Theis Tønnessen

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure

OBJECTIVES Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.

Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity

OBJECTIVE Heart failure is associated with alterations in contractile parameters and accompanied by abnormalities in intracellular calcium homeostasis. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and phospholamban (PLB) are important in intracellular calcium cycling. The aim of the present study was to examine mechanisms causing reductions in SERCA2 activity in the failing heart. METHODS Myocardial infarction (MI) was induced in male Wistar rats, and animals with congestive heart failure were examined 6 weeks after the primary operation. RESULTS Serine(16) monomeric and pentameric phosphorylated PLB were significantly downregulated (50 and 55%, respectively), whereas threonine(17) phosphorylated PLB was unchanged in failing compared to sham hearts. Protein phosphatases 1 and 2A were significantly upregulated (26 and 42%, respectively) and phosphatase 2C significantly downregulated (29%), whereas the level of protein kinase A regulatory subunit II remained unchanged during heart failure. Increasing PLB phosphorylation by forskolin in isolated cardiomyocytes after inhibition of the Na(+)-Ca(2+) exchanger activity had significantly greater effect on SERCA2 activity in failing than in sham cells (49 and 20% faster transient decline, respectively). Decreasing PLB phosphorylation by the protein kinase A inhibitor H89 had significantly less effect on SERCA2 activity in failing compared to sham cardiomyocytes (20 and 75% slower transient decline, respectively). CONCLUSION The observed changes in SERCA2 activity after increasing and decreasing serine(16) PLB phosphorylation in cardiomyocytes from sham and failing hearts, suggest that the observed reduction in serine(16) PLB phosphorylation is one major factor determining the reduced SERCA2 activity in heart failure after MI.

Increased In Vivo Expression and Production of Endothelin-1 by Porcine Cardiomyocytes Subjected to Ischemia

Circulating levels of the endothelium-derived vasoconstrictor peptide endothelin-1 (ET-1) are increased in association with myocardial ischemia and infarction. The present study investigates whether ET-1 is synthesized and produced locally in the ischemic heart. Sixteen pigs were divided into three groups. In the first group, the left anterior descending coronary artery was occluded for 90 minutes, followed by 150 minutes of reperfusion (group A, n = 8). Two additional groups were subjected to 90 minutes (group B, n = 4) or 240 minutes (group C, n = 4) of ischemia without reperfusion. Biopsies from the nonischemic and ischemic myocardium were rapidly obtained from the beating heart and subsequently examined by Northern blot, in situ hybridization, and immunohistochemistry. Arterial plasma ET-1 was measured before ischemia and at the end of the experiments. Northern blot revealed a twofold increase in ET-1 mRNA in the ischemic myocardium compared with the nonischemic myocardium. In situ hybridization showed a considerable increase in ET-1 mRNA over the ischemic cardiomyocytes. Substantial ET-1-like immunoreactivity (ET-1-ir) was detected in cardiomyocytes in the ischemic region. In contrast, little or no ET-1-ir or mRNA was detected in nonischemic cardiomyocytes. Both in the ischemic and nonischemic regions, little ET-1-ir was detected in vascular endothelial cells or vascular smooth muscle cells. There was no difference in the intensity and distribution of ET-1 mRNA expression or ET-1-ir among experimental groups A, B, and C. Arterial plasma ET-1 was increased only in group A, the reperfused group.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased cardiac IL-18 mRNA, pro-IL-18 and plasma IL-18 after myocardial infarction in the mouse; a potential role in cardiac dysfunction

OBJECTIVE Interleukin (IL)-18 has been reported to be an important predictor for mortality in ischemic heart disease. IL-18 has proinflammatory properties, induces cell death and stimulates nitric oxide production. We hypothesized that following myocardial infarction (MI) an increased myocardial IL-18 production occurs, which may be involved in the pathogenesis of post-ischemic heart failure. METHODS AND RESULTS Seven days after induction of MI in the mouse, myocardial hypertrophy and pulmonary edema were observed. RNase protection assay of tissue from the non-infarcted left ventricular myocardium revealed an increase in IL-18 (2.0-fold; P<0.001) and IL-1 beta (1.6-fold; P<0.001) mRNA after MI. Enhanced abundance of pro-IL-18 (1.4-fold; P<0.05), IL-18 receptor (3.5-fold; P<0.05) and IL-18 binding proteins (1.6-fold; P<0.05) was also demonstrated, whereas cardiac IL-18 protein decreased by 25% (P<0.05) following MI. However, the concentration of circulating IL-18 was significantly elevated (MI; 90.4+/-11.7 pg/ml, sham; 47.2+/-4.2 pg/ml; P<0.001). After MI, enhanced cardiac activity of the pro-IL-18 processing enzyme, caspase-1, was measured. Additionally, a 3.4-fold increase (P<0.001) in the activity of the IL-18 degrading enzyme, caspase-3, was found in cardiac tissue, which may explain the observed reduction of cardiac IL-18 protein abundance. Finally, IL-18 reduced shortening of electrically stimulated adult cardiomyocytes and left ventricular contractility in vivo. CONCLUSIONS After MI in the mouse, increased production of cardiac IL-18 mRNA and pro-IL-18, as well as circulating IL-18 occurs. Since IL-18 also reduced myocardial contractility, we suggest that IL-18 may be involved in the pathogenesis of contractile dysfunction following MI.

Cardiac O-GlcNAc signaling is increased in hypertrophy and heart failure.

Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

Increased cardiac expression of endothelin-1 mRNA in ischemic heart failure in rats

OBJECTIVES Plasma endothelin (ET) concentrations are increased in heart failure. The aims of the present study were to investigate to what extent cardiac ET mRNA expression is induced in ischemic heart failure and whether there may be compensatory downregulation of myocardial mRNA levels for the ETA and ETB receptor subtypes. METHODS In rats with ischemic heart failure (left ventricular end-diastolic pressure > 15 mmHg) due to left coronary artery ligation. Northern blot analyses were performed on mRNA isolated from cardiac tissues. RESULTS A substantial upregulation was revealed in all chambers of the failing hearts. Up to 27-fold upregulation (mean 10.6 +/- 4.0, P = 0.002) of left ventricular ET-1 mRNA levels was measured 1 week after myocardial infarction, whereas only a modest upregulation was detected after 6 weeks (mean 2.7 +/- 0.5, P < 0.05). Ribonuclease protection assay revealed 2.8 +/- 0.4-fold higher levels of ET-1 mRNA in the left ventricular area subjected to myocardial infarction compared to the non-infarcted tissue after 1 week. Left ventricular ET-1 mRNA correlated significantly with left ventricular end-diastolic pressure after 1 week (r2 = 0.86, P = 0.007). The ETA and ETB receptor mRNA levels tended to increase 1 week after myocardial infarction although these changes were not statistically significant. CONCLUSIONS Cardiac ET-1 mRNA levels are increased in ischemic heart failure and correlate significantly with left ventricular end-diastolic pressure 1 week after myocardial infarction. The increase in cardiac ET-1 mRNA is not accompanied by a decrease in ET receptor mRNA.

Syndecan-4 Is Essential for Development of Concentric Myocardial Hypertrophy via Stretch-Induced Activation of the Calcineurin-NFAT Pathway

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4−/− mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4−/−-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.

The effect of aortic valve replacement on plasma B-type natriuretic peptide in patients with severe aortic stenosis--one year follow-up.

B‐type natriuretic peptide (BNP) is synthesized in cardiac tissue in response to increased wall stress and myocardial hypertrophy.

Transient, isopeptide-specific induction of myocardial endothelin-1 mRNA in congestive heart failure in rats

Increased myocardial expression of preproendothelin-1 (ppET-1) mRNA has been associated with congestive heart failure (CHF) in rats. However, the time course and isoform pattern of ppET mRNA induction and the cellular localization of ET in failing hearts are unknown. Thus our aim was to investigate myocardial ppET mRNA expression in CHF rats during the first 6 wk after induction of myocardial infarction. Furthermore, performing immunohistochemical analysis, we also investigated the origin and localization of immunoreactive endothelin (ET) in different regions of the failing heart. Ribonuclease protection assays revealed a marked increase in ppET-1 mRNA levels in rat myocardial tissues during CHF. The induction of ppET-1 mRNA was isopeptide specific and transient. The most substantial upregulation was observed in the infarcted area, where maximal expression of ppET-1 mRNA was observed after 7 days (25-fold increase, P < 0.05). However, a marked and statistically significant induction of ppET-1 mRNA was also observed in the nonischemic myocardium. Immunohistochemical analysis revealed ET-1-like immunoreactivity in cardiomyocytes, vascular endothelial cells, macrophages, and proliferating fibroblasts. Thus immunohistochemistry revealed the structural basis for the dramatic upregulation of the myocardial ET system in the infarcted region, suggesting a role for ET in the healing process after myocardial infarction. However, the global upregulation of ppET-1 mRNA in the heart also suggests an autocrine/paracrine regulatory mechanism in the nonischemic myocardium during CHF.Increased myocardial expression of preproendothelin-1 (ppET-1) mRNA has been associated with congestive heart failure (CHF) in rats. However, the time course and isoform pattern of ppET mRNA induction and the cellular localization of ET in failing hearts are unknown. Thus our aim was to investigate myocardial ppET mRNA expression in CHF rats during the first 6 wk after induction of myocardial infarction. Furthermore, performing immunohistochemical analysis, we also investigated the origin and localization of immunoreactive endothelin (ET) in different regions of the failing heart. Ribonuclease protection assays revealed a marked increase in ppET-1 mRNA levels in rat myocardial tissues during CHF. The induction of ppET-1 mRNA was isopeptide specific and transient. The most substantial upregulation was observed in the infarcted area, where maximal expression of ppET-1 mRNA was observed after 7 days (25-fold increase, P < 0.05). However, a marked and statistically significant induction of ppET-1 mRNA was also observed in the nonischemic myocardium. Immunohistochemical analysis revealed ET-1-like immunoreactivity in cardiomyocytes, vascular endothelial cells, macrophages, and proliferating fibroblasts. Thus immunohistochemistry revealed the structural basis for the dramatic upregulation of the myocardial ET system in the infarcted region, suggesting a role for ET in the healing process after myocardial infarction. However, the global upregulation of ppET-1 mRNA in the heart also suggests an autocrine/paracrine regulatory mechanism in the nonischemic myocardium during CHF.

Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5.

Pressure overload-induced TGF-β signaling activates cardiac fibroblasts (CFB) and leads to increased extracellular matrix (ECM) protein synthesis including fibrosis. Excessive ECM accumulation may in turn affect cardiac function contributing to development of heart failure. The aim of this study was to examine the effects of SM16, an orally active small molecular inhibitor of ALK5, on pressure overload-induced cardiac fibrosis. One week after aortic banding (AB), C57Bl/6J mice were randomized to standard chow or chow with SM16. Sham operated animals served as controls. Following 4 weeks AB, mice were characterized by echocardiography and cardiovascular magnetic resonance before sacrifice. SM16 abolished phosphorylation of SMAD2 induced by AB in vivo and by TGF-β in CFB in vitro. Interestingly, Masson Trichrome and Picrosirius Red stained myocardial left ventricular tissue revealed reduced development of fibrosis and collagen cross-linking following AB in the SM16 treated group, which was confirmed by reduced hydroxyproline incorporation. Furthermore, treatment with SM16 attenuated mRNA expression following induction of AB in vivo and stimulation with TGF-β in CFB in vitro of Col1a2, the cross-linking enzyme LOX, and the pro-fibrotic glycoproteins SPARC and osteopontin. Reduced ECM synthesis by CFB and a reduction in myocardial stiffness due to attenuated development of fibrosis and collagen cross-linking might have contributed to the improved diastolic function and cardiac output seen in vivo, in combination with reduced lung weight and ANP expression by treatment with SM16. Despite these beneficial effects on cardiac function and development of heart failure, mice treated with SM16 exhibited increased mortality, increased LV dilatation and inflammatory heart valve lesions that may limit the use of SM16 and possibly also other small molecular inhibitors of ALK5, as future therapeutic drugs.