Polymorphisms in genes encoding nonsarcomeric proteins and their role in the pathogenesis of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), clinically characterized by contractile dysfunction and ventricular chamber enlargement, is a heterogeneous heart disease leading to progressive systolic heart failure and sudden cardiac death. The etiology of the disease is multifactorial and involves genetic factors, viral infections, autoimmune phenomena, and toxic agents. Within the last two decades, a growing body of evidence has suggested that single-gene mutations play a pivotal role in the development of familial forms of dilated cardiomyopathies. Numerous genes encoding cytoskeletal, sarcomeric, and nuclear proteins have been linked to the pathogenesis of DCM, and most of the respective mutants disrupt the structural integrity of sarcomeres in cardiac myocytes. Frequently, point mutations in cytoskeletal proteins critically diminish force generation and interfere with mechanical transduction within the contractile apparatus of the myocardium, thereby ultimately leading to impaired systolic function. However, hitherto reported sarcomeric gene defects explain the etiology of the disease only in some families, leaving other forms of DCM subentities unresolved. Since one of the major factors in DCM pathogenesis involves autoimmune-mediated damage to cardiac tissue, candidate genes that are involved in controlling immune reactions have currently come into focus in genetic research. We and others have shown that a single-nucleotide polymorphism (SNP) in the gene encoding cytotoxic T-lymphocyte antigen 4 (CTLA4) is associated with the diagnosis of DCM. Cytotoxic T-lymphocyte antigen 4 is an inhibitory receptor molecule expressed on activated T lymphocytes, where it functions as an important negative regulator of T-cell activation by competing with the costimulatory CD28 receptor to bind to B7 receptors localized on the surface of antigen-presenting cells. The observed association between CTLA4 genotypes and DCM suggests that genetic factors contribute to both unbalanced immune responses in the myocardium and the development of left ventricular dysfunction. In this review, we will briefly discuss how these findings may stimulate the search for novel DCM-associated SNPs in human genes expressed in noncardiomyocytes.

Protein kinase C zeta. A novel regulator of both phosphorylation and de-phosphorylation of cardiac sarcomeric proteins.

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.

Decreased p38 MAPK activity in end-stage failing human myocardium: p38 MAPK alpha is the predominant isoform expressed in human heart.

Short duration exposure to cellular stresses have been shown to activate p38 mitogen-activated protein kinase (MAPK) in cultured rat ventricular cardiomyocytes and isolated perfused hearts; however, effects of chronic stress on p38 MAPK are not well understood. This study determined whether alterations in the p38 MAPK pathway occurred prior to end-stage human heart failure. The p38 MAPK alpha isoform was detectable in human cardiac tissue. However, carefully controlled analysis of protein and message in this study demonstrated an absence of the p38 MAPK beta -isoform. Low levels of message for the non-SB203580 sensitive p38 MAPK gamma and delta isoforms were also detected in both normal and failing human myocardium. Ischemic and idiopathic end-stage failing human hearts were compared to non-failing hearts for both p38 alpha MAPK protein level and total p38 MAPK activity. Western blotting techniques demonstrated no significant changes in total p38 alpha MAPK content. However, approximately 75% decreases in active/phosphorylated p38 MAPK (P<0.005) were observed in both ischemic and idiopathic failing hearts compared to non-failing hearts. In-gel kinase assays confirmed that activated p38 MAPK, detected by Western blotting, phosphorylated its potential downstream targets. When compared to non-failing hearts, approximately 46% decreases in p38 MAPK phosphorylation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) were observed in ischemic and idiopathic failing hearts (P=0.03 and P=0.04 respectively). Active p38 MAPK was localized to sarcomeric structures in the cytosol of myocytes by confocal immunofluorescence microscopy. The correlation between decreased MAPKAPK-2 phosphorylation and loss of active p38 MAPK in failing human myocytes suggests that decreases in the activation of p38 MAPK alpha, the predominant cardiac isoform, occur prior to end-stage heart failure.

Mouse myoblasts can fuse and form a normal sarcomere in the absence of beta1 integrin expression.

Antibody perturbation experiments suggested that migration, terminal differentiation and fusion of myoblasts are dependent on beta1 integrin expression. In addition, several studies have postulated that beta1 integrins have a role in the formation of sarcomeres. In the present report we have analysed skeletal myogenesis in wild-type/beta1-null chimeric mice and beta1-null embryoid bodies. Trunk and limbs of beta1-null chimeric mice contained muscle tissue composed of normal and beta1-null myoblasts indicating that all myotomic sublineages can form, migrate to their peripheral targets and fuse in the absence of beta1 integrin expression. Pure populations of beta1-null myoblasts and satellite cells isolated from beta1-null chimeric embryos and chimeric newborn mice, respectively, were able to differentiate in vitro and to fuse into multinucleated myotubes. Quantitative and qualitative comparisons between normal and beta1-null myoblasts revealed no apparent difference in their capacity to terminally differentiate and fuse. Furthermore, beta1-null myotubes developed sarcomeres which were indistinguishable from wild-type controls. When normal and beta1-null ES cells were differentiated into embryoid bodies, they contained fully differentiated myotubes with normal sarcomeres and normal deposition of costameric components. However, formation of beta1-null myotubes was delayed and was less efficient in beta1-null embryoid bodies than in wild-type controls. High expression of alphav integrin subunit at the tips of normal as well as beta1-null myotubes indicated that the lack of beta1 integrins did not result in a significant redistribution of alphav-containing receptors.

Depressed unloaded sarcomere shortening velocity in acute murine coxsackievirus myocarditis: myocardial remodelling in the absence of necrosis or hypertrophy.

We used right ventricular papillary muscles to study cellular dysfunction in acute murine coxsackievirus myocarditis. We measured unloaded sarcomere shortening velocity (V0) with laser diffraction (HeNe, lambda = 623.8 nm) 7 days after coxsackievirus infection (M) (n = 7) and after infection + monoclonal antibodies to eliminate T cells (T) (n = 4) and in normals (N) (n = 8). A servomotor rapidly shortened a muscle until slack early in contraction and V0 was measured at the onset of zero force. V0 in N was 4.14 +/- 0.84 microns.s-1 at Sl = 2.08 +/- 0.09 microns, 1.70 +/- 0.33 microns.s-1 at 2.06 +/- 0.08 microns in M and, in preliminary experiments, 4.75 +/- 0.96 microns.s-1 at 2.06 +/- 0.07 microns in T. Resting force and stiffness were normal in M. Ventriculor weights in M and T were the same as N. There was an increase in mononuclear cells in M papillary muscles, but no fibrosis or necrosis. Thus, V0 was markedly reduced in acute viral myocarditis in the absence of tissue disruption or hypertrophy, but not if T cells were absent. Pyrophosphate gel electrophoresis showed a shift from predominantly fast in N to a slow myosin isoform in M. Myosin remodelling and reduced unloaded sarcomere shortening velocity occur early in acute coxsackievirus myocarditis and are dependent on immune responses to the virus, but are not a result of histopathological changes.

A population of myogenic cells derived from the mouse neural tube.

Embryonic mouse neural tubes produce a variety of terminally differentiated cells in vitro, mostly neurons and glia. We report here that some of these cells differentiate into skeletal muscle cells. The possibility of mesoderm contamination was ruled out as follows. First, Dil+ muscle cells were present in cultures from a Dil-labeled neuroepithelium. Second, a small fraction of cultured neural tube cells coexpressed muscle myosin and neuronal beta III tubulin within the same cell. Third, embryos generated from embryonic stem cells in which nlacZ was targeted into the myogenic gene myf-5 expressed nlacZ in a localized region of the neural tube. These myf-5+ cells coexpress neuronal and muscle markers in culture. The developmental significance of this phenomenon is discussed in the context of overlapping regulatory networks between myogenesis and neurogenesis.