Impact of delayed type hypersensitivity arthritis on development of heart failure by aortic constriction in mice.

AIMS: Patients with rheumatoid arthritis (RA) have increased risk of heart failure (HF). The mechanisms and cardiac prerequisites explaining this association remain unresolved. In this study, we sought to determine the potential cardiac impact of an experimental model of RA in mice subjected to HF by constriction of the ascending aorta. METHODS: Aorta was constricted via thoracotomy and placement of o-rings with inner diameter 0.55 mm or 0.66 mm, or sham operated. RA-like phenotype was instigated by delayed-type hypersensitivity arthritis (DTHA) two weeks after surgery and re-iterated after additional 18 days. Cardiac magnetic resonance imaging (MRI) was performed before surgery and at successive time points throughout the study. Six weeks after surgery the mice were euthanized, blood and tissue were collected, organ weights were documented, and expression levels of cardiac foetal genes were analysed. In a supplemental study, DTHA-mice were euthanized throughout 14 days after induction of arthritis, and blood was analysed for important markers and mediators of RA (SAP, TNF-α and IL-6). In order to put the latter findings into clinical context, the same molecules were analysed in serum from untreated RA patients and compared to healthy controls. RESULTS: Significant elevations of inflammatory markers were found in both patient- and murine blood. Furthermore, the DTHA model appeared clinically relevant when compared to the inflammatory responses observed in three prespecified RA severity disease states. Two distinct trajectories of cardiac dysfunction and HF development were found using the two o-ring sizes. These differences were consistent by both MRI, organ weights and cardiac foetal gene expression levels. Still, no difference within the HF groups, nor within the sham groups, could be found when DTHA was induced. CONCLUSION: DTHA mediated systemic inflammation did not cause, nor modify HF caused by aortic constriction. This indicates other prerequisites for RA-induced cardiac dysfunction.

Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.

Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.

Hepatic connective tissue growth factor expression and regulation differ between non-steatotic and non-alcoholic steatotic livers from brain-dead donor.

Accurate evaluation of liver steatosis is required from brain-dead donors (BDDs) with nonalcoholic fatty liver disease (NAFLD). Our purposes were to investigate expression and regulation of connective tissue growth factor (CTGF) expression in livers from human and rat after brain death, and further evaluate its potential application. NAFLD and brain death models were established in rats. LX2 cells were cultured under hypoxia/reoxygenation. CTGF protein and mRNA levels were measured in liver samples from BDDs of human and rat by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. YAP-regulated CTGF expression was investigated in LX2 cells via YAP small interfering RNA and Verteporfin treatment. Blood CTGF level from BDDs was measured by enzyme-linked immunosorbent assay. After brain death, CTGF, transforming growth factor-ß and YAP were overexpressed in non-alcoholic steatotic liver, whereas CTGF was downregulated in non-steatotic liver. Time-series analysis revealed that CTGF and YAP expression was comparable, as confirmed by inhibited YAP expression in LX2 cells. CTGF level and NAFLD activity were linearly correlated. CTGF expression and regulation differ between non-steatosis and nonalcoholic steatosis livers from BDDs. CTGF may be an important factor to evaluate graft quality from BDDs with NAFLD.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1ß release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKß, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.

CCN2/CTGF attenuates myocardial hypertrophy and cardiac dysfunction upon chronic pressure-overload.

BACKGROUND: Myocardial CCN2/CTGF (connective tissue growth factor) is strongly induced in heart failure (HF) and acts as a cardioprotective factor in ischemia/reperfusion injury. However, its functional role in myocardial hypertrophy remains unresolved. METHODS AND RESULTS: Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) and non-transgenic littermate control (NLC) mice were subjected to chronic pressure-overload by abdominal aortic banding. After 4weeks of persistent pressure-overload, a time point at which compensatory hypertrophy of the left ventricle (LV) prevails, Tg-CTGF mice displayed diminished increase of LV mass compared with NLC. At study end-point after 12 weeks of sustained aortic constriction, the mice displayed LV dilatation and reduced cardiac function. Repeated transthoracic echocardiography during the 12 weeks of chronic pressure-overload, revealed attenuation of LV dilatation and virtually sustained systolic function in Tg-CTGF mice compared with NLC mice. Also, increase of LV mass was blunted in Tg-CTGF versus NLC mice at study end-point. Consistently, increases of myocardial ANP, BNP and skeletal α-actin mRNA levels were blunted in Tg-CTGF mice subjected to chronic pressure-overload. Furthermore, cardiac myocytes from Tg-CTGF mice displayed increased phospho-NFATc2 levels and attenuated hypertrophic response upon stimulation with α1-adrenoceptor agonist, indicating that CTGF attenuates hypertrophic signaling in cardiac myocytes. Increase of myocardial collagen contents in mice subjected to aortic banding was similar in Tg-CTGF and NLC mice, indicating that CTGF have minimal impact on myocardial collagen deposition. CONCLUSION: This study provides novel evidence that CTGF attenuates cardiac hypertrophy upon chronic pressure-overload due to inhibition of signaling mechanisms that promote pathologic myocardial hypertrophy.

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload.

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 µl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of ß-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced ß(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.

Myocardial connective tissue growth factor (CCN2/CTGF) attenuates left ventricular remodeling after myocardial infarction.

AIMS: Myocardial CCN2/CTGF is induced in heart failure of various etiologies. However, its role in the pathophysiology of left ventricular (LV) remodeling after myocardial infarction (MI) remains unresolved. The current study explores the role of CTGF in infarct healing and LV remodeling in an animal model and in patients admitted for acute ST-elevation MI. METHODS AND RESULTS: Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) and non-transgenic littermate controls (NLC) were subjected to permanent ligation of the left anterior descending coronary artery. Despite similar infarct size (area of infarction relative to area at risk) 24 hours after ligation of the coronary artery in Tg-CTGF and NLC mice, Tg-CTGF mice disclosed smaller area of scar tissue, smaller increase of cardiac hypertrophy, and less LV dilatation and deterioration of LV function 4 weeks after MI. Tg-CTGF mice also revealed substantially reduced mortality after MI. Remote/peri-infarct tissue of Tg-CTGF mice contained reduced numbers of leucocytes, macrophages, and cells undergoing apoptosis as compared with NLC mice. In a cohort of patients with acute ST-elevation MI (n = 42) admitted to hospital for percutaneous coronary intervention (PCI) serum-CTGF levels (s-CTGF) were monitored and related to infarct size and LV function assessed by cardiac MRI. Increase in s-CTGF levels after MI was associated with reduced infarct size and improved LV ejection fraction one year after MI, as well as attenuated levels of CRP and GDF-15. CONCLUSION: Increased myocardial CTGF activities after MI are associated with attenuation of LV remodeling and improved LV function mediated by attenuation of inflammatory responses and inhibition of apoptosis.

Adrenomedullin is increased in alveolar macrophages and released from the lungs into the circulation in severe heart failure.

Adrenomedullin (AM) is a potent vasorelaxing peptide with natriuretic, diuretic, and growth inhibitory properties. Plasma concentrations and myocardial AM expression are increased in heart failure (HF). Since AM and AM binding sites are abundantly expressed in the lungs, we investigated to what extent pulmonary AM and AM receptor subtypes [CRLR/RAMP2 (AM1) and CRLR/RAMP3 (AM2)] are changed in HF and whether the lungs contribute to the increased plasma concentrations of AM reported in HF. Pulmonary AM mRNA and protein expression were increased by 2.8- and 2.6-fold, respectively, whereas mRNA expression of RAMP2 and CRLR was decreased in rats with HF 7 days after induction of MI compared to sham-operated rats (P < 0.05). Pulmonary AM receptor density was substantially decreased in HF rats compared to sham (3.7 +/-0.6 vs. 29.9 +/- 1.1 fmol/mg membrane protein; P < 0.05). Immunoreactivities against AM and the AM receptor components CRLR, RAMP2, and RAMP3 in the pulmonary tissue were seen in vascular smooth muscle cells, vascular endothelial cells, and in alveolar macrophages. AM mRNA expression in alveolar macrophages obtained from HF rats by bronchoalveolar lavage was 2.9-fold higher than in sham-operated rats (P < 0.05). An even more substantial increase of AM mRNA expression was found in alveolar macrophages from patients with HF (10-fold, P < 0.05), and this increase displayed a negative correlation to left ventricular systolic function (P < 0.05). Furthermore, a net release of AM from the lungs into the circulation was only found in HF patients with the most severe left ventricular systolic dysfunction. Thus, our data demonstrate increased expression and decreased receptor binding of AM in the lungs in severe HF. Furthermore, our data indicate that alveolar macrophages are an important source of pulmonary AM in both experimental and clinical HF. Finally, a net release of AM from the lungs into the circulation was only found in patients with severe systolic dysfunction.

Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function.

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac beta-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, alpha(1)-adrenergic, and beta(1)-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC(50) for GRK3, 5 +/- 0.7 pmol/mg of protein; EC(50) for GRK3ct, 2 +/- 0.2 pmol/mg of protein). The alpha(1)-adrenergic receptor was also a preferred substrate of GRK3 (IC(50),7 +/- 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and alpha(1)-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced beta(1)-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at beta(1)-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling beta(1)-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and alpha(1)-adrenergic receptors.

RAMP2 and RAMP3 mRNA levels are increased in failing rat cardiomyocytes and associated with increased responsiveness to adrenomedullin.

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 <0.05). Fairly high mRNA levels and immunoreactivity against the AM receptor chaperone receptor activity-modifying protein-2 (RAMP2) were also detected in the cardiomyocytes and non-cardiomyocytes. However, induction of RAMP2 mRNA expression was restricted to cardiomyocytes (1.8-fold increase in cardiomyocytes from CHF rats vs. sham rats; P <0.05). In contrast, very low levels of RAMP3 mRNA were observed. RAMP3 mRNA levels, however, were elevated in both cardiomyocytes and non-cardiomyocytes from CHF rats (6.5-fold and 2.4-fold increase vs. sham rats, respectively; P <0.05). Parallel increases of specific AM receptor binding sites and of AM-stimulated adenylyl cyclase activities were observed in failing cardiomyocytes compared to cardiomyocytes from sham rats (fivefold and sixfold increase, respectively; P <0.05). Thus, this study demonstrates that AM mRNA levels, AM receptor binding sites, and AM-stimulated adenylyl cyclase activities are increased in cardiomyocytes from failing rat hearts. Furthermore, our data suggest that induction of RAMP2 and RAMP3 contributes to the increased responsiveness to AM in failing cardiomyocytes.

Connective tissue growth factor--a novel mediator of angiotensin II-stimulated cardiac fibroblast activation in heart failure in rats.

The pathophysiologic mechanisms of myocardial remodeling in heart failure (HF) remain poorly understood. Using differential mRNA display of myocardial tissue from rats with ischemic HF vs. controls we identified robust myocardial induction of the mRNA encoding connective tissue growth factor (CTGF). The aim of this study was to investigate the sites of synthesis and the mechanisms of induction of CTGF in failing myocardial tissue. The study demonstrates that myocardial expression of CTGF mRNA and protein is substantially elevated in non-ischemic tissue from both the left and the right ventricles of rats with experimentally induced myocardial infarction (MI). The induction of myocardial CTGF mRNA was shown to transcend from early post-infarction HF to chronic HF. In situ hybridization and immunohistochemical analysis of myocardial tissue sections demonstrated expression of CTGF confined to fibroblasts and endothelial cells of non-ischemic myocardial tissue. In subsequent experiments rats subjected to MI were randomized to treatment with the AT1 angiotensin receptor antagonist losartan (12.5 mg/kg b.i.d. per os) or vehicle. Losartan attenuated ventricular hypertrophy, improved hemodynamics, and prevented the induction of myocardial CTGF mRNA observed in rats post-MI. To provide the cellular basis of Ang II-stimulated CTGF mRNA expression, primary cultures of rat myocardial fibroblasts were stimulated with Ang II (10(-7) M). Real-time reverse transcription-polymerase chain reaction and western blot analysis demonstrate that Ang II induces rapid, AT1 receptor-mediated elevations of CTGF mRNA and protein in rat cardiac fibroblasts. Furthermore, CTGF was shown to stimulate fibroblast proliferation in vitro. In conclusion, this study demonstrates that CTGF is a myocardial effector of Ang II-induced myocardial remodeling in HF mediated via AT1 receptors situated on cardiac fibroblasts.

Induction of myocardial biglycan in heart failure in rats--an extracellular matrix component targeted by AT(1) receptor antagonism.

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<0.05), substantial elevations of biglycan mRNA were observed throughout the study period. Analysis of cellular distribution revealed that biglycan expression was confined to myocardial fibroblasts and vascular endothelial cells. In cardiac fibroblasts isolated from failing hearts, biglycan mRNA levels were markedly elevated compared with fibroblasts from sham-operated rats. In addition, in rats with ischemic heart failure treatment with the AT(1) receptor antagonist losartan (12.5 mg.kg(-1) b.i.d. per os, for 25 days) prevented the increase of myocardial biglycan as well as TGF-beta(1) mRNA. CONCLUSION: This report demonstrates global induction of myocardial biglycan mRNA in heart failure. Myocardial biglycan expression could be targeted by AT(1) receptor antagonism, an intervention well documented to halt cardiac remodelling in heart failure. Furthermore, the study provides evidence that angiotensin II is a regulator of biglycan expression in cardiac fibroblasts.