Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

HDAC inhibition helps post-MI healing by modulating macrophage polarization.

AIMS: Following an acute myocardial infarction (MI) the extracellular matrix (ECM) undergoes remodeling in order to prevent dilation of the infarct area and maintain cardiac output. Excessive and prolonged inflammation following an MI exacerbates adverse ventricular remodeling. Macrophages are an integral part of the inflammatory response that contribute to this remodeling. Treatment with histone deacetylase (HDAC) inhibitors preserves LV function and myocardial remodeling in the post-MI heart. This study tested whether inhibition of HDAC activity resulted in preserving post-MI LV function through the regulation of macrophage phenotype and early resolution of inflammation. METHODS AND RESULTS: HDAC inhibition does not affect the recruitment of CD45+ leukocytes, CD45+/CD11b+ inflammatory monocytes or CD45+/CD11b+CD86+ inflammatory macrophages for the first 3 days following infarct. Further, HDAC inhibition does not change the high expression level of the inflammatory cytokines in the first days following MI. However, by day 7, there was a significant reduction in the levels of CD45+/Cd11b+ and CD45+/CD11b+/CD86+ cells with HDAC inhibition. Remarkably, HDAC inhibition resulted in the dramatic increase in the recruitment of CD45+/CD11b+/CD206+ alternatively activated macrophages as early as 1 day which remained significantly elevated until 5 days post-MI. qRT-PCR revealed that HDAC inhibitor treatment shifts the cytokine and chemokine environment towards an M2 phenotype with upregulation of M2 markers at 1 and 5 days post-MI. Importantly, HDAC inhibition correlates with significant preservation of both LV ejection fraction and end-diastolic volume and is associated with a significant increase in micro-vessel density in the border zone at 14 days post-MI. CONCLUSION: Inhibition of HDAC activity result in the early recruitment of reparative CD45+/CD11b+/CD206+ macrophages in the post-MI heart and correlates with improved ventricular function and remodeling. This work identifies a very promising therapeutic opportunity to manage macrophage phenotype and enhance resolution of inflammation in the post-MI heart.

Evidence for a non-canonical role of HDAC5 in regulation of the cardiac Ncx1 and Bnp genes.

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5((-/-)) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies.

Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide.

Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells. We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47-/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45moderate/HSP47+/macrophage marker- cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.

Inhibition of class I histone deacetylase activity represses matrix metalloproteinase-2 and -9 expression and preserves LV function postmyocardial infarction.

Left ventricular (LV) remodeling, after myocardial infarction (MI), can result in LV dilation and LV pump dysfunction. Post-MI induction of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated as causing deleterious effects on LV and extracellular matrix remodeling in the MI region and within the initially unaffected remote zone. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We assessed the efficacy of both class I/IIb- and class I-selective HDAC inhibitors on MMP-2 and MMP-9 abundance and determined if treatment resulted in the attenuation of adverse LV and extracellular matrix remodeling and improved LV pump function post-MI. MI was surgically induced in MMP-9 promoter reporter mice and randomized for treatment with a class I/IIb HDAC inhibitor for 7 days post-MI. After MI, LV dilation, LV pump dysfunction, and activation of the MMP-9 gene promoter were significantly attenuated in mice treated with either the class I/IIb HDAC inhibitor tichostatin A or suberanilohydroxamic acid (voronistat) compared with MI-only mice. Immunohistological staining and zymographic levels of MMP-2 and MMP-9 were reduced with either tichostatin A or suberanilohydroxamic acid treatment. Class I HDAC activity was dramatically increased post-MI. Treatment with the selective class I HDAC inhibitor PD-106 reduced post-MI levels of both MMP-2 and MMP-9 and attenuated LV dilation and LV pump dysfunction post-MI, similar to class I/IIb HDAC inhibition. Taken together, these unique findings demonstrate that selective inhibition of class I HDACs may provide a novel therapeutic means to attenuate adverse LV remodeling post-MI.

Transcriptional pathways and potential therapeutic targets in the regulation of Ncx1 expression in cardiac hypertrophy and failure.

Changes in cardiac gene expression contribute to the progression of heart failure by affecting cardiomyocyte growth, function, and survival. The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. Several transcriptional pathways mediate Ncx1 expression in pathological cardiac remodeling. Both α-adrenergic receptor (α-AR) and ß-adrenergic receptor (ß-AR) signaling can play a role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Our studies have even demonstrated that NCX1 can directly act as a regulator of "activity-dependent signal transduction" mediating changes in its own expression. Finally, we present evidence that histone deacetylases (HDACs) and histone acetyltransferases (HATs) act as master regulators of Ncx1 expression. We show that many of the transcription factors regulating Ncx1 expression are important in cardiac development and also in the regulation of many other genes in the so-called fetal gene program, which are activated by pathological stimuli. Importantly, studies have revealed that the transcriptional network regulating Ncx1 expression is also mediating many of the other changes in genetic remodeling contributing to the development of cardiac dysfunction and revealed potential therapeutic targets for the treatment of hypertrophy and failure.

Cytoskeletal role in protection of the failing heart by ß-adrenergic blockade.

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac ß-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because ß-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on ß-adrenergic overdrive and thus could be reversed by ß-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, ß(1)- (but not ß(2)-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had ß-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB ß-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy.

Time course of right ventricular pressure-overload induced myocardial fibrosis: relationship to changes in fibroblast postsynthetic procollagen processing.

Myocardial fibrillar collagen is considered an important determinant of increased ventricular stiffness in pressure-overload (PO)-induced cardiac hypertrophy. Chronic PO was created in feline right ventricles (RV) by pulmonary artery banding (PAB) to define the time course of changes in fibrillar collagen content after PO using a nonrodent model and to determine whether this time course was dependent on changes in fibroblast function. Total, soluble, and insoluble collagen (hydroxyproline), collagen volume fraction (CVF), and RV end-diastolic pressure were assessed 2 days and 1, 2, 4, and 10 wk following PAB. Fibroblast function was assessed by quantitating the product of postsynthetic processing, insoluble collagen, and levels of SPARC (secreted protein acidic and rich in cysteine), a protein that affects procollagen processing. RV hypertrophic growth was complete 2 wk after PAB. Changes in RV collagen content did not follow the same time course. Two weeks after PAB, there were elevations in total collagen (control RV: 8.84 ± 1.03 mg/g vs. 2-wk PAB: 11.50 ± 0.78 mg/g); however, increased insoluble fibrillar collagen, as measured by CVF, was not detected until 4 wk after PAB (control RV CVF: 1.39 ± 0.25% vs. 4-wk PAB: 4.18 ± 0.87%). RV end-diastolic pressure was unchanged at 2 wk, but increased until 4 wk after PAB. RV fibroblasts isolated after 2-wk PAB had no changes in either insoluble collagen or SPARC expression; however, increases in insoluble collagen and in levels of SPARC were detected in RV fibroblasts from 4-wk PAB. Therefore, the time course of PO-induced RV hypertrophy differs significantly from myocardial fibrosis and diastolic dysfunction. These temporal differences appear dependent on changes in fibroblast function.

Remodeling of the peripheral cardiac conduction system in response to pressure overload.

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.

Modulation of Hypoxia-Induced Pulmonary Vascular Leakage in Rats by Seabuckthorn (Hippophae rhamnoides L.).

Cerebral and pulmonary syndromes may develop in unacclimatized individuals shortly after ascent to high altitude resulting in high altitude illness, which may occur due to extravasation of fluid from intra to extravascular space in the brain, lungs and peripheral tissues. The objective of the present study was to evaluate the potential of seabuckthorn (SBT) (Hippophae rhamnoides L.) leaf extract (LE) in curtailing hypoxia-induced transvascular permeability in the lungs by measuring lung water content, leakage of fluorescein dye into the lungs and further confirmation by quantitation of albumin and protein in the bronchoalveolar lavage fluid (BALF). Exposure of rats to hypoxia caused a significant increase in the transvascular leakage in the lungs. The SBT LE treated animals showed a significant decrease in hypoxia-induced vascular permeability evidenced by decreased water content and fluorescein leakage in the lungs and decreased albumin and protein content in the BALF. The SBT extract was also able to significantly attenuate hypoxia-induced increase in the levels of proinflammatory cytokines and decrease hypoxia-induced oxidative stress by stabilizing the levels of reduced glutathione and antioxidant enzymes. Pretreatment of the extract also resulted in a significant decrease in the circulatory catecholamines and significant increase in the vasorelaxation of the pulmonary arterial rings as compared with the controls. Further, the extract significantly attenuated hypoxia-induced increase in the VEGF levels in the plasma, BALF (ELISA) and lungs (immunohistochemistry). These observations suggest that SBT LE is able to provide significant protection against hypoxia-induced pulmonary vascular leakage.

beta-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes.

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.

Beta3 integrin-mediated ubiquitination activates survival signaling during myocardial hypertrophy.

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a beta(3) integrin (-/-) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate beta(3) integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-kappaB transcription of the E3 ligase, cIAP1. In PO beta(3)(-/-) mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a beta(3) integrin/Ub/NF-kappaB pathway contributes to compensatory hypertrophic growth.

Histone deacetylases facilitate sodium/calcium exchanger up-regulation in adult cardiomyocytes.

It is becoming increasingly evident that histone deacetylases (HDACs) have a prominent role in the alteration of gene expression during the growth remodeling process of cardiac hypertrophy. HDACs are generally viewed as corepressors of gene expression. However, we demonstrate that class I and class II HDACs play an important role in the basal expression and up-regulation of the sodium calcium exchanger (Ncx1) gene in adult cardiomyocytes. Treatment with the HDAC inhibitor trichostatin A (TSA) prevented the pressure-overload-stimulated up-regulation of Ncx1 expression. Overexpression of HDAC5 resulted in the dose-dependent up-regulation of basal and alpha-adrenergic stimulated Ncx1 expression. We show that Nkx2.5 recruits HDAC5 to the Ncx1 promoter, where HDAC5 complexes with HDAC1. Nkx2.5 also interacts with transcriptional activator p300, which is recruited to the Ncx1 promoter. We demonstrate that when Nkx2.5 is acetylated, it is found associated with HDAC5, whereas deacetylated Nkx2.5 is in complex with p300. Notably, TSA treatment prevents p300 from being recruited to the endogenous Ncx1 promoter, resulting in the repression of Ncx1 expression. We propose a novel model for Ncx1 regulation in which deacetylation of Nkx2.5 is required for the recruitment of p300 and results in up-regulation of exchanger expression.

Lack of beta3 integrin signaling contributes to calpain-mediated myocardial cell loss in pressure-overloaded myocardium.

Although cardiac hypertrophy initially ensues as a compensatory mechanism, it often culminates in congestive heart failure. Based on our earlier studies that calpain and beta3 integrin play cell death and survival roles, respectively, during pressure-overload (PO) hypertrophy, we investigated if the loss of beta3 integrin signaling is a potential mechanism for calpain-mediated cardiomyocyte death during PO. beta3 Integrin knockout (beta3) and wild-type mice were used to induce either moderate or severe PO in vivo for short-term (72-hour) and long-term (4-week) transverse aortic constriction. Whereas wild-type mice showed no changes during moderate PO at both time points, beta3 mice exhibited both enrichment of the mu-calpain isoform and programmed cell death of cardiomyocytes after 4-week PO. However, with severe PO that caused increased mortality in both mice groups, cell death was observed in wild-type mice also. To study calpain's role, calpeptin, a specific inhibitor of calpain, was administered through an osmotic mini-pump at 2.5 mg/kg per day beginning 3 days before moderate transverse aortic constriction or sham surgery. Calpeptin administration blocked both calpain enrichment and myocardial cell death in the 4-week PO beta3 mice. Because beta3 integrin contributes to cardioprotective signaling, these studies indicate that the loss of specific integrin function could be a key mechanism for calpain-mediated programmed cell death of cardiomyocytes in PO myocardium.

Novel l-adenosine analogs as cardioprotective agents.

Two l-nucleosides, l-3'-amino-3'-deoxy-N(6)-dimethyladenosine (l-3'-ADMdA) 1, previously synthesized in our laboratory, and the novel l-3'-amino-3'-deoxy-N(6)-methyladenosine-5'-N-methyluronamide (l-3'-AM-MECA) 2 were evaluated in an ischemia/reperfusion model on Langendorff perfused mouse heart. l-3'-ADMdA 1 was found to enhance functional recovery from ischemia (32.2+/-3.7cm H(2)O/s % rate pressure product, compared to 21.3+/-1.4 for the control and 30.7+/-3.4 for adenosine) and increase the time to onset of ischemic contracture (14.5+/-0.9min, compared to 10.5+/-1.0min for the control and 13.6+/-0.6min for adenosine) comparable to adenosine. Consistent with the functional recovery data, decreased infarction area was seen in the case of 1 (19.1+/-8.4, compared to 40.5+/-7.2% for the control and 11.5+/-2.1% for adenosine). In contrast, l-3'-AM-MECA 2 did not show significant functional recovery, increased onset of contracture, nor decreased infarction area compared to control. Unlike adenosine, neither 1 nor 2 induced cardiac standstill in mouse heart.

Angiokine Wisp-1 is increased in myocardial infarction and regulates cardiac endothelial signaling.

Myocardial infarctions (MIs) cause the loss of myocytes due to lack of sufficient oxygenation and latent revascularization. Although the administration of histone deacetylase (HDAC) inhibitors reduces the size of infarctions and improves cardiac physiology in small-animal models of MI injury, the cellular targets of the HDACs, which the drugs inhibit, are largely unspecified. Here, we show that WNT-inducible secreted protein-1 (Wisp-1), a matricellular protein that promotes angiogenesis in cancers as well as cell survival in isolated cardiac myocytes and neurons, is a target of HDACs. Further, Wisp-1 transcription is regulated by HDACs and can be modified by the HDAC inhibitor, suberanilohydroxamic acid (SAHA/vorinostat), after MI injury. We observe that, at 7 days after MI, Wisp-1 is elevated 3-fold greater in the border zone of infarction in mice that experience an MI injury and are injected daily with SAHA, relative to MI alone. Additionally, human coronary artery endothelial cells (HCAECs) produce WISP-1 and are responsive to autocrine WISP-1-mediated signaling, which functionally promotes their proangiogenic behavior. Altering endogenous expression of WISP-1 in HCAECs directly impacts their network density in vitro. Therapeutic interventions after a heart attack define the extent of infarct injury, cell survival, and overall prognosis. Our studies shown here identify a potentially novel cardiac angiokine, Wisp-1, that may contribute to beneficial post-MI treatment modalities.

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model.

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.

HDACs Regulate miR-133a Expression in Pressure Overload-Induced Cardiac Fibrosis.

BACKGROUND: MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. METHODS AND RESULTS: Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload hypertrophy. Mice were treated with class I and IIb HDAC inhibitor (HDACi) via drinking water for 2 and 4 weeks post TAC. miRNA expression was determined by real-time polymerase chain reaction. Echocardiography was performed at baseline and post TAC end points for structural and functional assessment. Chromatin immunoprecipitation was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7- and 0.5-fold at 2 and 4 weeks post TAC, respectively, when compared with vehicle control (P<0.05). HDAC inhibition prevented this significant decrease 2 weeks post TAC and maintained miR-133a expression near vehicle control levels, which coincided with (1) a decrease in connective tissue growth factor expression, (2) a reduction in cardiac fibrosis and left atrium diameter (marker of end-diastolic pressure), suggesting an improvement in diastolic function. Chromatin immunoprecipitation analysis revealed that HDAC1 and HDAC2 are present on the miR-133a enhancer regions. CONCLUSIONS: The results reveal that HDACs play a role in the regulation of pressure overload-induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload-induced cardiac fibrosis.

Ceramide synthase 5 mediates lipid-induced autophagy and hypertrophy in cardiomyocytes.

Diabetic cardiomyopathy (DbCM), which consists of cardiac hypertrophy and failure in the absence of traditional risk factors, is a major contributor to increased heart failure risk in type 2 diabetes patients. In rodent models of DbCM, cardiac hypertrophy and dysfunction have been shown to depend upon saturated fatty acid (SFA) oversupply and de novo sphingolipid synthesis. However, it is not known whether these effects are mediated by bulk SFAs and sphingolipids or by individual lipid species. In this report, we demonstrate that a diet high in SFA induced cardiac hypertrophy, left ventricular systolic and diastolic dysfunction, and autophagy in mice. Furthermore, treatment with the SFA myristate, but not palmitate, induced hypertrophy and autophagy in adult primary cardiomyocytes. De novo sphingolipid synthesis was required for induction of all pathological features observed both in vitro and in vivo, and autophagy was required for induction of hypertrophy in vitro. Finally, we implicated a specific ceramide N-acyl chain length in this process and demonstrated a requirement for (dihydro)ceramide synthase 5 in cardiomyocyte autophagy and myristate-mediated hypertrophy. Thus, this report reveals a requirement for a specific sphingolipid metabolic route and dietary SFAs in the molecular pathogenesis of lipotoxic cardiomyopathy and hypertrophy.

ß3 integrin in cardiac fibroblast is critical for extracellular matrix accumulation during pressure overload hypertrophy in mouse.

The adhesion receptor ß3 integrin regulates diverse cellular functions in various tissues. As ß3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of ß3 integrin in cardiac fibrosis by using wild type (WT) and ß3 integrin null (ß3-/-) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, ß3-/- mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from ß3-/- mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in ß3-/- mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and ß3-/- mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that ß3-/- cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in ß3-/- cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that ß3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.