T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs),

Postnatal cardiac myocytes respond to stress signals by hypertrophic growth and activation of a fetal gene program. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy, and mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals. To further define the roles of HDACs in cardiac hypertrophy, we analyzed the effects of HDAC inhibitors on the responsiveness of primary cardiomyocytes to hypertrophic agonists. Paradoxically, HDAC inhibitors imposed a dose-dependent blockade to hypertrophy and fetal gene activation. We conclude that distinct HDACs play positive or negative roles in the control of cardiomyocyte hypertrophy. HDAC inhibitors are currently being tested in clinical trials as anti-cancer agents. Our results suggest that these inhibitors may also hold promising clinical value as therapeutics for cardiac hypertrophy and heart failure.Postnatal cardiac myocytes undergo hypertrophic growth in response to a variety of stress signals (reviewed in Ref. 1). The hypertrophic response is characterized by increases in myocyte size and protein synthesis, assembly and organization of sarcomeres, and activation of a fetal gene program. Although traditionally considered an adaptive response to pathological signaling, chronic expression of fetal cardiac genes in the heart can result in maladaptive changes in cardiac contractility and calcium handling that culminate in dilated cardiomyopathy, heart failure, and sudden death from arrhythmias (2). Moreover, increasing evidence in rodent models indicates that cardiac function is preserved when hypertrophy is inhibited in the face of stress signaling, pointing to the potential importance of therapeutic strategies for modulating the hypertrophic process (3-9).Recent studies have revealed key roles for chromatin-modifying enzymes in the control of cardiac hypertrophy (10 -12). The structure of chromatin is governed by the acetylation state of nucleosomal histones (13,14). Acetylation of histone tails by histone acetyltransferases (HATs) 1 results in relaxation of nucleosomal structure and transcriptional activation. Acetylated histones also serve as targets for binding of bromo-domain proteins that possess HAT activity and act as transcriptional activators. The actions of HATs are opposed by histone deacetylases (HDACs), which deacetylate nucleosomal histones, thereby promoting chromatin condensation and transcriptional repression.Mammalian HDACs can be divided into three classes based on their similarity with three yeast HDACs (reviewed in Refs. 15 and 16). Class I HDACs (HDACs 1, 2, 3, and 8) are expressed ubiquitously and consist mainly of a deacetylase domain. Members of class II (HDACs 4, 5, 7, and 9) are highly expressed in striated muscle and brain and have an extended N terminus in addition to the catalytic domain. Class III HDACs resemble the yeast HDAC Sir2, which is activated by nicotinamide adenine dinucleotide (17).Class II HDACs interact with a variety of positive and negative cofactors as well as other ...

Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased ␤-myosin heavy chain (␤MyHC) gene expression. Our previous work showed that Sp3 contributes to decreased ␤MyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Pur␣, and Pur␤ proteins mediate repression of ␤MyHC expression under NWB conditions. Binding of Pur␣ or Pur␤ to the single-stranded ␤MyHC distal negative regulatory element-sense strand (d␤NRE-S) element is markedly increased under NWB conditions. Ectopic expression of Pur␣ and Pur␤ decreased ␤MyHC reporter gene expression, while mutation of the d␤NRE-S element increased expression in C2C12 myotubes. The d␤NRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Pur␣ and Pur␤ decreased endogenous Sp3 and Pur protein levels and increased ␤MyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Pur␣, Pur␤, and Sp3, while chromatin immunoprecipitation assays demonstrated Pur␣, Pur␤, and Sp3 binding to the ␤MyHC proximal promoter region harboring the d␤NRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

Class II histocompatibility antigens are composed of polymorphic alpha and beta polypeptides which associate in the endoplasmic reticulum (ER) with a third, non-polymorphic invariant polypeptide (Ii). The alpha beta Ii complexes are subsequently transported through the Golgi to the endosomes, where the Ii chain dissociates before the alpha beta complex is transported to the cell surface. Results from transport-defective class II expression variant studies suggest that class II intracellular transport is regulated in multiple intracellular compartments. Consistent with this, a large number of studies have demonstrated that protein folding and/or oligomerization is facilitated in the ER by a class of proteins collectively known as molecular chaperones. In this report, we show that the ER-resident protein calnexin associates with human and murine class II antigens. Specifically, calnexin associates in the ER with free Ii polypeptides and partially assembled wild-type class II complexes, including A alpha and/or A alpha Ii complexes, as well as with alpha beta dimers isolated from class II transport-defective cells. Calnexin also physically associates with alpha beta Ii complexes, but not with mature alpha beta dimers. These results suggest that calnexin may regulate class II intracellular transport by facilitating the production of transport competent molecules out of the ER. In addition, we report that the nucleotide sequence of the gene encoding murine calnexin shows a high degree of homology to human IP90 and dog calnexin at both the nucleotide and deduced amino acid sequence level. The isolation of cDNA fragments encoding murine calnexin will allow us to further evaluate the functional consequences of calnexin-class II interaction.

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.