Load regulation of the properties of adult feline cardiocytes. The role of substrate adhesion.

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac ␣-and ␤-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 3 Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.Although many important alterations have been described in the properties of hypertrophied myocardium, the mechanisms responsible for contractile dysfunction and many other maladaptive changes of cardiac muscle cells, or cardiocytes, have yet to be fully defined. Although most research in this area has focused on structural and regulatory changes within the myofilament, it has also been found that changes in the microtubule component of the extra-myofilament cytoskeleton may lead both to contractile dysfunction by increasing the internal resistance to sarcomere motion (1, 2) and to disordered cellular homeostasis by impeding cytoskeleton-based intracellular transport (3-6).Our major original finding was that, in severe pressure overload cardiac hypertrophy with increased ventricular wall stress, there is the early appearance and then the persistence of a dense microtubule network and associated contractile dysfunction (7,8). We have now found several synergistic bases for this dense microtubule network. First, during hypertrophy there is persistent tran...

Section: Journal Of Biological Chemistrysupporting

confidence: 81%

Site-specific Microtubule-associated Protein 4 Dephosphorylation Causes Microtubule Network Densification in Pressure Overload Cardiac Hypertrophy

Panneerselvam

Samanna

Cheng

et al. 2010

“…Adult Cardiocyte Culture Model-Adult feline cardiocytes were isolated from normal cats and cultured on laminin coated four-well culture trays as described previously (45). Isolated cardiocytes were suspended in a 1.8 mM Ca 2ϩ containing mitogen-free M-199 medium at pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

c-Raf/MEK/ERK Pathway Controls Protein Kinase C-mediated p70S6K Activation in Adult Cardiac Muscle Cells

Iijima¹,

Laser²,

Shiraishi³

et al. 2002

137

104

Hypertrophic cardiac growth is a major compensatory response of the heart to an increased mechanical (hemodynamic) load in the form of either pressure or volume overload. Although this response is initially compensatory, a transition from this state to failure occurs when further growth of the heart is not sufficient to normalize the wall stress and maintain contractile function (1). Therefore, a major research interest in cardiovascular disease is to understand how the increase in hemodynamic load is transmitted intracellularly for mediating hypertrophic growth. Although the mechanical load appears to directly regulate the hypertrophic growth initiation, the signaling mechanism that connects load to such growth is not well understood.A major cellular event during cardiac hypertrophy is increased protein synthesis (1-5). Enhanced protein synthesis can occur via accelerated protein translation, increased biogenesis of translational components, or both. A significant amount of mRNA of vertebrate cells possesses a unique 5Ј-terminal oligopyrimidine (5Ј-TOP) 1 sequence in the 5Ј-untranslated region (5Ј-UTR), and these mRNA species generally code for specific ribosomal proteins (6, 7). Their translation is largely controlled via phosphorylation of the 40 S ribosomal S6 protein (S6 protein) at its C terminus (8) by p70/85 S6 kinase (S6K1) (9 -12). There are two isoforms of S6K1: the 70-kDa isoform was first isolated from mouse 3T3 cells (13), and the 85-kDa isoform of this kinase was then identified (14). The p85 isoform is expressed from the same transcript as the p70 isoform through an alternative translational initiation start site, which adds a 23-amino acid nuclear localization signal to the N terminus (15,16). Therefore, the 85-kDa isoform is predominantly in the nucleus, whereas the 70-kDa isoform is present mostly in the cytoplasm. Both the S6K isoforms are collectively called p70/85S6K, p70S6K, or S6K1 and have been shown to phosphorylate the S6 protein and mediate the biogenesis of the translational components, including several of the ribosomal proteins and elongation factors (12). The p85 isoform has been shown to have additional roles in translational control, G 1 to S phase transition, and increased DNA synthesis (17). Recent studies using S6K1 knockout mice (18) demonstrate no appreciable change in S6 protein phosphorylation, 5Ј-TOP mRNA translation, or cell growth, although these mice exhibited a small mouse phenotype. These studies (18) and other independent studies (19 -21) resulted in the discovery of another S6K (S6K2), which possesses 70% homology with the p70 isoform of

“…Electrical Stimulation Model-Adult feline cardiocytes were obtained by collagenase digestion in combination with mechanical agitation as described previously (6,8,9). Following cell isolation, the Ca 2ϩ -tolerant cardiocytes were placed in Life Technologies, Inc. M199 serumfree medium with Earle's salts at a concentration of 50,000 rod-shaped cells/ml.…”

Section: Methodsmentioning

confidence: 99%

“…These findings have been extended to isolated adult cardiocytes in culture. Rates of protein synthesis were accelerated in response to electrically stimulated cardiocyte contraction (6,7), in response to a basal load as defined by adherence of the cardiocytes to the culture dish and establishment of a resting length (8) and in response to passive stretch of cardiocytes adherent to a deformable membrane (9). Thus, the integrity of adult cardiocytes in primary culture is maintained with respect to the ability to transduce changes in mechanical load into an anabolic response such as accelerated protein synthesis rate.…”

mentioning

confidence: 99%

Contraction Accelerates Myosin Heavy Chain Synthesis Rates in Adult Cardiocytes by an Increase in the Rate of Translational Initiation

Ivester

Tuxworth

Cooper

et al. 1995

The purpose of this study was to determine the mechanism by which contraction acutely accelerates the synthesis rate of the contractile protein myosin heavy chain (MHC). Laminin-adherent adult feline cardiocytes were maintained in a serum-free medium and induced to contract at 1 Hz via electrical field stimulation. Electrical stimulation of contraction accelerated rates of MHC synthesis 28%, p < 0.05 by 4 h as determined by incorporation of [ 3 H]phenylalanine into MHC. MHC mRNA expression as measured by RNase protection was unchanged after 4 h of electrical stimulation. MHC mRNA levels in messenger ribonucleoprotein complexes and translating polysomes were examined by sucrose gradient fractionation. The relative percentage of polysomebound MHC mRNA was equal at 47% in both electrically stimulated and control cardiocytes. However, electrical stimulation of contraction resulted in a reproducible shift of MHC mRNA from smaller polysomes into larger polysomes, indicating an increased rate of initiation. This shift resulted in significant increases in MHC mRNA levels in the fractions containing the larger polysomes of electrically stimulated cardiocytes as compared with nonstimulated controls. These data indicate that the rate of MHC synthesis is accelerated in contracting cardiocytes via an increase in translational efficiency.Hypertrophic growth occurs in terminally differentiated adult cardiocytes by an increase in cellular mass via a relatively coordinate increase in the proteins comprising each of the cellular components (1). This accumulation of cardiocyte proteins occurs by an increase in rates of protein synthesis relative to rates of protein degradation (2). The anabolic changes that occur during hypertrophy of the adult myocardium are generally considered to be a compensatory response to an increase in hemodynamic load (3). As demonstrated in studies employing isolated papillary muscle preparations, both the active tension and passive strain components of load have been identified as mechanical stimuli for accelerating protein synthesis rates in adult myocardium (4, 5). These findings have been extended to isolated adult cardiocytes in culture. Rates of protein synthesis were accelerated in response to electrically stimulated cardiocyte contraction (6, 7), in response to a basal load as defined by adherence of the cardiocytes to the culture dish and establishment of a resting length (8) and in response to passive stretch of cardiocytes adherent to a deformable membrane (9). Thus, the integrity of adult cardiocytes in primary culture is maintained with respect to the ability to transduce changes in mechanical load into an anabolic response such as accelerated protein synthesis rate.The specific mechanisms by which cardiocyte protein synthesis is regulated probably occur at many levels, including transcriptional, post-transcriptional, and translational processes (10). It is well established, for example, that transcriptional and post-transcriptional mechanisms regulate steady state mRNA levels and are respon...