain for greater than 40 days. Naked pDNA-IL-10 reversed chronic constriction injury (CCI)-induced allodynia both shortly after nerve injury as well as 2 months later. This supports that spinal proinflammatory cytokines are important in both the initiation and maintenance of neuropathic pain. Importantly, pDNA-IL-10 gene therapy reversed mechanical allodynia induced by CCI, returning rats to normal pain responsiveness, without additional analgesia. Together, these data suggest that intrathecal IL-10 gene therapy may provide a novel approach for prolonged clinical pain control.

Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10.

Gene therapy for the control of pain has, to date, targeted neurons. However, recent evidence supports that spinal cord glia are critical to the creation and maintenance of pain facilitation through the release of proinflammatory cytokines. Because of the ability of interleukin-10 (IL-10) to suppress proinflammatory cytokines, we tested whether an adenoviral vector encoding human IL-10 (AD-h-IL10) would block and reverse pain facilitation. Three pain models were examined, all of which are mediated by spinal pro-inflammatory cytokines. Acute intrathecal administration of rat IL-10 protein itself briefly reversed chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia. The transient reversal caused by IL-10 protein paralleled the half-life of human IL-10 protein in the intrathecal space (t(1/2) approximately 2 h). IL-10 gene therapy both prevented and reversed thermal hyperalgesia and mechanical allodynia, without affecting basal responses to thermal or mechanical stimuli. Extra-territorial, as well as territorial, pain changes were reversed by this treatment. Intrathecal AD-h-IL10 injected over lumbosacral spinal cord led to elevated lumbosacral cerebrospinal fluid (CSF) levels of human IL-10, with far less human IL-10 observed in cervical CSF. In keeping with IL-10's known anti-inflammatory actions, AD-h-IL10 lowered CSF levels of IL-1, relative to control AD. These studies support that this gene therapy approach provides an alternative to neuronally focused drug and gene therapies for clinical pain control.

Acute effects of desmin mutations on cytoskeletal and cellular integrity in cardiac myocytes.

Mutations in desmin have been associated with a subset of human myopathies. Symptoms typically appear in the second to third decades of life, but in the most severe cases can manifest themselves earlier. How desmin mutations lead to aberrant muscle function, however, remains poorly defined. We created a series of four mutations in rat desmin and tested their in vitro filament assembly properties. RDM-G, a chimera between desmin and green fluorescent protein, formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro assembly at the unit-length filament stage, while RDM-3 had more subtle effects on assembly. When expressed in cultured rat neonatal cardiac myocytes via adenovirus infection, these mutant proteins disrupted the endogenous desmin filament to an extent that correlated with their defects in in vitro assembly properties. Disruption of the desmin network by RDM-1 was also associated with disruption of plectin, myosin, and alpha-actinin organization in a significant percentage of infected cells. In contrast, expression of RDM-2, which is similar to previously characterized human mutant desmins, took longer to disrupt desmin and plectin organization and had no significant effect on myosin or alpha-actinin organization over the 5-day time course of our studies. RDM-3 had the mildest effect on in vitro assembly and no discernable effect on either desmin, plectin, myosin, or alpha-actinin organization in vivo. These results indicate that mutations in desmin have both direct and indirect effects on the cytoarchitecture of cardiac myocytes.

Rational promoter selection for gene transfer into cardiac cells.

Cardiomyocytes (CMCs) are extremely difficult to transfect with non-viral techniques, but they are efficiently infected by adenoviruses. The most commonly used promoters to drive protein expression in cardiac myocytes are of viral origin, since they are believed to be constitutively active and minimally regulated by physiological or pharmacological challenge of cells. In recombinant adenoviruses, we systematically compared three different promoters: the cytomegalovirus (CMV), the Rous sarcoma virus (RSV), and a synthetic promoter with three MEF2 transcription factor-binding sites upstream of the heat-shock protein 68 minimal promoter. We determined their basal activity in primary cardiac cells as well as their possible stimulation by commonly used agonists. The CMV promoter was activated up to 60-fold by the phorbol ester phorbol myristate acetate (PMA) and/or forskolin in neonatal rat CMCs and cardiac fibroblasts. Primary adult rat CMCs had higher basal expression from the CMV promoter that was not activated by PMA or forskolin. The RSV promoter was less affected by agonists and was more active in cardiac myocytes compared to cardiac fibroblasts. The MEF2-responsive promoter showed high basal expression in both myocytes and fibroblasts, and minimal induction by phorbol esters and forskolin. The relevance of reporter gene induction was confirmed with a contractile protein, troponin T (TnT). The CMV promoter driving TnT could be induced more than 15-fold with phenylephrine or forskolin to replace the endogenous protein almost to completion at a multiplicity of infection of 10. These results suggest the following use of the tested promoters: an inducible system (CMV), a myocyte-enriched system (RSV), or a stable control system (MEF2).