These results suggest that inhibition of HDACs triggers pharmacologic preconditioning to protect the ischemic heart, which involves p38 activation.

We have previously shown that the inhibition of histone deacetylases (HDACs) protects the heart against acute myocardial ischemia and reperfusion injury. We also demonstrated that HDAC inhibition stimulates myogenesis and angiogenesis in a cultured embryonic stem cell model. We investigate whether in vivo inhibition of HDAC preserves cardiac performance and prevents cardiac remodeling in mouse myocardial infarction (MI) through the stimulation of endogenous regeneration. MI was created by ligation of the left descending artery. Animals were divided into three groups: 1) sham group, animals that underwent thoracotomy without MI; 2) MI, animals that underwent MI; and 3) MI ϩ trichostatin A (TSA), MI animals that received a daily intraperitoneal injection of TSA. In addition, infarcted mice received a daily intraperitoneal injection of TSA (0.1 mg/kg), a selective HDAC inhibitor. 5-Bromo-2-deoxyuridine (50 mg/kg) was delivered every other day to pulse-chase label in vivo endogenous cardiac replication. Eight weeks later, the MI hearts showed a reduction in ventricular contractility. HDAC inhibition increased the improvement of myocardial functional recovery after MI, which was associated with the prevention of myocardial remodeling and reduction of myocardial and serum tumor necrosis factor ␣. HDAC inhibition enhanced the formation of new myocytes and microvessels, which was consistent with the robust increase in proliferation and cytokinesis in the MI hearts. An increase in angiogenic response was demonstrated in MI hearts receiving TSA treatment. It is noteworthy that TSA treatment significantly inhibited HDAC activity and increased phosphorylation of Akt-1, but decreased active caspase 3. Taken together, our results indicate that HDAC inhibition preserves cardiac performance and mitigates myocardial remodeling through stimulating cardiac endogenous regeneration.

The cardiac Na ؉ -Ca 2؉ exchanger (NCX1) is the principal Ca 2؉ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and ␣-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from ؊184 to ؉172 is sufficient for cardiac expression and ␣-adrenergic stimulation. Mutational analysis revealed that both the CArG element at ؊80 and the GATA element at ؊50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the ؊172 E-box demonstrated that it was required for ␣-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (؉103 to ؉134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the ؊172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.The Na ϩ -Ca 2ϩ exchanger (NCX1) 1 catalyzes the electrogenic exchange of one intracellular calcium ion for three extracellular sodium ions across the plasma membrane in many mammalian cells. Transport is reversible and can facilitate calcium entry, which in the heart is capable of triggering calcium release from the sarcoplasmic reticulum (1). The exchanger is most abundant in the heart, where it regulates Ca 2ϩ fluxes across the sarcolemma and serves a critical role in the maintenance of the cellular calcium balance for excitation-contraction coupling. Na ϩ -Ca 2ϩ exchanger activity in cardiomyocytes is regulated by several factors. It is activated by cytosolic Ca 2ϩ and MgATP (2) and inhibited by cytosolic sodium (3) and ATP depletion (4). A high affinity Ca 2ϩ -binding domain has been identified in the large cytoplasmic loop (residues 371-508) that is believed to be responsible for calcium regulation (5). It is also inhibited by the exchanger inhibitory peptide, which corresponds to a 20-amino acid segment at the N terminus of the large cytoplasmic loop (6). A recent study has demonstrated that the exchanger is phosphorylated via a protein kinase C-dependent pathway and that NCX1 phosphorylation appears to coincide with up-regulation of exchanger activity (7).In addition, the exchanger is regulated at the transcriptional level in cardiac hypertrophy, ischemia, and failure. In the feline model of acute right ventricular hypertrophy, NCX1 message levels are rapidly up-regulated following pressure overload (8,9). An increase in NCX1 mRNA expression is also observed in cultured cardiac myocytes following ␣-adrenergic stimulation by phenylephrine or exposure to veratridine. Importantly, the exchanger is also...

The Na ؉-Ca 2؉ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (؊2000 to ؊1250 base pairs (bp)) and one or more negative elements (؊1250 to ؊250 bp), a minimum promoter (؊250 to ؉200 bp) is sufficient for cardiac expression and ␣-adrenergic stimulation.

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...

Increased activity of Ser/Thr protein phosphatases types 1 (PP1) and 2A (PP2A) during maladaptive cardiac hypertrophy contributes to cardiac dysfunction and eventual failure, partly through effects on calcium metabolism. A second maladaptive feature of pressure overload cardiac hypertrophy that instead leads to heart failure by interfering with cardiac contraction and intracellular transport is a dense microtubule network stabilized by decoration with microtubule-associated protein 4 (MAP4). In an earlier study we showed that the major determinant of MAP4-microtubule affinity, and thus microtubule network density and stability, is site-specific MAP4 dephosphorylation at Ser-924 and to a lesser extent at Ser-1056; this was found to be prominent in hypertrophied myocardium. Therefore, in seeking the etiology of this MAP4 dephosphorylation, we looked here at PP2A and PP1, as well as the upstream p21-activated kinase 1, in maladaptive pressure overload cardiac hypertrophy. The activity of each was increased persistently during maladaptive hypertrophy, and overexpression of PP2A or PP1 in normal hearts reproduced both the microtubule network phenotype and the dephosphorylation of MAP4 Ser-924 and Ser-1056 seen in hypertrophy. Given the major microtubule-based abnormalities of contractile and transport function in maladaptive hypertrophy, these findings constitute a second important mechanism for phosphatase-dependent pathology in the hypertrophied and failing heart. Pathological cardiac hypertrophy may be accompanied by increased density and MAP4 2 decoration of the cardiomyocyte microtubule network (1, 2), which causes defects in cellular contractile (3, 4) and transport (5, 6) function. We recently have described, in pathological but not physiological cardiac hypertrophy, site-specific dephosphorylation of three MAP4 serine residues (7); one site is in the MAP4 projection domain, and two are in the microtubule-binding domain. Of these, the striking dephosphorylation at feline MAP4 Ser-924 corresponding to human MAP4 Ser-914 within the first of the four KXGS repeats of the MAP4 microtubule-binding domain was especially interesting, because adenoviral expression of a dephosphomimetic Ser-924 3 Ala feline MAP4 mutant in normal cardiomyocytes phenocopied the features of microtubule network densification, stabilization, and MAP4 overdecoration seen in pathological cardiac hypertrophy. Conversely, adenoviral expression of a phosphomimetic Ser-924 3 Asp feline MAP4 mutant in normal cells caused microtubule depolymerization.This first MAP4 microtubule-binding domain KXGS motif repeat is closely homologous to the corresponding first repeat in the neuronal MAP Tau, with feline MAP4 Ser-924 corresponding to human full-length Tau Ser-262, and it is known that phosphorylation of both MAP4 and Tau at the respective serine residues virtually abolishes MAP binding to microtubules, with consequent microtubule network destabilization (8, 9). Indeed, hyperphosphorylation of Tau Ser-262, leading to aggregation of the detached Tau into...

We have recently demonstrated that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia-reperfusion (I/R) injury. The mechanism by which HDAC inhibition confers myocardial protection remains unknown. The purpose of this study is to investigate whether the disruption of NF-kappaB p50 would eliminate the protective effects of HDAC inhibition. Wild-type and NF-kappaB p50-deficient mice were treated with trichostatin A (TSA; 0.1 mg/kg ip), a potent inhibitor of HDACs. Twenty-four hours later, the hearts were perfused in Langendorff model and subjected to 30 min of ischemia and 30 min of reperfusion. Inhibition of HDACs by TSA in wild-type mice produced marked improvements in left ventricular end-diastolic pressure, left ventricular rate pressure product, and the reduction of infarct size compared with non-TSA-treated group. TSA-induced cardioprotection in wild-type animals was absent with genetic deletion of NF-kappaB p50 subunit. Notably, Western blot displayed a significant increase in nuclear NF-kappaB p50 and the immunoprecipitation demonstrated a remarkable acetylation of NF-kappaB p50 at lysine residues following HDAC inhibition. EMSA exhibited a subsequent increase in NF-kappaB DNA binding activity. Luciferase assay demonstrated an activation of NF-kappaB by HDAC inhibition. The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased in cell viability in simulated ischemia. The resistance of H9c2 cardiomyoblasts to simulated ischemia by HDAC inhibition was eliminated by genetic knockdown of NF-kappaB p50 with transfection of NF-kappaB p50 short interfering RNA but not scrambled short interfering RNA. These results suggest that NF-kappaB p50 acetylation and activation play a pivotal role in HDAC inhibition-induced cardioprotection.

We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91−/− mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30 min of ischemia and 30 min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/L) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91+/+cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91phox-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91+/+cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect.