Striated muscle-specific β1D-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway

Alterations in the extracellular matrix occur during the cardiac hypertrophic process. Because integrins mediate cell-matrix adhesion and β1D-integrin (β1D) is expressed exclusively in cardiac and skeletal muscle, we hypothesized that β1D and focal adhesion kinase (FAK), a proximal integrin-signaling molecule, are involved in cardiac growth. With the use of cultured ventricular myocytes and myocardial tissue, we found the following: 1) β1D protein expression was upregulated perinatally; 2) α1-adrenergic stimulation of cardiac myocytes increased β1D protein levels 350% and altered its cellular distribution; 3) adenovirally mediated overexpression of β1D stimulated cellular reorganization, increased cell size by 250%, and induced molecular markers of the hypertrophic response; and 4) overexpression of free β1D cytoplasmic domains inhibited α1-adrenergic cellular organization and atrial natriuretic factor (ANF) expression. Additionally, FAK was linked to the hypertrophic response as follows: 1) coimmunoprecipitation of β1D and FAK was detected; 2) FAK overexpression induced ANF-luciferase; 3) rapid and sustained phosphorylation of FAK was induced by α1-adrenergic stimulation; and 4) blunting of the α1-adrenergically modulated hypertrophic response was caused by FAK mutants, which alter Grb2 or Src binding, as well as by FAK-related nonkinase, a dominant interfering FAK mutant. We conclude that β1D and FAK are both components of the hypertrophic response pathway of cardiac myocytes.

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Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.1.c175 ◽

2001 ◽

Vol 280 (1) ◽

pp. C175-C182 ◽

Cited By ~ 9

Author(s):

Michihiro Tateyama ◽

Shuqin Zong ◽

Tsutomu Tanabe ◽

Rikuo Ochi

Keyword(s):

Cardiac Myocytes ◽

Fluorescent Protein ◽

Ventricular Myocytes ◽

Foreign Gene ◽

Adrenergic Stimulation ◽

Patch Clamp Technique ◽

Voltage Range ◽

Whole Cell Patch Clamp ◽

Green Fluorescent ◽

Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

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Moderate Calcium Channel Dysfunction in Adult Mice with Inducible Cardiomyocyte-specific Excision of the cacnb2 Gene

Journal of Biological Chemistry ◽

10.1074/jbc.m111.227819 ◽

2011 ◽

Vol 286 (18) ◽

pp. 15875-15882 ◽

Cited By ~ 34

Author(s):

Marcel Meissner ◽

Petra Weissgerber ◽

Juan E. Camacho Londoño ◽

Jean Prenen ◽

Sabine Link ◽

...

Keyword(s):

Cardiac Myocytes ◽

Cardiac Myocyte ◽

Heart Function ◽

Cardiac Fibroblasts ◽

Calcium Currents ◽

Protein Fractions ◽

Protein Levels ◽

Adult Mice ◽

Embryonic Cardiomyocytes ◽

The Difference

The major L-type voltage-gated calcium channels in heart consist of an α1C (CaV1.2) subunit usually associated with an auxiliary β subunit (CaVβ2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of CaVβ2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the CaVβ2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, CaVβ2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include CaVβ2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other CaVβ proteins or changes of CaV1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, CaVβ2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, CaVβ2 down-regulation in cardiomyocytes is well tolerated by the adult mice.

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Confocal and Electron Microscopy of cultured cardiac myocytes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147661 ◽

1993 ◽

Vol 51 ◽

pp. 364-365

Author(s):

D.G. Simpson ◽

R.L. Price ◽

M. Terracio ◽

L. Terracio ◽

T.K. Borg

Keyword(s):

Cardiac Myocytes ◽

Heart Development ◽

Cardiac Myocyte ◽

Striated Muscle ◽

Intercellular Junctions ◽

Membrane Receptors ◽

Cell Junctions ◽

The Many

Early in heart development cardiac myocytes are spherical in shape, intercellular junctions are distributed at irregular intervals around the periphery of the cell, and myofibrillar organization is essentially random. As myocytes mature, they undergo extensive morphogenesis during which the phenotype changes to a tubular rodlike shape, cell junctions congregate at the distal ends of cells to form intercalated disks, and myofibrils become organized in parallel arrays typical of striated muscle. Although not fully understood, it is known that these changes are a result of interactive processes between intracellular components of the cytoskeleton, integrin membrane receptors, and the extracellular matrix (ECM).In vivo studies on the process of cardiac myocyte maturation and myofibrillogenesis are difficult because of the complex biochemical environment of the intact animal and the many extra- and intracellular interactions which are required for proper development and myofibrillogenesis. Unfortunately, in previously available in vitro modelling systems, isolated myocytes spread out over the culture substratum, assume a stellate nonpolar shape, and myofibril organization remains essentially random.

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Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.3.h1189 ◽

1999 ◽

Vol 277 (3) ◽

pp. H1189-H1199 ◽

Cited By ~ 9

Author(s):

David J. Pinsky ◽

Walif Aji ◽

Matthias Szabolcs ◽

Eleni S. Athan ◽

Youping Liu ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Death ◽

Programmed Cell Death ◽

Cardiac Myocytes ◽

Cardiac Myocyte ◽

Ventricular Myocytes ◽

No Production ◽

P53 Expression ◽

Rat Ventricular Myocytes ◽

Adult Rat

Excessive nitric oxide (NO) production within the heart is implicated in the pathogenesis of myocyte death, but the mechanism whereby NO kills cardiac myocytes is not known. To determine whether NO may trigger programmed cell death (apoptosis) of adult rat ventricular myocytes in culture, the NO donor S-nitroso- N-acetylpenicillamine (SNAP) was shown to kill purified cardiac myocytes in a dose-dependent fashion. In situ analysis of ventricular myocytes plated on chamber slides using nick-end labeling of DNA demonstrated that SNAP induces cardiac myocyte apoptosis, which was confirmed by the identification of oligonucleosomal DNA fragmentation on agarose gel electrophoresis. Similarly, treatment of cardiac myocytes with cytokines that induce inducible NO synthase was shown to cause an NO-dependent induction of apoptosis. Addition of reduced hemoglobin to scavenge NO liberated by SNAP extinguished both the increase in percentage of apoptotic cells and the appearance of DNA ladders. Treatment with SNAP (but not with N-acetylpenicillamine or SNAP + hemoglobin) not only induced apoptosis but resulted in a marked increase in p53 expression in cardiac myocytes detected by Western blotting and immunohistochemistry. These data indicate that NO has the capacity to kill cardiac myocytes by triggering apoptosis and suggest the involvement of p53 in this process.

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Abstract 169: Elucidating the role of GRK5 in Physiological Hypertrophy

Circulation Research ◽

10.1161/res.115.suppl_1.169 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Christopher J Traynham ◽

Alessandro Cannavo ◽

Jonathan Hullmann ◽

Jessica L Gold ◽

Walter J Koch

Keyword(s):

Exercise Training ◽

Cardiac Hypertrophy ◽

Cardiac Function ◽

Cardiac Myocyte ◽

Ventricular Myocytes ◽

Neonatal Rat ◽

Heart Weight ◽

Hypertrophic Response ◽

Pathological Cardiac Hypertrophy ◽

Physiological Hypertrophy

Cardiac function is dynamically regulated by various G protein-coupled receptors (GPCRs). GPCR kinases (GRKs) are important in cardiac GPCR regulation through phosphorylation and desensitization of these receptors. GRK2 and GRK5 are the predominant GRKs in the heart, and the most widely characterized as they are both up-regulated in the failing heart. Prior studies from our Lab have determined that GRK5 plays a crucial role in pathological cardiac hypertrophy. Another type of hypertrophy termed, “physiological hypertrophy” occurs with exercise training and is defined as an enlargement in cardiac myocyte size leading to favorable cardiac adaptations. At present, it is unclear if GRK5 is a regulator of physiological hypertrophy in addition to its role in maladaptive hypertrophy. We hypothesize that GRK5 will not regulate physiological hypertrophy such that mice with cardiac-specific overexpression of GRK5 (TgGRK5) will yield a similar post-exercise cardiac physiological hypertrophic response as that of control wild-type (WT) mice. To test this hypothesis, TgGRK5 and WT mice were exposed to a 21 day high-intensity swimming exercise protocol. For each line, sham mice, which did not swim served as appropriate controls. At the conclusion of this protocol, mice were sacrificed and heart weight (HW), body weight (BW), and tibia length (TL) measured. TgGRK5 and WT mice both exhibited a characteristic 10-15% increase in HW/BW and HW/TL ratios, which are standard measures of cardiac hypertrophy. In addition, hearts were sectioned and H&E stained to evaluate myocyte size. Both TgGRK5 and WT mice exhibited a significant increase in myocyte size. Cardiac function was evaluated via echocardiography both prior to and after exercise training, and no changes were observed between TgGRK5 and WT mice after training. These data were re-affirmed in H9C2 cells and neonatal rat ventricular myocytes overexpressing either GFP or GRK5, which exhibited similar increases in cell size and AKT phosphorylation after IGF-1 treatment, a physiological hypertrophy stimulus. Taken together, these data suggest that physiological hypertrophy is similar in both control and TgGRK5 mice, confirming that GRK5 is solely a regulator of pathological cardiac hypertrophy.

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A Ras-Dependent Pathway Regulates RNA Polymerase II Phosphorylation in Cardiac Myocytes: Implications for Cardiac Hypertrophy

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6729 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6729-6736 ◽

Cited By ~ 54

Author(s):

Maha Abdellatif ◽

Sharon E. Packer ◽

Lloyd H. Michael ◽

Dou Zhang ◽

Min Ji Charng ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cardiac Myocytes ◽

Cardiac Myocyte ◽

Pressure Overload ◽

Dominant Negative ◽

Adrenergic Stimulation ◽

Pol Ii ◽

Terminal Domain ◽

Cardiac Myocyte Hypertrophy

ABSTRACT Despite extensive evidence implicating Ras in cardiac muscle hypertrophy, the mechanisms involved are unclear. We previously reported that Ras, through an effector-like function of Ras GTPase-activating protein (GAP) in neonatal cardiac myocytes (M. Abdellatif et al., J. Biol. Chem. 269:15423–15426, 1994; M. Abdellatif and M. D. Schneider, J. Biol. Chem. 272:527–533, 1997), can up-regulate expression from a comprehensive set of promoters, including both cardiac cell-specific and constitutive ones. To investigate the mechanism(s) underlying these earlier findings, we have used recombinant adenoviruses harboring a dominant negative Ras (17N Ras) allele or the N-terminal domain of GAP (nGAP), responsible for the Ras-like effector function. Inhibition of endogenous Ras reduced basal levels of [3H]uridine and [3H]phenylalanine incorporation into total RNA, mRNA, and protein, with parallel changes in apparent cell size. In addition, 17N Ras markedly inhibited phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II), known to regulate transcript elongation, accompanied by down-regulation of its principal kinase, cyclin-dependent kinase 7 (Cdk7). In contrast, nGAP elicited the opposite effects on each of these parameters. Furthermore, cotransfection of constitutively active Ras (12R Ras) with wild-type pol II, rather than a truncated mutant lacking the CTD, demonstrated that Ras activation of transcription was dependent on the pol II CTD. Consistent with a potential role for this pathway in the development of cardiac myocyte hypertrophy, α1-adrenergic stimulation similarly enhanced pol II phosphorylation and Cdk7 expression, where both effects were inhibited by dominant negative Ras, while pressure overload hypertrophy led to an increase in both hyperphosphorylated and hypophosphorylated pol II in addition to Cdk7.

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Phorbol ester stimulates cyclooxygenase-2 expression and prostanoid production in cardiac myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.2.h719 ◽

2000 ◽

Vol 279 (2) ◽

pp. H719-H725 ◽

Cited By ~ 5

Author(s):

Ralph Schuette ◽

Margot C. LaPointe

Keyword(s):

Protein Synthesis ◽

Cardiac Myocytes ◽

Ventricular Myocytes ◽

Fold Increase ◽

Cyclooxygenase 2 ◽

Leucine Incorporation ◽

P38 Kinase ◽

Protein Levels ◽

Cox 2 ◽

Stimulation Of

Phorbol-12-myristate- 13-acetate (PMA) has been shown to induce hypertrophy of cardiac myocytes. The prostaglandin endoperoxide H synthase isoform 2 (cyclooxygenase-2, COX-2) has been associated with enhanced growth and/or proliferation of several types of cells. Thus we studied whether PMA induces COX-2 and prostanoid products PGE2 and PGF2α in neonatal ventricular myocytes and whether endogenous COX-2 products participate in their growth. In addition, we examined whether PMA affects interleukin-1β (IL-1β) stimulation of COX-2 and PGE2production. PMA (0.1 μmol/l) stimulated growth, as indicated by a 1.6-fold increase in [3H]leucine incorporation. PMA increased COX-2 protein levels 2.8-fold, PGE2 3.7-fold, and PGF2α 2.9-fold. Inhibition of either p38 kinase or protein kinase C (PKC) prevented PMA-stimulated COX-2. Inhibition of COX-2 with either indomethacin or NS-398 had no effect on PMA-stimulated [3H]leucine incorporation. Exogenous administration of PGF2α, but not PGE2, stimulated protein synthesis. Treatment with IL-1β (5 ng/ml) increased COX-2 protein levels 42-fold, whereas cotreatment with IL-1β and PMA stimulated COX-2 protein only 32-fold. IL-1β did not affect control or PMA-stimulated protein synthesis. These findings indicate that: 1) PMA, acting through PKC and p38 kinase, enhances COX-2 expression, but chronic treatment with PMA partially inhibits IL-1β stimulation of COX-2; and 2) exogenous PGF2α is involved in neonatal ventricular myocyte growth but endogenous COX-2 products are not.

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Novel 3D culture system for study of cardiac myocyte development

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01027.2002 ◽

2003 ◽

Vol 285 (2) ◽

pp. H570-H578 ◽

Cited By ~ 59

Author(s):

Heather J. Evans ◽

Janea K. Sweet ◽

Robert L. Price ◽

Michael Yost ◽

Richard L. Goodwin

Keyword(s):

Cardiac Myocytes ◽

Molecular Mechanisms ◽

Cardiac Myocyte ◽

Ventricular Myocytes ◽

3D Culture ◽

Health Concern ◽

Type I ◽

Myocardial Repair ◽

Proliferation And Differentiation

Insufficient myocardial repair after pathological processes contributes to cardiovascular disease, which is a major health concern. Understanding the molecular mechanisms that regulate the proliferation and differentiation of cardiac myocytes will aid in designing therapies for myocardial repair. Models are needed to delineate these molecular mechanisms. Here we report the development of a model system that recapitulates many aspects of cardiac myocyte differentiation that occur during early cardiac development. A key component of this model is a novel three-dimensional tubular scaf-fold engineered from aligned type I collagen strands. In this model embryonic ventricular myocytes undergo a transition from a hyperplastic to a quiescent phenotype, display significant myofibrillogenesis, and form critical cell-cell connections. In addition, embryonic cardiac myocytes grown on the tubular substrate have an aligned phenotype that closely resembles in vivo neonatal ventricular myocytes. We propose that embryonic cardiac myocytes grown on the tube substrate develop into neonatal cardiac myocytes via normal in vivo mechanisms. This model will aid in the elucidation of the molecular mechanisms that regulate cardiac myocyte proliferation and differentiation, which will provide important insights into myocardial development.

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The Extracellular Matrix: Not Just Pretty Fibrils

Science ◽

10.1126/science.1176009 ◽

2009 ◽

Vol 326 (5957) ◽

pp. 1216-1219 ◽

Cited By ~ 1799

Author(s):

Richard O. Hynes

Keyword(s):

Extracellular Matrix ◽

Solid Phase ◽

Genetic Diseases ◽

Structural Support ◽

Cell Matrix ◽

Ecm Proteins ◽

Developmental Patterning ◽

Mediate Cell ◽

Cell Matrix Adhesion ◽

Stem Cell Niches

The extracellular matrix (ECM) and ECM proteins are important in phenomena as diverse as developmental patterning, stem cell niches, cancer, and genetic diseases. The ECM has many effects beyond providing structural support. ECM proteins typically include multiple, independently folded domains whose sequences and arrangement are highly conserved. Some of these domains bind adhesion receptors such as integrins that mediate cell-matrix adhesion and also transduce signals into cells. However, ECM proteins also bind soluble growth factors and regulate their distribution, activation, and presentation to cells. As organized, solid-phase ligands, ECM proteins can integrate complex, multivalent signals to cells in a spatially patterned and regulated fashion. These properties need to be incorporated into considerations of the functions of the ECM.

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