NLRP3 inflammasome activation during myocardial ischemia reperfusion is cardioprotective.

BACKGROUND: The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1ß mediates release of bioactive IL-1ß. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. METHODS AND RESULTS: The left coronary artery (LCA) of WT, NLRP3(-/-) and ASC(-/-) mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. CONCLUSION: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

The cardiokine secreted Frizzled-related protein 3, a modulator of Wnt signalling, in clinical and experimental heart failure.

OBJECTIVES: Experimental studies have shown involvement of Wnt signalling in heart failure (HF). We hypothesized that secreted frizzled-related protein 3 (sFRP3), a modulator of Wnt signalling, is related to the progression of HF. DESIGN: Circulating sFRP3 was measured in 153 HF patients and compared with 25 healthy controls. The association of sFRP3 with mortality was evaluated in 1202 patients (GISSI-HF trial). sFRP3 mRNA expression was assessed in failing human and murine left ventricles (LV), and cellular localization was determined after fractioning of myocardial tissue. In vitro studies were carried out in cardiac fibroblasts subjected to cyclic mechanical stretch. RESULTS: (i) Heart failure patients had significantly raised serum sFRP3 levels compared with controls, (ii) during a median follow-up of 47 months, 315 patients died in the GISSI-HF substudy. In univariable Cox regression, tertiles of baseline sFRP3 concentration were significantly associated with all-cause and cardiovascular mortality. After adjustment for demographic and clinical variables, but not for CRP and NT-proBNP, the associations with mortality remained significant for the third tertile (all-cause, HR 1.45, P = 0.011; cardiovascular, HR 1.66, P = 0.003), (iii) sFRP3 mRNA expression was increased in failing human LV, with a decline following LV assist device therapy. LV from post-MI mice showed an increased sFRP3 mRNA level, particularly in cardiac fibroblasts, and (iv) mechanical stretch enhanced sFRP3 expression and release in myocardial fibroblasts. CONCLUSION: There is an association between increased sFRP3 expression and adverse outcome in HF, suggesting that the failing myocardium itself contributes to an increase in circulating sFRP3.

Induction of a myocardial adrenomedullin signaling system during ischemic heart failure in rats.

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<0.05) compared with the sham group. Parallel elevations of myocardial ADM receptor and RAMP-2 mRNA levels were also observed (2.3- and 1.5-fold increase, respectively; P<0.05). In addition, high levels of ADM mRNA were seen in the ischemic region. Immunohistochemical analysis revealed a substantial increase of ADM-ir in microvascular endothelium and perivascular interstitial cells of myocardial tissue contiguous to the ischemic region. In addition, radioligand binding studies demonstrated a 1.6-fold increase of specific ADM binding sites in the failing left ventricle (P<0.05). Intervention with the mixed ET(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.

Myocardial distribution and regulation of GRK and beta-arrestin isoforms in congestive heart failure in rats.

Myocardial G protein-coupled receptor kinase 2 (GRK2) has been shown to be involved in the pathophysiology of congestive heart failure (CHF). However, the cellular distribution of this isoform, as well as the other isoforms of the GRK-arrestin system, has not been studied in myocardial tissue. Thus myocardial expression and cellular distribution of the different GRK and arrestin isoforms were investigated in a rat model of CHF. Rats subjected to ligation of the left coronary artery or sham operation were euthanized 2, 7, or 42 days after the surgical procedure. Myocardial GRK2, GRK5, beta-arrestin-1, and beta-arrestin-2 mRNA levels, but not that of GRK3, were induced in the failing hearts. Consistently, Western blot analysis of tissue extracts from the nonischemic region of the left ventricle revealed 3.0-, 2.6-, and 1.5-fold elevations of GRK2, GRK5, and beta-arrestin-1, respectively, 7 days after induction of myocardial infarction compared with the sham-operated rats (P < 0.05). Immunohistochemical analysis of myocardial tissue sections and Western blot analysis of isolated cells revealed localization of GRK2 and beta-arrestin-1 predominantly in endothelial cells. Conversely, GRK3 was confined to cardiac myocytes. GRK5 immunostaining appeared to be homogeneously distributed in the cellular elements of the myocardium. In conclusion, myocardial mRNA and protein levels of GRK2, GRK5, and beta-arrestin-1 are induced in postinfarction failure in rats. The immunohistochemical analysis suggests that GRK2 and beta-arrestin-1 may act as primary regulators of endothelial function. Conversely, the cellular distribution of GRK3 and GRK5 implicates these isoforms as putative regulators of cardiac myocyte function.

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.