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180 results found
Shou W, Aghdasi B, Armstrong DL, et al., 1998, Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12., Nature, Vol: 391, Pages: 489-492, ISSN: 0028-0836
FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems. Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial. FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine receptor (RyR1). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6. To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium. About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.
Agah R, Kirshenbaum LA, Abdellatif M, et al., 1997, Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo., J Clin Invest, Vol: 100, Pages: 2722-2728, ISSN: 0021-9738
Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.
MacLellan WR, Schneider MD, 1997, Death by design. Programmed cell death in cardiovascular biology and disease., Circ Res, Vol: 81, Pages: 137-144, ISSN: 0009-7330
Programmed cell death (apoptosis) is recognized, increasingly, as a contributing cause of cardiac myocyte loss with ischemia/reperfusion injury, myocardial infarction, and long-standing heart failure. Although the exact mechanisms initiating apoptosis in these in vivo settings remain unproven, insights into the molecular circuitry controlling apoptosis more widely suggest the potential to protect mammalian ventricular muscle from apoptosis through one or more of these pathways, by pharmacological means or, conceivably, gene transfer.
Agah R, Frenkel PA, French BA, et al., 1997, Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo., J Clin Invest, Vol: 100, Pages: 169-179, ISSN: 0021-9738
Mouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations. However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology. Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells. Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alphaMyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alphaMyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein. Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells. Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.
Abdellatif M, Schneider MD, 1997, An effector-like function of Ras GTPase-activating protein predominates in cardiac muscle cells., J Biol Chem, Vol: 272, Pages: 525-533, ISSN: 0021-9258
In contrast to familiar role for Ras in proliferation, we and others previously suggested that Ras also mediates hypertrophy, the increase in cell mass characteristic of post-natal ventricular muscle. We showed that activated (G12R) and dominant-negative (S17N) Ha-Ras regulate "constitutive" and growth factor-responsive genes equivalently, in both cardiac myocytes and non-cardiac, Mv1Lu cells. Here, we attempt to delineate pathways by which Ras exerts this global effect. The E63K mutation, which impairs binding of guanine nucleotide releasing factor to Ras, alleviated suppression by S17N, consistent with sequestration of exchange factors as the mechanism for inhibition. To compare potential Ras effector proteins, we first engineered G12R/D38N, to abolish binding of Raf and phosphatidylinositol-3-kinase and established that this site was indispensable for augmenting gene expression. To distinguish between inhibition of Ras by Ras GTPase-activating protein (GAP) versus a potential effector function of GAP, we tested the effector domain substitution P34R: this mutation, which abolishes GAP binding, enhanced Ras-dependent transcription in Mv1Lu cells, yet interfered with Ras-dependent expression in ventricular myocytes. To examine the dichotomous role of Ras-GAP predicted from these P34R results, we transfected both cell types with full-length GAP, the C-terminal catalytic domain (cGAP), or N-terminal Src homology domains (nGAP). In Mv1Lu cells, cGAP markedly inhibited both reporter genes, whereas GAP and nGAP had little effect. Antithetically, in ventricular myocytes, GAP and nGAP activated gene expression, whereas cGAP was ineffective. Thus, Ras activates gene expression through differing effectors contingent on cell type, and an effector-like function of GAP predominates in ventricular muscle.
Schneider MD, 1996, Myocardial infarction as a problem of growth control: cell cycle therapy for cardiac myocytes?, J Card Fail, Vol: 2, Pages: 259-263, ISSN: 1071-9164
Pump failure after myocardial infarction ultimately can be ascribed, in large part, to the inability of ventricular muscle to regenerate functional mass through cell proliferation. Recent studies using adenoviral gene transfer have provided direct evidence for the operation of two growth-suppressing pathways in cardiac muscle, via "pocket proteins," including the retinoblastoma gene product, and via a less well understood protein, p300. An understanding of molecular mechanisms that confer a virtually irreversible lock to the proliferative cell cycle in "postmitotic" cardiac muscle, together with improved means for delivery of exogenous genes to the heart, suggests the long-term potential for manipulating cardiac growth to achieve a therapeutic benefit.
Paradis P, MacLellan WR, Belaguli NS, et al., 1996, Serum response factor mediates AP-1-dependent induction of the skeletal alpha-actin promoter in ventricular myocytes., J Biol Chem, Vol: 271, Pages: 10827-10833, ISSN: 0021-9258
"Fetal" gene transcription, including activation of the skeletal alpha-actin (SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic ligands. Induction of the promoter by transforming growth factor beta or norepinephrine requires serum response factor (SRF) and TEF-1; expression is inhibited by YY1. We and others postulated that immediate-early transcription factors might couple trophic signals to this fetal program. However, multiple Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We therefore cotransfected ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to SREs, nor physical interaction between Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene via SRF, without detectable binding to the promoter or to SRF.
Brand T, Schneider MD, 1996, Transforming growth factor-beta signal transduction, CIRCULATION RESEARCH, Vol: 78, Pages: 173-179, ISSN: 0009-7330
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- Citations: 39
Brand T, Schneider MD, 1995, Inactive type II and type I receptors for TGF beta are dominant inhibitors of TGF beta-dependent transcription., J Biol Chem, Vol: 270, Pages: 8274-8284, ISSN: 0021-9258
Although transforming growth factor-beta (TGF beta) is implicated in differentiation and disease, proof of in vivo function requires specific inhibitors of the TGF beta cascade. TGF beta binds a family of type I and type II receptors (T beta RI, T beta RII), containing a cytoplasmic serine/threonine kinase domain. We previously reported that kinase-deficient T beta RII (delta kT beta RII) blocks TGF beta-dependent transcription in cardiac myocytes. It is controversial whether both receptors are needed in all cells for gene regulation by TGF beta or whether they mediate distinct subsets of TGF beta-dependent events. To resolve this uncertainty, TGF beta-dependent transcription was investigated in cardiac myocytes versus mink lung epithelial cells. 1) delta kT beta RII inhibits induction of a TGF beta-responsive reporter gene, in both cell backgrounds. 2) Charged-to-alanine mutations of key residues of the T beta RII kinase, including consensus ATP binding and amino acid recognition motifs, are competent for binding but not transcriptional activation. Each inactive receptor inhibits TGF beta-dependent transcription in both cell types. 3) Kinase-deficient T beta RI (delta kT beta RI) likewise impairs TGF beta-dependent transcription, less completely than delta kT beta RII; kinase-deficient activin type I receptor has no effect. 4) TGF beta-binding proteins in cardiac cells and Mv1Lu cells are comparable by affinity labeling and immunoprecipitation; however, Mv1Lu cells express up to 3-fold higher levels of T beta RII and T beta RI. Thus, the model inferred from TGF beta-resistant cell lines (that T beta RII and T beta RI are necessary in tandem for the TGF beta-signaling complex to regulate transcription) is valid for cardiac myocytes, the cell type most prominently affected in TGF beta-deficient animals.
Kirshenbaum LA, Schneider MD, 1995, Adenovirus E1A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains., J Biol Chem, Vol: 270, Pages: 7791-7794, ISSN: 0021-9258
To examine the potential impact of disrupting "pocket" protein function on cardiac differentiation and growth, we introduced 12 S E1A genes into neonatal ventricular myocytes, by adenoviral gene transfer. In the absence of E1B, E1A was cytotoxic, with features typical of apoptosis. In the presence of E1B, E1A preferentially inhibited transcription of cardiac-restricted alpha-actin promoters, and reactivated DNA synthesis in cardiac myocytes, without cell death. Mutations that abrogate known activities of the amino terminus of E1A, versus conserved region 2, demonstrate that the "pocket" protein- and p300-binding domains each suffice, in the absence of the other, for transcriptional repression and re-entry into S phase.
Schneider MD, Brand T, 1995, Molecular analysis of TGF beta signal transduction. Dominant-inhibitory mutations of the type II and type I TGF beta receptor., Ann N Y Acad Sci, Vol: 752, Pages: 309-316, ISSN: 0077-8923
BRAND T, SCHNEIDER MD, 1995, THE TGF-BETA SUPERFAMILY IN MYOCARDIUM - LIGANDS, RECEPTORS, TRANSDUCTION, AND FUNCTION, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, Vol: 27, Pages: 5-18, ISSN: 0022-2828
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- Citations: 84
Kirshenbaum LA, Angelides KJ, Schneider MD, 1994, Images in cardiovascular medicine. Detection of exogenous gene expression in live adult ventricular myocytes after adenoviral gene delivery., Circulation, Vol: 90, Pages: 2124-2125, ISSN: 0009-7322
MacLellan WR, Lee TC, Schwartz RJ, et al., 1994, Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1., J Biol Chem, Vol: 269, Pages: 16754-16760, ISSN: 0021-9258
Skeletal alpha-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, down-regulated after birth, and reactivated by trophic signals including type beta-transforming growth factors (TGF beta). To investigate the molecular basis for cardiac-restricted and TGF beta-induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtually abolish SkA transcription in both TGF beta- and vehicle-treated cells; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases both basal and TGF beta-dependent expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. However, efficient SkA transcription requires combinatorial action of SRE1 with consensus sites for Sp1 and the SV40 enhancer-binding protein, TEF-1. As isolated motifs, either SRE1- or TEF-1-binding sites function as TGF beta response elements. Induction of the SkA promoter by TGF beta required SRF and TEF-1 in concert, unlike other pathways for TGF beta-dependent gene expression.
Abdellatif M, MacLellan WR, Schneider MD, 1994, p21 Ras as a governor of global gene expression., J Biol Chem, Vol: 269, Pages: 15423-15426, ISSN: 0021-9258
The molecular mechanisms for signaling by receptor serine/threonine kinases are incompletely understood. To test the potential involvement of p21 H-Ras proteins in signal transduction for type beta transforming growth factors (TGF beta), TGF beta-responsive and constitutive reporter genes were cotransfected into cardiac myocytes and mink lung epithelial cells, with dominant inhibitory (Asn-17) or activated (Arg-12) Ras expression vectors. Asn-17 Ras inhibited both TGF beta-dependent and basal expression of inducible promoters (skeletal alpha-actin and plasminogen activator inhibitor-1), with equivalent dose-response relations. All seven reporter constructs were comparably sensitive to down-regulation by Asn-17 Ras, including those driven by nominally constitutive viral control regions or a TATA-less initiator element. All constructs were up-regulated by Arg-12 Ras more variably. Wild-type Ras had intermediate effects and could rescue a minimal thymidine kinase promoter from inhibition by dominant negative Ras. Thus, a Ras-dependent event is required for efficient expression of an unexpectedly global or inclusive set of genes.
Schneider MD, Abdellatif M, Kirshenbaum LA, et al., 1994, Regulatory circuits for myocardial hypertrophy, Heart Failure, Vol: 10, Pages: 166-176, ISSN: 8755-7673
Adaptation of ventricular muscle to mechanical load has been postulated to involve plasticity of expression in cardiac genes encoding components of the sarcomere, proteins affecting calcium homeostasis, acutely inducible transcription factors, and myocardial growth factors including transforming growth factor-β (TGFβ). Evidence is reviewed for the hypothesis that TGFβ and other cardiac growth factors function in a hierarchical pathway that can largely account for the characteristic changes produced by load. Strengths and limitations of genetic methods are discussed, in relation to the potential use of a dominant-negative receptor for TGFβ as an organ-specific inhibitor of TGFβ effects.
MACLELLAN WR, BRAND T, SCHNEIDER MD, 1993, TRANSFORMING GROWTH-FACTOR-BETA IN CARDIAC ONTOGENY AND ADAPTATION, CIRCULATION RESEARCH, Vol: 73, Pages: 783-791, ISSN: 0009-7330
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- Citations: 77
MacLellan WR, Brand T, Schneider MD, 1993, Transforming growth factor-beta in cardiac ontogeny and adaptation., Circ Res, Vol: 73, Pages: 783-791, ISSN: 0009-7330
The transforming growth factor-beta (TGF-beta) superfamily comprises a set of regulatory peptides with multiple effects on cell growth and differentiation. The elaborate regulation of TGF-beta s during embryonic development of the heart, the upregulation of TGF-beta after hemodynamic stress, and the impact of TGF-beta on cardiac gene expression together imply a prominent functional role for this family of growth factors in cardiac organogenesis and hypertrophy. Basal and TGF-beta-induced expression of skeletal alpha-actin, one of several genes specifically associated with developing or hypertrophied myocardium, each are contingent on transcriptional activation by serum response factor. A truncated form of the type II TGF-beta receptor, created by deletion of the cytoplasmic kinase domain, acts as a dominant suppressor of TGF-beta signal transduction in cultured cardiac muscle cells and may provide a suitable means to establish the functions of TGF-beta in vivo.
Schneider MD, French BA, 1993, The advent of adenovirus. Gene therapy for cardiovascular disease., Circulation, Vol: 88, Pages: 1937-1942, ISSN: 0009-7322
Kirshenbaum LA, MacLellan WR, Mazur W, et al., 1993, Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus., J Clin Invest, Vol: 92, Pages: 381-387, ISSN: 0021-9738
Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells.
BRAND T, MACLELLAN WR, SCHNEIDER MD, 1993, A DOMINANT-NEGATIVE RECEPTOR FOR TYPE-BETA TRANSFORMING GROWTH-FACTORS CREATED BY DELETION OF THE KINASE DOMAIN, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 268, Pages: 11500-11503, ISSN: 0021-9258
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- Citations: 111
Parker TG, Chow KL, Schwartz RJ, et al., 1992, Positive and negative control of the skeletal alpha-actin promoter in cardiac muscle. A proximal serum response element is sufficient for induction by basic fibroblast growth factor (FGF) but not for inhibition by acidic FGF., J Biol Chem, Vol: 267, Pages: 3343-3350, ISSN: 0021-9258
Like mechanical load in vivo, basic fibroblast growth factor (bFGF) selectively provokes cardiac expression of "fetal" genes including skeletal alpha-actin (SkA). Antithetically, acidic FGF (aFGF) suppresses SkA transcription. To define sites controlling SkA transcription in cardiac muscle cells, rat cardiac myocytes were transfected with internal-deletion and block-substitution mutations in the SkA promoter, including three motifs resembling the fos serum response element (SRE). The upstream, central, and proximal SREs each contributed to basal expression in cardiac myocytes. To determine whether identical elements mediate induction by bFGF versus inhibition by aFGF, the proximal SRE (SRE1) and fos SRE were positioned upstream from a neutral promoter. In cardiac myocytes, both the SRE1 and fos SRE were expressed at levels up to one-third that of the SkA promoter (nucleotides -202 to -11). Neither was expressed in quiescent cardiac fibroblasts. bFGF augmented SRE1-CAT activity, whereas aFGF produced no change; the fos SRE was induced by both. The transcriptional and mitogenic actions of aFGF were contingent on the presence of a putative nuclear translocation motif. Thus 1) the SkA SRE1 and fos SRE each suffice for tissue specificity in cardiac myocytes; 2) unlike the c-fos SRE, the SkA SRE1 is induced selectively by bFGF yet not aFGF; 3) sequences alternative or in addition to the SRE1 are obligatory for aFGF to suppress the SkA promoter; and 4) possible differences in intracellular localization are one basis for divergent actions of aFGF and bFGF in cardiac muscle cells.
Black FM, Packer SE, Parker TG, et al., 1991, The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load., J Clin Invest, Vol: 88, Pages: 1581-1588, ISSN: 0021-9738
Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; however, it is not known whether reactivation of the smooth muscle alpha-actin gene occurs in ventricular hypertrophy. We therefore investigated myocardial expression of the smooth muscle alpha-actin gene after hemodynamic overload. Smooth muscle alpha-actin mRNA was discernible 24 h after coarctation and was persistently expressed for up to 30 d. In hypertrophied hearts, the prevalence of smooth muscle alpha-actin gene induction was 0.909, versus 0.545 for skeletal muscle alpha-actin (P less than 0.05). Ventricular mass after 2 d or more of aortic constriction was more highly correlated with smooth muscle alpha-actin gene activation (r = 0.852; P = 0.0001) than with skeletal muscle alpha-actin (r = 0.532; P = 0.009); P less than 0.0005 for the difference in the correlation coefficients. Thus, smooth muscle alpha-actin is a molecular marker of the presence and extent of pressure-overload hypertrophy, whose correlation with cardiac growth at least equals that of skeletal alpha-actin. Induction of smooth muscle alpha-actin was delayed and sustained after aortic constriction, whereas the nuclear oncogenes c-jun and junB were expressed rapidly and transiently, providing potential dimerization partners for transcriptional control by c-fos.
Parker TG, Chow KL, Schwartz RJ, et al., 1991, TGF-beta 1 and fibroblast growth factors selectively up-regulate tissue-specific fetal genes in cardiac muscle cells., Ciba Found Symp, Vol: 157, Pages: 152-160, ISSN: 0300-5208
TGF-beta 1, like basic and acidic fibroblast growth factor (FGF), inhibits differentiated gene expression in skeletal myoblasts. It potentiates FGF-beta 1 down-regulated expression of the alpha-myosin heavy chain gene and the sarcoplasmic reticulum calcium ATPase gene, yet up-regulated expression of the genes for beta-myosin heavy chain, atrial natriuretic factor, and both skeletal and smooth muscle alpha-actin-four transcripts associated with the embryonic heart. TGF-beta 1 did not affect cardiac alpha-actin gene expression. These responses resemble the generalized 'fetal' phenotype seen during hypertrophy triggered by a haemodynamic load. Chick skeletal and cardiac alpha-actin promoter-driven reported genes were transfected into neonatal rat cardiac myocytes. TGF-beta 1 stimulated skeletal alpha-actin transcription, but not transcription from the cardiac alpha-actin promoter. Basic FGF produced the same results as TGF-beta 1, but acidic FGF suppressed expression of both alpha-actin genes; these results were true for purified and recombinant FGFs. Modulation of alpha-actin transcription by growth factors corresponded accurately to control of the endogenous genes. Three positive cis-acting elements were critical for skeletal alpha-actin transcription in cardiac, as well as skeletal, myocytes, particularly the downstream CCAAT box-associated repeat. Thus, TGF-beta 1 and FGFs selectively induce an ensemble of 'fetal' genes and differentially regulate alpha-actin transcription in cardiac muscle cells.
Parker TG, Schneider MD, 1991, Growth factors, proto-oncogenes, and plasticity of the cardiac phenotype., Annu Rev Physiol, Vol: 53, Pages: 179-200, ISSN: 0066-4278
Parker TG, Chow KL, Schwartz RJ, et al., 1990, Differential regulation of skeletal alpha-actin transcription in cardiac muscle by two fibroblast growth factors., Proc Natl Acad Sci U S A, Vol: 87, Pages: 7066-7070, ISSN: 0027-8424
In cardiac muscle, acidic and basic fibroblast growth factors (aFGF and bFGF) regulate at least five genes in common (including alpha and beta myosin heavy chains, atrial natriuretic factor, and the sarcoplasmic reticulum calcium ATPase), provoking a generalized "fetal" phenotype similar to events in pressure-overload hypertrophy; however, aFGF and bFGF differentially control the striated alpha-actins. bFGF stimulates and aFGF inhibits skeletal alpha-actin transcripts associated with the embryonic heart, whereas cardiac alpha-actin mRNA is inhibited by aFGF but not bFGF. To elucidate mechanisms for these selective and discordant actions of aFGF and bFGF on cardiac muscle, chicken skeletal and cardiac alpha-actin promoter-driven reporter genes were introduced into neonatal rat cardiac myocytes by electroporation. Skeletal alpha-actin transcription was selectively stimulated by bFGF, whereas the cardiac alpha-actin promoter was unaffected. In contrast, aFGF suppressed both transfected alpha-actin genes. The differential regulation of skeletal alpha-actin transcription was equivalent with either purified or recombinant FGFs and was observed with 5' flanking sequences from either nucleotide -202 or -2000 to nucleotide -11. Positive and negative modulation of alpha-actin transcription by growth factors corresponded accurately to the endogenous genes in all permutations studied. These investigations provide a model for reciprocal control of gene transcription by aFGF vs. bFGF.
Schneider MD, Parker TG, 1990, Cardiac myocytes as targets for the action of peptide growth factors., Circulation, Vol: 81, Pages: 1443-1456, ISSN: 0009-7322
Shih HT, Wathen MS, Marshall HB, et al., 1990, Dihydropyridine receptor gene expression is regulated by inhibitors of myogenesis and is relatively insensitive to denervation., J Clin Invest, Vol: 85, Pages: 781-789, ISSN: 0021-9738
To evaluate developmental and physiological signals that may influence expression of the dihydropyridine-sensitive "slow" Ca2+ channel, we analyzed dihydropyridine receptor (DHPR) mRNA abundance in mouse skeletal muscle. Using synthetic oligonucleotide probes corresponding to the rabbit skeletal muscle DHPR, a 6.5 kb DHPR transcript was identified in postnatal skeletal muscle and differentiated C2 or BC3H1 myocytes, but not cardiac muscle or brain. DHPR gene expression was reversibly suppressed by 0.4 nM transforming growth factor beta-1 or by transfection with a mutant c-H-ras allele, nominal inhibitors of myogenesis that block the appearance of slow channels and DHPR. In contrast, both BC3H1 and C2 myocytes containing the activated ras vector expressed the gene encoding the nicotinic acetylcholine receptor delta subunit, demonstrating that not all muscle-specific genes are extinguished by ras. Denervation stimulated DHPR gene expression less than 0.6-fold, despite 8-fold upregulation of delta-subunit mRNA and reciprocal effects on the skeletal and cardiac alpha-actin genes. Thus, DHPR gene induction is prevented by inhibitors of other muscle-specific genes, whereas, at most, relatively small changes in DHPR mRNA abundance occur during adaptation to denervation.
Parker TG, Packer SE, Schneider MD, 1990, Peptide growth factors can provoke "fetal" contractile protein gene expression in rat cardiac myocytes., J Clin Invest, Vol: 85, Pages: 507-514, ISSN: 0021-9738
Cardiac-specific gene expression is intricately regulated in response to developmental, hormonal, and hemodynamic stimuli. To test whether cardiac muscle might be a target for regulation by peptide growth factors, the effect of three growth factors on the actin and myosin gene families was investigated by Northern blot analysis in cultured neonatal rat cardiac myocytes. Transforming growth factor-beta 1 (TGF beta 1, 1 ng/ml) and basic fibroblast growth factor (FGF, 25 ng/ml) elicited changes corresponding to those induced by hemodynamic load. The "fetal" beta-myosin heavy chain (MHC) was up-regulated about four-fold, whereas the "adult" alpha MHC was inhibited greater than 50-60%; expression of alpha-skeletal actin increased approximately two-fold, with little or no change in alpha-cardiac actin. Thus, peptide growth factors alter the program of differentiated gene expression in cardiac myocytes, and are sufficient to provoke fetal contractile protein gene expression, characteristic of pressure-overload hypertrophy. Acidic FGF (25 ng/ml) produced seven- to eightfold reciprocal changes in MHC expression but, unlike either TGF-beta 1 or basic FGF, inhibited both striated alpha-actin genes by 70-90%. Expression of vascular smooth muscle alpha-actin, the earliest alpha-actin induced during cardiac myogenesis, was increased by all three growth factors. Thus, three alpha-actin genes demonstrate distinct responses to acidic vs. basic FGF.
Payne PA, Olson EN, Hsiau P, et al., 1987, An activated c-Ha-ras allele blocks the induction of muscle-specific genes whose expression is contingent on mitogen withdrawal., Proc Natl Acad Sci U S A, Vol: 84, Pages: 8956-8960, ISSN: 0027-8424
During myogenesis, induction of muscle-specific genes is subject to negative control by polypeptide mitogens and type-beta transforming growth factor. Since transduction of growth factor signals may require proteins encoded by cellular ras oncogenes, we have tested whether a mutationally altered Harvey ras expression vector, by itself, can prevent establishment of a differentiated phenotype in BC3H1 mouse myoblasts. Transfection with the valine-12 allele of the human Harvey ras gene, under the control of its own promoter, was sufficient to prevent the induction of both muscle creatine kinase activity and the nicotinic acetylcholine receptor following mitogen withdrawal but did not inhibit withdrawal from the cell cycle. The loss of creatine kinase activity resulted from a corresponding block to induction of muscle creatine kinase mRNA. Similarly, mitogen withdrawal elicited little or no alpha-actin mRNA in ras-transfected cells. These results suggest that an activated ras allele can inhibit myogenesis through a mechanism independent of cell proliferation and can preclude activation of genes whose up-regulation normally accompanies mitogen withdrawal.
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