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G protein-coupled receptor kinase-2 and -3 (GRK2 and GRK3) in cardiac myocytes catalyze phosphorylation and desensitization of different G protein-coupled receptors through specificity controlled by their carboxyl-terminal pleckstrin homology domain. Although GRK2 has been extensively investigated, the function of cardiac GRK3 remains unknown. Thus, in this study cardiac function of GRK3 was investigated in transgenic (Tg) mice with cardiac-restricted expression of a competitive inhibitor of GRK3, i.e. the carboxyl-terminal plasma membrane targeting domain of GRK3 (GRK3ct). Cardiac myocytes from Tg-GRK3ct mice displayed significantly enhanced agonist-stimulated alpha(1)-adrenergic receptor-mediated activation of ERK1/2 versus cardiac myocytes from nontransgenic littermate control (NLC) mice consistent with inhibition of GRK3. Tg-GRK3ct mice did not display alterations of cardiac mass or left ventricular dimensions compared with NLC mice. Tail-cuff plethysmography of 3- and 9-month-old mice revealed elevated systolic blood pressure in Tg-GRK3ct mice versus control mice (3-month-old mice, 136.8 +/- 3.6 versus 118.3 +/- 4.7 mm Hg, p0.001), an observation confirmed by radiotelemetric recording of blood pressure of conscious, unrestrained mice. Simultaneous recording of left ventricular pressure and volume in vivo by miniaturized conductance micromanometry revealed increased systolic performance with significantly higher stroke volume and stroke work in Tg-GRK3ct mice than in NLC mice. This phenotype was corroborated in electrically paced ex vivo perfused working hearts. However, analysis of left ventricular function ex vivo as a function of increasing filling pressure disclosed significantly reduced (dP/dt)(min) and prolonged time constant of relaxation (tau) in Tg-GRK3ct hearts at elevated supraphysiological filling pressure compared with control hearts. Thus, inhibition of GRK3 apparently reduces tolerance to elevation of preload. In conclusion, inhibition of cardiac GRK3 causes hypertension because of hyperkinetic myocardium and increased cardiac output relying at least partially on cardiac myocyte alpha(1)-adrenergic receptor hyper-responsiveness. The reduced tolerance to elevation of preload may cause impaired ability to withstand pathophysiological mechanisms of heart failure.

Evidence has accumulated to suggest that cardiomyocyte apoptosis contributes significantly to the progression of heart failure. A salient characteristic of the failing heart is an upregulation of the adenylyl cyclase inhibitory G protein alpha subunit, Galpha(i2); however this upregulation has yet to be established as beneficial or deleterious. Of note, G protein-coupled receptor (GPCR) signaling through Gi has been implicated in the cardioprotective properties of adenosine, opioids, and bradykinin; however to date there has been no in vivo evidence that increased Gi in ischemic myocardium is cardioprotective. We have created a novel molecular tool to specifically address this question and other Gi-related mechanisms in the heart. We have developed a Gi inhibitor, GiCT - the carboxyl-terminal 63 amino acids of Galphai(i2), which represents the region of Gi that interacts specifically with GPCRs. Gi selectivity of GiCT has been shown in vitro as Gi signals are inhibited while Gs and Gq signals are not. Thus, a class-specific Gi inhibitor has been developed. We next created a transgenic mouse line with inducible, cardiac-specific expression of GiCT (Tg-GiCT mice). At baseline, Tg-GiCT mice display a structural and functional phenotype consistent with non-transgenic (nTg) mice. However, when subjected to stress in the form of myocardial ischemia/reperfusion (I/R) injury, Tg-GiCT mice demonstrate a significant increase in infarct size as compared to nTg mice. Furthermore, Tg-GiCT mice demonstrate increased myocardial apoptosis in response to I/R, with a resultant decrease in contractile performance. These results indicate first that ischemic damage induces apoptotic signals that can be countered by GPCR signaling specifically through Gi. Moreover, the beneficial survival effects of Gi signaling on the prevention of myocardial apoptosis in ischemia appears to outweigh any potential detrimental effects that Gi upregulation may have on contractility through inhibition of cAMP. Overall, our results demonstrate the in vivo utility of GiCT - to elucidate the novel finding that upregulation of Gi seen in dysfunctional myocardium induces survival signals and appears to be an adaptive mechanism post-ischemia to protect myocytes from apoptosis.

Objectives : Pulmonary remodeling is a well recognized consequence of heart failure (HF). However, the cellular and molecular mechanisms orchestrating the structural alterations of the lungs in HF are poorly understood. We have previously reported induction of the profibrotic peptide connective tissue growth factor (CTGF) in myocardial tissue of rats with HF, suggesting a role of CTGF during myocardial remodeling. The aim of the present study was to explore the potential role of CTGF in pulmonary remodeling in HF. Methods : Pulmonary tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Real-time quantitative RT-PCR was employed to investigate mRNA levels. The cellular distribution of CTGF was analysed by immunohistochemistry. Results : Seven days after induction of myocardial infarction (MI) and HF in rats we found 2.3-fold and 1.9-fold increase of pulmonary transforming growth factor-β1 and procollagen α1(I) mRNA levels, respectively, and typical morphological characteristics of pulmonary remodeling including interstitial fibrosis and medial thickening of pulmonary arteries. Pulmonary CTGF mRNA levels were substantially elevated in HF rats compared to sham-operated rats (4-fold; P

Background Inflammation plays a pathogenic role in the development of heart failure (HF). The aim of this study was to examine the effect of treatment with the immunomodulating drug thalidomide in a rat model for post-myocardial infarction (MI) HF. Methods Rats were subjected to MI by left coronary artery ligation or sham-operated. Seven days after surgical intervention rats were randomised to treatment with thalidomide or vehicle for 8 weeks. Results Our main findings were: (i) thalidomide treatment did not affect cardiac function or the hypertrophic response, as determined by haemodynamic measurements and heart chamber weights, respectively. (ii) HF rats treated with thalidomide had a minor reduction in septum and relative wall thickness (p

Adrenomedullin (AM) is a potent vasorelaxing peptide with natriuretic and diuretic actions. Recent data indicate that AM may function as an endogenous regulator of cardiac function. We investigated to what extent AM, the AM receptor subtypes, and AM receptor-associated proteins were regulated in cardiomyocytes and non-cardiomyocytes of rats with congestive heart failure (CHF), and whether such regulation was paralleled by corresponding alterations of functional responses to AM. Cardiomyocytes and non-cardiomyocytes were isolated from myocardial tissue of rats 7 days after induction of myocardial infarction or sham operation. AM immunoreactivity was found in cardiomyocytes, endothelial cells, and fibroblasts. Robust increase of AM mRNA levels was observed both in the cardiomyocytes and in the non-cardiomyocytes of CHF rats compared to that of sham-operated rats (2.7-fold and 3.7-fold, respectively, P

Objective: Cardiac remodelling associated with congestive heart failure typically involves dilatation of the ventricular cavities, cardiomyocyte hypertrophy and alterations of extracellular matrix. Biglycan is an extracellular proteoglycan with several recently appreciated functions including cell adhesion, collagen fibril assembly, and growth factor interactions. The aims of this study were to investigate the regulation of biglycan expression and to elucidate the site(s) of synthesis of biglycan in myocardial tissue in an experimental model of heart failure (HF). Methods: Myocardial tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Northern blot analysis and real-time quantitative RT-PCR were employed to investigate mRNA levels. The cellular distribution of biglycan was analysed by in situ hybridisation and immunohistochemistry. Results: Myocardial biglycan mRNA levels in non-ischemic tissue of both left and right ventricles of heart failure rats were substantially elevated as compared to sham-operated rats. Although expression levels peaked 7 days after MI (13-fold increase compared to the sham group, P

Objective: The pulmonary circulation may contribute to elevated plasma levels of endothelin-1 (ET-1) in congestive heart failure (CHF). The aims of the present study were to determine the mechanisms of increased secretion of ET-1 from the pulmonary circulation in CHF. Methods: Juvenile pigs were subjected to sham operation ( n =9) or rapid cardiac pacing-induced CHF (215–240 bpm, n =15). Results: Three weeks of rapid pacing led to significant left ventricular dilatation, increased cardiac filling pressures, and reduced contractility (CHF pigs). Arterial plasma ET-1 levels in the CHF pigs were increased 4-fold compared to sham pigs ( P

Purpose: Type 2 diabetes (T2D) is an important risk factor for heart failure. T2D is associated with the metabolic syndrome, encompassing cardiovascular risk factors such as obesity, insulin resistance and dyslipidemia, factors also associated with left ventricular diastolic dysfunction. Patients with T2D have elevated plasma levels of free fatty acids and these are proposed to activate inflammation. Interleukin-1β (IL-1β) is activated by the NLRP3 inflammasome and a central mediator in T2D. Palmitate, the most abundant saturated fatty acid in plasma was recently shown to activate the NLRP3 inflammasome in macrophages. The aim of this study was to investigate if this mechanism operates within myocardial cells. Methods and Results: We first replicated previous findings by incubating bone marrow-derived macrophages with palmitate, and found release of active IL-1β. We went on to study primary mouse cardiac fibroblasts (CF). CF were stimulated with LPS, causing priming of the inflammasome, and then with palmitate. This caused release of active IL-1β. Importantly, this was not found in CF from NLRP3 and ASC deficient mice, demonstrating that the effect of palmitate on IL-1β release is mediated through the NLRP3 inflammasome. Other fatty acids such as eicosapentaenoic acid (EPA), oleic acid (OA), docosahexaenoic acid did not cause inflammasome activation. The omega-3 fatty EPA is known to suppress palmitate-induced metabolic stress, and when combining palmitate and EPA, we found markedly reduced release of IL-1β. By viewing CF stained with Bodipy under fluorescence microscopy, we observed an inhibitory effect of EPA on PA-induced lipid accumulation. AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis and also suppresses reactive oxygen species (ROS) production. We found that palmitate markedly reduced AMPK phosphorylation and Mitosox staining revealed increased mitochondrial ROS. Addition of the pharmacological AMPK activator AICAR completely blocked palmitate-induced IL-1β production. AMPK is also involved in regulation of autophagy, and we found that palmitate reduced levels of beclin-1, indicating that reduced autophagy could represent a mechanism for palmitate-induced inflammasome activation. Conclusion: Palmitate causes activation of the NLRP3 inflammasome in cardiac fibroblasts and this may involve increased mitochondrial ROS and defective autophagy. These findings indicate that NLRP3 inflammasome may act as a sensor of metabolic stress in the heart. EPA inhibits PA-induced NLRP3 inflammasome activation, possibly via reduced lipid accumulation.

Purpose: Immune activation plays a nodal role in the pathogenesis of heart failure (HF) and Toll-like receptor 9 (TLR9) signaling significantly impacts the development of systolic HF. However, the consequence of altered TLR9 activity in diastolic HF is unknown. Methods: We engaged in a 3-generation breeding strategy using αMHC-MerCreMer Serca2a flox/flox mice crossed with TLR9-/- mice to generate several comparable mouse lines. Employing these various genetic mouse lines we addressed the importance of TLR9 on progression of diastolic HF, the latter by temporally restricted depletion of cardiac myocyte SERCA2a. Mice were allocated to two substudies; 1) a 12 week survival study registering spontaneous death or signs of severe morbidity according to pre-specified criteria (leading to euthanasia) and 2) a study examining mice at baseline, 3 weeks and 6 weeks by MRI and echocardiography. After imaging at 6 weeks mice were euthanized and tissue and blood were harvested. Results: All mice depleted of cardiomyocyte SERCA2a, but none with the SERCA2a gene intact, reached our pre-specified end-parameter within 73 days. The presence of TLR9 in this model of diastolic HF led to significant improvement in survival with a median life expectancy of 62.5 days as compared to 58 days when the TLR9 gene was deleted (p=0.007). In accordance with these findings serial imaging demonstrated an earlier onset of left ventricular restrictive filling abnormalities in the HF group depleted of TLR9. Significantly larger left atria, lower end-systolic and end-diastolic left ventricular volumes and lower stroke volumes could be detected. Conclusions: The presence of TLR9 augments the development of diastolic HF induced by SERCA2a depletion. This observation may pave the road for novel diastolic HF treatment strategies. ![Figure][1] Survival in days after HF induction [1]: pending:yes

Background: Experimental studies have shown a central role for Toll-like receptor 9 (TLR9) in the pathogenesis of heart failure (HF). There is also evidence that mitochondrial DNA (mtDNA) not confined within the mitochondrion may act as a TLR9 ligand. However, it is unknown whether these findings also have a human correlate. Methods: Plasma from patients with ischemic or non-ischemic HF (n=86), as well as plasma from age and gender matched controls were analyzed for mtDNA by qPCR. Tissue from the left ventricular apical region was collected upon implantation of a Left Ventricular Assist Device (LVAD) in patients with end-stage HF. Upon heart transplantation of these patients, apical cardiac tissue was harvested from the explanted hearts. RNA and protein were isolated from these samples for subsequent analyses of TLR9 mRNA and protein expression. The antibody used for TLR9 immunoblot analyses was targeted against non-activated (non-cleaved) TLR9 and could therefore be used as an indirect marker for activated TLR9 receptors. Results: We found increased circulating levels of mtDNA in patients with symptomatic HF compared with controls (p=0.003). Higher level of mtDNA was found in plasma from symptomatic HF patients (NYHA III and IV, p=0.01) compared with asymptomatic patients (NYHA I and II, p=0.06). In cardiac tissue from patients treated with LVAD there was a reduction of TLR9 mRNA expression upon treatment with LVAD (Fig 1, p= 0.002). Reduced endogenous TLR9 activation was confirmed in the same samples as a higher level of non-cleaved receptor was shown after LVAD treatment. Conclusion: Human HF is associated with increased cardiac TLR9. Furthermore, the TLR9 ligand, mtDNA, is increased within the circulation of HF patients. These data suggest a pathophysiological role for TLR9 in human HF. ![Figure][1] mRNA TLR9 in LVAD treated patients [1]: pending:yes

Purpose: Systemic inflammation in established HF is clinically relevant as many HF patients have concomitant chronic inflammatory conditions. Repetitive activation of Toll-like receptor 9 (TLR9) induces chronic inflammation. We used a pharmacological protocol to induce systemic, chronic inflammation in murine cardiomyopathy induced by cardiomyocyte specific deletion of SERCA2a. Methods: Cardiac expression of SERCA2a was deleted in 10 week old male αMHC-MerCreMer Serca2a flox/flox mice by one intraperitoneal injection of Tamoxifen, resulting in a lethal diastolic HF phenotype after 7 weeks. 4 weeks after conditional knock out (KO), mice were randomized to TLR9 agonist (CpG-B;50 mg i.p.) or PBS every 3 days. Echocardiography was performed 8 weeks after induction of HF, followed by euthanization and analyses of tissue and blood. The groups were followed for 12 weeks, registering death and morbidity (by pre-specified criteria). Non-HF control groups treated with CpG-B or PBS served as controls. Results: TLR9 stimulated mice showed alterations in levels of leukocytes, erythrocytes and platelets as measured by flow cytometry and peripheral blood count, as well as pulmonary inflammation and hypersplenism (histology). The alterations were not found in controls. Echocardiographic analyses revealed aggravation in diastolic parameters in HF mice treated with CpG B. The latter group had reduced life expectancy compared with HF controls (median 59.5 and 68.5 days respectively, p=0.003). Non-HF, CpG B treated mice did not die or display signs of disease within the observation period. Conclusions: Chronic inflammation promoted by continuous TLR9 stimulation worsens diastolic HF caused by SERCA2a deletion. This study may provide insight into the consequences of chronic inflammatory conditions on HF development. ![Figure][1] Survival in days after HF induction [1]: pending:yes

Background: NLRP3 is an innate immune receptor that recognizes tissue damage and initiate inflammatory processes: chiefly, upon activation, NLRP3 forms multiprotein complexes with the adaptor protein ASC and caspase-1, termed inflammasomes, which cleaves pro-IL-1β and mediates release of bioactive IL-1β. Thus, NLRP3 activation may lead to further tissue damage. Hence, we hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction and sought to investigate the mechanisms herein. Methods: The LAD of WT, NLRP3 KO and ASC KO mice of both genders were sham-operated or ligated in vivo for 30 minutes followed by 3 or 24 hours reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3Cys or PBS was injected intraperitoneally 60 minutes prior to LAD ligation. In animals subjected to 24 hours reperfusion, the LAD was re-ligated in vivo and non-ischemic tissue stained by retrograde perfusion of Evans Blue in the aorta. The area at risk was stained after slicing with TTC, revealing the area of non-stained tissue as the infarct size. For further mechanistic investigations, blood plasmas and left ventricle tissues were collected and a hypothesis-driven selection of protein or mRNA targets were investigated with Western blot, ELISA, bioplex or PCR. Results: No difference in the area at risk was observed between any groups. Surprisingly, hearts from NLRP3 -and ASC deficient mice featured larger infarct size than that of WT mice (p=0.0048 and not statistically significant [p=0.0840], respectively). Furthermore, preconditioning with the TLR2 agonist Pam3Cys reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. There was no difference in plasma troponin I, IL-1β or IL-1α between any groups at 3 or 24 hours. In accordance with this, there were similar corresponding mRNA levels in cardiac tissue. However, there were significantly higher levels of CXCL1 in plasma from NLRP3-deficient mice at 3 hours. Preconditioning with Pam3Cys and MI for 30 min, resulted in increased phosphorylation of the cardioprotective kinase AKT in WT mice. However, this was not observed in NLRP3 -or ASC deficient mice, indicating a role for the NLRP3 inflammasome in cardioprotection. Conclusion: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardio-protective p-AKT signalling pathway involved in preconditioning. Furthermore, increased CXCL1 chemokine release may result in more tissue damage due to recruitment of neutrophils. Hence, our data imply that NRLP3 contribute to cardio-protection during ischemia-reperfusion.

Accumulation of DNA damage due to impaired base excision repair (BER) may play a role in the pathogenesis of myocardial failure. The DNA glycosylase Neil3 is known to carry out BER during oxidative stress induced DNA damage, but is also thought to have a role in stem cell biology. Based on this, we hypothesized that Neil3 plays a role in myocardial remodeling during heart failure (HF). qPCR showed an increased expression of Neil3 in myocardial biopsies from patients with severe HF, and that the Neil3 expression significantly decreased following improvement of myocardial function following treatment with left ventricular assist device. In a murine model of myocardial infarction (MI) we found a strong upregulation of Neil3 on day 3, 7 and 21 after MI. At day 7, myocardial cell isolation revealed a low Neil3 expression in cardiomyocytes, while a 2.5- and a 4.5-fold increased expression was found in endothelial cells and fibroblasts, respectively, following MI compared to sham. In leukocytes an equally high expression of Neil3 was observed in both sham and MI. Using Neil3 knock-out (KO) mice we found a significantly higher mortality in KO compared to wild type (WT) mice after MI ( 73% vs. 40%), despite smaller infarct sizes in the Neil3 KO, as determined by MRI. The first week post MI, all animals died from cardiac rupture. Zymography on protein extracts from the infarcted tissue revealed significantly increased levels of MMP2 in the Neil3 KO, 3 days after MI. Immunohistochemistry (IHC), at the same time point, showed similar numbers of inflammatory cells measured by the presence of Ly6G+ neutrophils, Mac2+ monocytes/macrophages and CD45+ leukocytes. At day 6 a significantly higher number of Ki67+ proliferating cells were observed in the Neil3 KO infarcted areas. In accordance with this, primary cardiac fibroblasts lacking Neil3 showed a higher proliferation rate, measured both by BrdU and scrape wound assay. Furthermore, we found a significantly higher mRNA expression of both the myofibroblast marker α-SMA, and collagen type III in cardiac fibroblasts isolated from Neil3 KO mice 7 days post MI, indicating increased levels of activated fibroblasts and altered fibroblast function. IHC 6 days post MI showed the same tendency towards more α-SMA-positive fibroblasts and more collagen by Sirius red in the Neil3 KO infarcts, however these differences were not statistically significant. In conclusion, our data suggest that after MI, Neil3 plays a role in regulating fibroblast proliferation and function, affecting wound healing and extracellular matrix composition and quality, altogether reducing mortality.

Background —Increased plasma adrenomedullin (ADM) levels have been reported in congestive heart failure (HF). The present study was designed to investigate myocardial regulation of the different components of the ADM signaling system (ADM, ADM receptor, and receptor-activity–modifying protein-2, RAMP-2) during ischemic HF in rats and to identify the cells in the myocardium displaying ADM-like immunoreactivity (ADM-ir). Furthermore, the effects of endothelin (ET) receptor antagonism on expression of the myocardial ADM system during HF were investigated. Methods and Results —Northern blot analysis revealed increased ADM mRNA expression in the nonischemic left ventricle, with maximal levels 28 days after induction of myocardial infarction (1.5-fold, P P P A /ET B receptor antagonist bosentan (100 mg · kg −1 · day −1 PO) for 15 days prevented the increase of RAMP-2 mRNA. Conclusions —The study demonstrates a concerted induction of several components of the myocardial ADM signaling system during postinfarction failure and that the vessels are the main source of myocardial ADM. Our observations indicate a role for ADM as an autocrine/paracrine factor during ventricular remodeling after myocardial infarction.