Innate Immune Nod1/RIP2 Signaling Is Essential for Cardiac Hypertrophy but Requires Mitochondrial Antiviral Signaling Protein for Signal Transductions and Energy Balance.

BACKGROUND: Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone. METHODS: To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1-/-, RIP2-/-, or wild-type mice to transverse aortic constriction or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. RESULTS: Nod1 and RIP2 proteins were upregulated in the heart after transverse aortic constriction, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1-/- and RIP2-/- mice subjected to transverse aortic constriction exhibited better survival, improved cardiac function, and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis, including NF (nuclear factor) κB and MAPK (mitogen-activated protein kinase)-GATA4/p300, were reduced in both Nod1-/- and RIP2-/- mice after transverse aortic constriction compared with wild-type mice. Coimmunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF κB signaling, and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions. CONCLUSIONS: We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling after stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response, and mitochondrial energy metabolism in stressed cardiomyocytes. Thus, Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.

Endoplasmic reticulum resident protein 44 (ERp44) deficiency in mice and zebrafish leads to cardiac developmental and functional defects.

BACKGROUND: Endoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isomerase family, is induced during ER stress, and may be involved in regulating Ca(2+) homeostasis. However, the role of ERp44 in cardiac development and function is unknown. The aim of this study was to investigate the role of ERp44 in cardiac development and function in mice, zebrafish, and embryonic stem cell (ESC)-derived cardiomyocytes to determine the underlying role of ERp44. METHODS AND RESULTS: We generated and characterized ERp44(-/-) mice, ERp44 morphant zebrafish embryos, and ERp44(-/-) ESC-derived cardiomyocytes. Deletion of ERp44 in mouse and zebrafish caused significant embryonic lethality, abnormal heart development, altered Ca(2+) dynamics, reactive oxygen species generation, activated ER stress gene profiles, and apoptotic cell death. We also determined the cardiac phenotype in pressure overloaded, aortic-banded ERp44(+/-) mice: enhanced ER stress activation and increased mortality, as well as diastolic cardiac dysfunction with a significantly lower fractional shortening. Confocal and LacZ histochemical staining showed a significant transmural gradient for ERp44 in the adult heart, in which high expression of ERp44 was observed in the outer subepicardial region of the myocardium. CONCLUSIONS: ERp44 plays a critical role in embryonic heart development and is crucial in regulating cardiac cell Ca(2+) signaling, ER stress, ROS-induced oxidative stress, and activation of the intrinsic mitochondrial apoptosis pathway.

HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress.

The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1(-/-) mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein-protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

The primary benefits of angiotensin-converting enzyme inhibition on cardiac remodeling occur during sleep time in murine pressure overload hypertrophy.

OBJECTIVES: Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. BACKGROUND: Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. METHODS: We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wake time for 8 weeks starting 1 week after surgery. RESULTS: TAC mice given captopril at sleep time had improved cardiac function and significantly decreased heart: body weight ratios, myocyte cross-sectional areas, intramyocardial vascular medial wall thickness, and perivascular collagen versus TAC mice given captopril or placebo during wake time. Captopril induced similar drops in blood pressure at sleep or wake time, suggesting that time-of-day differences were not attributable to blood pressure changes. These beneficial effects of captopril were correlated with diurnal changes in ACE mRNA expression in the heart. CONCLUSIONS: The ACE inhibitor captopril benefited cardiovascular remodeling only when administered during sleep; wake-time captopril ACE inhibition was identical to that of placebo. These studies support the hypothesis that the heart (and vessels) remodel during sleep time and also illustrate the importance of diurnal timing for some cardiovascular therapies.

Cathepsin-L contributes to cardiac repair and remodelling post-infarction.

AIMS: Cathepsin-L (CTSL) is a member of the lysozomal cysteine protease family, which participates in remodelling of various tissues. Herein, we sought to examine the potential regulation of CTSL in cardiac remodelling post-infarction. METHODS AND RESULTS: Experimental myocardial infarction (MI) was created in CTSL-deficient (Ctsl(-/-)) mice (B6 × FSB/GnEi a/a Ctsl(fs)/J) and wild-type littermates (Ctsl(+/+)) by left coronary artery ligation. At days 3, 7, 14, and 28 post-MI, we monitored survival rate and evaluated cardiac function, morphology, and molecular endpoints of repair and remodelling. Survival was 56% in Ctsl(-/-) mice in contrast to 80% (P < 0.05) in Ctsl(+/+) mice post-MI by day 28. The Ctsl(-/-) mice exhibited greater scar dilatation, wall thinning, and worse cardiac dysfunction when compared with Ctsl(+/+) mice. Cardiac matrix metallopeptidase-9 (MMP-9) activity was also diminished, and c-kit-positive cells, natural killer cells, fibrocytes, and monocytes mobilized to peripheral blood and deposited to the infarcted myocardium were significantly decreased in Ctsl(-/-) mice. Furthermore, the local inflammatory response, and granulocyte-colony stimulating factor, stem cell factor (SCF), and stromal cell-derived factor-1 (SDF-1α) expression, as well as cell proliferation, revascularization, and myofibroblast deposition were significantly decreased in Ctsl(-/-) mice compared with Ctsl(+/+) mice. CONCLUSION: Our data indicate that CTSL regulates cardiac repair and remodelling post-MI through a mechanism with multiple pathways.

Survival and cardiac remodeling after myocardial infarction are critically dependent on the host innate immune interleukin-1 receptor-associated kinase-4 signaling: a regulator of bone marrow-derived dendritic cells.

BACKGROUND: The innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor-associated kinase-4 (IRAK-4), downstream of Toll/interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects left ventricular remodeling after MI. METHODS AND RESULTS: Experimental MI was created in IRAK-4(-/-) and wild-type mice by left coronary ligation. Mice with a targeted deletion of IRAK-4 had an improved survival rate at 4 weeks after MI. IRAK-4(-/-) mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less proinflammatory and Th1 cytokine expression mediated by suppression of nuclear factor-kappaB and c-Jun N-terminal kinase activation. IRAK-4(-/-) mice had fewer infiltrations of CD45+ leukocytes and CD11c+ dendritic cells, inhibition of apoptosis, and reduced fibrosis and nitric oxide production. Cardiac dendritic cells in IRAK-4(-/-) mice were relatively immature or functionally naïve after MI in that they demonstrated less cytokine and costimulatory molecule gene expression. Furthermore, IRAK-4(-/-) dendritic cells have less mobilization capacity. Transfer of wild type-derived bone marrow dendritic cells into IRAK-4(-/-) mice for functional dendritic cell reconstitution negated the survival advantage and reduced the cardiac dilation observed with IRAK-4(-/-) mice at 28 days after MI. CONCLUSIONS: Deletion of IRAK-4 has favorable effects on survival and left ventricular remodeling after MI through modification of the host inflammatory process by blunting the detrimental bone marrow dendritic cells mobilization after myocardial ischemia.

Simultaneous transforming growth factor beta-tumor necrosis factor activation and cross-talk cause aberrant remodeling response and myocardial fibrosis in Timp3-deficient heart.

The pleiotropic cytokines, transforming growth factor beta1 (TGFbeta1), and tumor necrosis factor (TNF) play critical roles in tissue homeostasis in response to injury and are implicated in multiple human diseases and cancer. We reported that the loss of Timp3 (tissue inhibitor of metalloproteinase 3) leads to abnormal TNF signaling and cardiovascular function. Here we show that parallel deregulation of TGFbeta1 and TNF signaling in Timp3(-/-) mice amplifies their cross-talk at the onset of cardiac response to mechanical stress (pressure overload), resulting in fibrosis and early heart failure. Microarray analysis showed a distinct gene expression profile in Timp3(-/-) hearts, highlighting activation of TGFbeta1 signaling as a potential mechanism underlying fibrosis. Neonatal cardiomyocyte-cardiofibroblast co-cultures were established to measure fibrogenic response to agonists known to be induced following mechanical stress in vivo. A stronger response occurred in neonatal Timp3(-/-) co-cultures, as determined by increased Smad signaling and collagen expression, due to increased TNF processing and precocious proteolytic maturation of TGFbeta1 to its active form. The relationship between TGFbeta1 and TNF was dissected using genetic and pharmacological manipulations. Timp3(-/-)/Tnf(-/-) mice had lower TGFbeta1 than Timp3(-/-), and anti-TGFbeta1 antibody (1D11) negated the abnormal TNF response, indicating their reciprocal stimulatory effects, with each manipulation abolishing fibrosis and improving heart function. Thus, TIMP3 is a common innate regulator of TGFbeta1 and TNF in tissue response to injury. The matrix-bound TIMP3 balances the anti-inflammatory and proinflammatory processes toward constructive tissue remodeling.

Gelsolin regulates cardiac remodeling after myocardial infarction through DNase I-mediated apoptosis.

Gelsolin, a calcium-regulated actin severing and capping protein, is highly expressed in murine and human hearts after myocardial infarction and is associated with progression of heart failure in humans. The biological role of gelsolin in cardiac remodeling and heart failure progression after injury is not defined. To elucidate the contribution of gelsolin in these processes, we randomly allocated gelsolin knockout mice (GSN(-/-)) and wild-type littermates (GSN(+/+)) to left anterior descending coronary artery ligation or sham surgery. We found that GSN(-/-) mice have a surprisingly lower mortality, markedly reduced hypertrophy, smaller late infarct size, less interstitial fibrosis, and improved cardiac function when compared with GSN(+/+) mice. Gene expression and protein analysis identified significantly lower levels of deoxyribonuclease (DNase) I and reduced nuclear translocation and biological activity of DNase I in GSN(-/-) mice. Absence of gelsolin markedly reduced DNase I-induced apoptosis. The association of hypoxia-inducible factor (HIF)-1alpha with gelsolin and actin filaments cleaved by gelsolin may contribute to the higher activation of DNase. The expression pattern of HIF-1alpha was similar to that of gelsolin, and HIF-1alpha was detected in the gelsolin complex by coprecipitation and HIF-1alpha bound to the promoter of DNase I in both gel-shift and promoter activity assays. Furthermore, the phosphorylation of Akt at Ser473 and expression of Bcl-2 were significantly increased in GSN(-/-) mice, suggesting that gelsolin downregulates prosurvival factors. Our investigation concludes that gelsolin is an important contributor to heart failure progression through novel mechanisms of HIF-1alpha and DNase I activation and downregulation of antiapoptotic survival factors. Gelsolin inhibition may form a novel target for heart failure therapy.

Direct injection of kit ligand-2 lentivirus improves cardiac repair and rescues mice post-myocardial infarction.

Myocardial infarction (MI) and subsequent adverse remodeling cause heart failure. Previously we demonstrated a role for Kit ligand (KL) in improving cardiac function post-MI. KL has two major isoforms; KL-1 is secreted whereas KL-2 is predominantly membrane bound. We demonstrate here first that KL-2-deficient mice have worse survival and an increased heart/bodyweight ratio post-MI compared to mice with reduced c-Kit receptor expression. Next we synthesized recombinant lentiviral vectors (LVs) that engineered functional expression of murine KL-1 and KL-2. For in vivo analyses, we directly injected these LVs into the left ventricle of membrane-bound KL-deficient Sl/Sl(d) or wild-type (WT) mice undergoing MI. Control LV/enGFP injection led to measurable reporter gene expression in hearts. Injection of LV/KL-2 attenuated adverse left ventricular remodeling and dramatically improved survival post-MI in both Sl/Sl(d) and WT mice (from 12 to 71% and 35 to 73%, respectively, versus controls). With regard toward beginning to understand the possible salutary mechanisms involved in this effect, differential staining patterns of Sca-1 and Ly49 on peripheral blood (PB) cells from therapeutically treated animals was found. Our data show that LV/KL-2 gene therapy is a promising treatment for MI.

Curcumin prevents and reverses murine cardiac hypertrophy.

Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. We hypothesized that curcumin, a natural polyphenolic compound abundant in the spice turmeric and a known suppressor of histone acetylation, would suppress cardiac hypertrophy through the disruption of p300 histone acetyltransferase-dependent (p300-HAT-dependent) transcriptional activation. We tested this hypothesis using primary cultured rat cardiac myocytes and fibroblasts as well as two well-established mouse models of cardiac hypertrophy. Curcumin blocked phenylephrin-induced (PE-induced) cardiac hypertrophy in vitro in a dose-dependent manner. Furthermore, curcumin both prevented and reversed mouse cardiac hypertrophy induced by aortic banding (AB) and PE infusion, as assessed by heart weight/BW and lung weight/BW ratios, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that curcumin abrogated histone acetylation, GATA4 acetylation, and DNA-binding activity through blocking p300-HAT activity. Curcumin also blocked AB-induced inflammation and fibrosis through disrupting p300-HAT-dependent signaling pathways. Our results indicate that curcumin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through suppression of p300-HAT activity and downstream GATA4, NF-kappaB, and TGF-beta-Smad signaling pathways.

Loss of PTEN attenuates the development of pathological hypertrophy and heart failure in response to biomechanical stress.

AIMS: The maladaptive response to biomechanical stress is a fundamental response in heart disease. Loss of the 3'-lipid phosphatase, phosphatase and tensin homolog deleted on chromosome ten (PTEN), is associated with increased phosphorylation of Akt/protein kinase B and glycogen synthase kinase-beta. We hypothesize that these key changes will halt the development of pathological hypertrophy and the progression to heart failure in response to pressure overload. METHODS AND RESULTS: In mice, muscle-specific knockout of PTEN, mckCRE-PTEN(flox/flox) (PTEN KO), resulted in basal hypertrophy and mild reduction in left ventricular (LV) systolic function. Male mice were subjected to aortic banding (AB) or sham operation. In contrast to mckCRE-PTEN(+/+) control mice, pressure overload in PTEN KO mice resulted in reduced pathological hypertrophy, less interstitial fibrosis, and reduced apoptosis with a marked preservation of LV function. Western blot analysis of mitogen-activated protein kinase (MAPK) signalling showed equivalent phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 with markedly reduced phosphorylation of jun N-terminal kinase (JNK)1 and JNK2, and p38 in PTEN KO mice subjected to AB. Loss of PTEN was associated with increased expression of the proangiogenic factors, vascular endothelial growth factor-A and angiopoietin-2, with preservation of the myocardial capillary density in response to pressure overload. Moreover, banded PTEN KO mice maintained the expression of several key metabolic genes that are known to be dysregulated in heart failure. In contrast, a subpressor dose of the G protein-coupled receptor (GPCR) agonist angiotensin II (Ang II) leads to increased pathological hypertrophy and MAPK activation in PTEN KO mice. CONCLUSION: Loss of PTEN prevents the development of maladaptive ventricular remodelling with preservation of angiogenesis and metabolic gene expression in response to pressure overload but not in response to the GPCR agonist, Ang II. Inhibition of PTEN signalling in the heart may represent a novel approach to slow the progression of heart failure in response to pathological biomechanical stress.

Impaired heart contractility in Apelin gene-deficient mice associated with aging and pressure overload.

Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin-converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene-targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload-induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin(-/y) and Apelin(+/y) mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging.

Tumor necrosis factor-alpha mediates cardiac remodeling and ventricular dysfunction after pressure overload state.

BACKGROUND: Pressure overload is accompanied by cardiac myocyte apoptosis, hypertrophy, and inflammatory/fibrogenic responses that lead to ventricular remodeling and heart failure. Despite incomplete understanding of how this process is regulated, the upregulation of tumor necrosis factor (TNF)-alpha after aortic banding in the myocardium is known. In the present study, we tested our hypothesis that TNF-alpha regulates the cardiac inflammatory response, extracellular matrix homeostasis, and ventricular hypertrophy in response to mechanical overload and contributes to ventricular dysfunction. METHODS AND RESULTS: C57/BL wild-type mice and TNF-knockout (TNF-/-) mice underwent descending aortic banding or sham operation. Compared with sham-operated mice, wild-type mice with aortic banding showed a significant increase in cardiac TNF-alpha levels, which coincided with myocyte apoptosis, inflammatory response, and cardiac hypertrophy in week 2 and a significant elevation in matrix metalloproteinase-9 activity and impaired cardiac function in weeks 2 and 6. Compared with wild-type mice with aortic banding, TNF-/- mice with aortic banding showed attenuated cardiac apoptosis, hypertrophy, inflammatory response, and reparative fibrosis. These mice also showed reduced cardiac matrix metalloproteinase-9 activity and improved cardiac function. CONCLUSIONS: Findings from the present study have suggested that TNF-alpha contributes to adverse left ventricular remodeling during pressure overload through regulation of cardiac repair and remodeling, leading to ventricular dysfunction.

Disturbed diurnal rhythm alters gene expression and exacerbates cardiovascular disease with rescue by resynchronization.

Day/night rhythms are recognized as important to normal cardiovascular physiology and timing of adverse cardiovascular events; however, their significance in disease has not been determined. We demonstrate that day/night rhythms play a critical role in compensatory remodeling of cardiovascular tissue, and disruption exacerbates disease pathophysiology. We use a murine model of pressure overload cardiac hypertrophy (transverse aortic constriction) in a rhythm-disruptive 20-hour versus 24-hour environment. Echocardiography reveals increased left ventricular end-systolic and -diastolic dimensions and reduced contractility in rhythm-disturbed transverse aortic constriction animals. Furthermore, cardiomyocytes and vascular smooth muscle cells exhibit reduced hypertrophy, despite increased pressure load. Microarray and real-time PCR demonstrate altered gene cycling in transverse aortic constriction myocardium and hypothalamic suprachiasmatic nucleus. With rhythm disturbance, there is a consequent altered cellular clock mechanism (per2 and bmal), whereas key genes in hypertrophic pathways (ANF, BNP, ACE, and collagen) are downregulated paradoxical to the increased pressure. Phenotypic rescue, including reversal/attenuation of abnormal pathology and genes, only occurs when the external rhythm is allowed to correspond with the animals' innate 24-hour internal rhythm. Our study establishes the importance of diurnal rhythm as a vital determinant in heart disease. Disrupted rhythms contribute to progression of organ dysfunction; restoration of normal diurnal schedules appears to be important for effective treatment of disease.

Correction of cardiac abnormalities in fabry mice by direct intraventricular injection of a recombinant lentiviral vector that engineers expression of alpha-galactosidase A.

BACKGROUND: Recombinant lentiviral vectors (LVs) offer the possibility of stable, long-term expression of transgenes even in non-dividing cells. In the present study this vector system was applied to a clinically relevant cardiovascular problem. METHODS AND RESULTS: Fabry disease results from deficient activity of alpha-galactosidase A (alpha-gal A) and cardiac abnormalities are a common and an important cause of death in patients with the disease. A therapeutic LV that delivers the alpha-gal A cDNA has been synthesized. In vitro studies established efficient transduction of the H9c2 rat cardiomyocytes and showed overexpression of enGFP (control) and alpha-gal A. In in vivo studies, the enGFP cDNA was transferred into C57BL/6 mouse hearts by direct intraventricular injection. Next, in a mouse model of Fabry disease, the recombinant therapeutic construct was delivered analogously. In cardiac tissue, alpha-gal A activity rose to 23% of normal levels at day 7 after LV injection, which is encouraging because levels of correction approximating 5% of normal may be curative for this disorder. There was also a corresponding reduction in globotriaosylceramide accumulation. Other organs assayed showed no detectable changes in alpha-gal A activity levels in injected animals. CONCLUSION: A localized benefit of directly injecting a therapeutic LV into the heart has been shown, confirming the utility of this delivery system for research and therapy for a variety of cardiovascular disorders.

Stem cell factor receptor induces progenitor and natural killer cell-mediated cardiac survival and repair after myocardial infarction.

Inappropriate cardiac remodeling and repair after myocardial infarction (MI) predisposes to heart failure. Studies have reported on the potential for lineage negative, steel factor positive (c-kit+) bone marrow-derived hematopoetic stem/progenitor cells (HSPCs) to repair damaged myocardium through neovascularization and myogenesis. However, the precise contribution of the c-kit signaling pathway to the cardiac repair process has yet to be determined. In this study, we sought to directly elucidate the mechanistic contributions of c-kit+ bone marrow-derived hematopoetic stem/progenitor cells in the maintenance and repair of damaged myocardium after MI. Using c-kit-deficient mice, we demonstrate the importance of c-kit signaling in preventing ventricular dilation and hypertrophy, and the maintenance of cardiac function after MI in c-kit-deficient mice. Furthermore, we show phenotypic rescue of cardiac repair after MI of c-kit-deficient mice by bone marrow transplantation of wild-type HSPCs. The transplanted group also had reduced apoptosis and collagen deposition, along with an increase in neovascularization. To better understand the mechanisms underlying this phenotypic rescue, we investigated the gene expression pattern within the infarcted region by using microarray analysis. This analysis suggested activation of inflammatory pathways, specifically natural killer (NK) cell-mediated mobilization after MI in rescued hearts. This finding was confirmed by immunohistology and by using an NK blocker. Thus, our investigation revealed a previously uncharacterized role for c-kit signaling after infarction by mediating bone marrow-derived NK and angiogenic cell mobilization, which contributes to improved remodeling and cardiac function after MI.

The remote ischemic preconditioning stimulus modifies gene expression in mouse myocardium.

BACKGROUND: We have recently demonstrated that remote ischemic preconditioning reduces ischemia-reperfusion injury in animal models. The mechanisms by which the remote ischemic preconditioning stimulus exerts its effect remain to be fully defined, and its effect on myocardial gene expression is unknown. We tested the hypothesis that remote ischemic preconditioning modifies myocardial gene expression immediately after the remote ischemic preconditioning stimulus (early phase) and 24 hours later (late phase). METHODS: Twenty male (C57BL/6) 10- to 12-week-old mice were randomized into 4 groups: group 1 (control, early phase; n = 5), group 2 (remote ischemic preconditioning, early phase; n = 5), group 3 (control, late phase; n = 5), and group 4 (remote ischemic preconditioning, late phase; n = 5). Groups 2 and 4 underwent remote ischemic preconditioning induced by 6 cycles of 4 minutes of occlusion and 4 minutes of reperfusion of the femoral artery. Groups 1 and 2 were killed 15 minutes after completion of sham procedure or remote ischemic preconditioning, and the hearts were removed and frozen in liquid nitrogen. Groups 3 and 4 were killed 24 hours after remote ischemic preconditioning, and the hearts were harvested in the same fashion. Gene expression was assessed by using the Affymetrix MG-430A chip (Affymetrix, Santa Clara, Calif). RESULTS: Data filtering (P < .05, analysis of variance) and hierarchic 2-way clustering identified significant differences in gene expression among the 4 groups. Genes involved in protection against oxidative stress (eg, Hadhsc, Prdx4, and Fabp4) and cytoprotection (Hsp73) were upregulated, whereas many proinflammatory genes (eg, Egr-1 and Dusp 1 and 6) were suppressed. CONCLUSION: A simple remote ischemic preconditioning stimulus modifies myocardial gene expression by upregulating cardioprotective genes and suppressing genes potentially involved in the pathogenesis of ischemia-reperfusion injury.

Combination of tumor necrosis factor-alpha ablation and matrix metalloproteinase inhibition prevents heart failure after pressure overload in tissue inhibitor of metalloproteinase-3 knock-out mice.

Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3(-/-) mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNFalpha converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-alpha (TNFalpha) processing. In addition, TNFalpha production increased in timp-3(-/-)-AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3(-/-)-AB myocardium. Timp-3(-/-)/tnfalpha(-/-) mice were generated and subjected to AB for comparative analyses with timp-3(-/-)-AB mice. This revealed the critical role of TNFalpha in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNFalpha, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNFalpha, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3(-/-)-AB mice. Notably, combining TNFalpha ablation with MMP inhibition completely rescued heart disease in timp-3(-/-)-AB mice. This study provides a basis for anti-TNFalpha and MMP inhibitor combination therapy in heart disease.

S100B expression modulates left ventricular remodeling after myocardial infarction in mice.

BACKGROUND: S100B, a 20-kDa, Ca2+-binding dimer, is a putative intrinsic negative regulator of myocardial hypertrophy expressed after myocardial infarction. S100B-overexpressing transgenic (TG) and S100B-knockout (KO) mice have been generated to assess the consequences of S100B expression and altered hypertrophy after infarction. METHODS AND RESULTS: We compared 21 wild-type (WT), 20 TG, and 24 KO mice over 35 days after experimental myocardial infarction with sham-operated controls (n=56). Of those, 4 WT-infarcted mice, 7 TG-infarcted mice, and 1 KO-infarcted mouse and no sham-operated mice died during the observation period. Among survivors, echocardiography, hemodynamic studies, and postmortem examination indicated that the WT and KO groups of infarcted mice mounted a hypertrophic response that was augmented in KO mice. The S100B-overexpressing TG group did not develop hypertrophy but demonstrated increased apoptosis. The postinfarct end-diastolic pressure was lower in KO mice than in WT mice, in accordance with other structural, hemodynamic, and functional parameters, which suggests that abrogation of S100B expression augmented hypertrophy, decreased apoptosis, and was beneficial to preservation of cardiac function within this time frame. CONCLUSIONS: S100B regulates the hypertrophic response and remodeling in the early postinfarct period and represents a potential novel therapeutic target.