A role of heparin-binding epidermal growth factor-like growth factor in cardiac remodeling after myocardial infarction

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is known to induce cell growth in various cell types via transactivation of epidermal growth factor receptor (EGFR). To investigate the involvement of HB-EGF and EGFR in cardiac remodeling after myocardial infarction (MI), we examined the expressions of mRNA and protein in rat hearts 6 weeks after MI-induction. Where increased expressions of HB-EGF mRNA and protein were observed, infarcted myocardium was replaced by extracellular matrix and interstitial fibroblasts. EGFR mRNA and protein expression did not show significant changes in sham-operated heart tissues, non-infarcted region, and infarcted region. In vitro study demonstrated that HB-EGF mRNA was expressed mainly in cultured fibroblasts rather than in myocytes. We suggest that the interaction between HB-EGF and EGFR transactivation is closely related to the proliferation of cardiac fibroblasts and cardiac remodeling after MI in an autocrine, paracrine, and juxtacrine manner.     

Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats

The pro-inflammatory cytokines interleukin (IL)-1 and IL-6 have been shown to be upregulated in the myocardium after injury and after adrenergic receptor stimulation. Together with other cytokines, such as the transforming growth factor (TGF)-, the pro-inflammatory cytokines have been implicated in the initiation of tissue repair and wound healing after myocardial infarction (MI). In the present study, the effect of -adrenergic receptor blockade with propranolol (2 mg/kg·h s.c. by miniosmotic pumps) on cardiac cytokine expression and on wound healing was analyzed in rats from 6–72 h after MI. IL-1 and IL-6 gene expression strongly increased in the infarcted myocardium 6 h after MI and peaked after 12 h, while TGF-, progressively increased from 12 h onwards. Also, TGF-₂ increased after 12 h, peaked after 24 h and declined thereafter, while TGF-, was only elevated after 72 h. Treatment with propranolol had a negative chronotropic effect throughout the observation period of 72 h. It attenuated the initial elevation in LVEDP and increased cardiac output ultimately. Furthermore, propranolol attenuated IL-1 mRNA expression, but had not effect on the other cytokines. Moreover, MMP-9 gelatinolytic activity was markedly attenuated by propranolol indicating a delayed resorption of the necrotic tissue and, possibly, collagen turnover. Replacement by scar tissue, however, was not affected as indicated by normal collagen expression.     

Pro-Inflammatory Action of MIF in Acute Myocardial Infarction via Activation of Peripheral Blood Mononuclear Cells

Objectives

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, has been implicated in the pathogenesis of multiple inflammatory disorders. We determined changes in circulating MIF levels, explored the cellular source of MIF, and studied the role of MIF in mediating inflammatory responses following acute myocardial infarction (MI).

Methods and Results

We recruited 15 patients with MI, 10 patients with stable angina and 10 healthy volunteers and measured temporal changes of MIF in plasma. Expression of MIF, matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) in cultured peripheral blood mononuclear cells (PBMCs) and the media were measured by ELISA or real-time PCR. Compared to controls, plasma levels of MIF and IL-6 were significantly elevated at admission and 72 h post-MI. In contrast, expression of MIF, MMP-9 and IL-6 by PBMCs from MI patients was unchanged at admission, but significantly increased at 72 h. Addition of MIF activated cultured PBMCs by upregulating expression of inflammatory molecules and also synergistically enhanced stimulatory action of IL-1β which were inhibited by anti-MIF interventions. In a mouse MI model we observed similar changes in circulating MIF as seen in patients, with reciprocal significant increases in plasma MIF and reduction of MIF content in the infarct myocardium at 3 h after MI. MIF content in the infarct myocardium was restored at 72 h post-MI and was associated with robust macrophage infiltration. Further, anti-MIF intervention significantly reduced inflammatory cell infiltration and expression of monocyte chemoattractant protein-1 at 24 h and incidence of cardiac rupture in mice post-MI.

Conclusion

MI leads to a rapid release of MIF from the myocardium into circulation. Subsequently MIF facilitates PBMC production of pro-inflammatory mediators and myocardial inflammatory infiltration. Attenuation of these events, and post-MI cardiac rupture, by anti-MIF interventions suggests that MIF could be a potential therapeutic target following MI.     

Up‐regulated TLR4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll‐like receptors (TLRs) that respond to pathogen‐ and damage‐associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4‐mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (B_max) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4‐blocking antibodies. We conclude that the expression, ligand‐binding capacity and pro‐inflammatory function of cardiomyocyte TLR4 are up‐regulated after long‐term MI, which promote inflammation and exacerbate heart failure.     

A chronic mouse model of myocardial ischemia-reperfusion: essential in cytokine studies

Reperfusion of the ischemic myocardium is associated with a cytokine cascade that reflects a cellular response to injury. We studied this cascade in the mouse and found that acute surgical trauma in sham-operated animals obscured early changes in cytokine induction that occur during myocardial ischemia-reperfusion (MI/R). Therefore, we utilized a new implantable device that allows occlusion and reperfusion of the left anterior descending coronary artery in a closed-chest mouse at any time after instrumentation. Induction of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha mRNA in the whole heart was examined by RNase protection assay and quantitated by Phosphor- Imager. At 3 h after instrumentation, levels of IL-6 mRNA in sham-operated animals increased above those of control naive hearts, whereas this increase did not occur until after 1 day for TNF-alpha mRNA. The surgical trauma led to exaggeration of I/R cytokine induction with greater variance in response. At 3 days and 1 wk after instrumentation, levels of both IL-6 and TNF-alpha mRNA in sham-operated animals were comparable to those of naive hearts and induction responses in I/R were much less variant. We also found that 1 h of ischemia and 2 h of reperfusion at all time points of recovery (i.e., 3 h and 1, 3, and 7 days after instrumentation) led to a significant increase in IL-6 and TNF-alpha mRNA levels. In addition, 3 h of permanent occlusion, which did not induce any mRNA increase after 1 wk postinstrumentation, caused marked upregulation of IL-6 mRNA in an acutely prepared animal. This study of early cytokine responses evoked by MI/R highlights the need for dissipation of acute surgical trauma by using a chronic, closed-chest mouse preparation.     

Down-regulation of mitofusin-2 expression in cardiac hypertrophy in vitro and in vivo

Mitofusin-2 (Mfn2) suppresses smooth muscle cell proliferation through inhibition of the Ras-extracellular signal-regulated kinases (ERK1/2) pathway. Since the ERK1/2 pathway is implicated in mediating hypertrophic signaling, we studied the changes in Mfn2 in cardiac hypertrophy using in vitro and in vivo models. Phenylephrine was used to induce hypertrophy in neonatal rat ventricular myocytes (NRVMs). In vivo hypertrophy models included spontaneously hypertensive rats (SHR), pressure-overload hypertrophy by transverse aortic constriction (TAC), hypertrophy of non-infarcted myocardium following myocardial infarction (MI), and cardiomyopathy due to cardiac-restricted overexpression of beta(2)-adrenergic receptors (beta(2)-TG). We determined hypertrophic parameters and analysed expression of atrial natriuretic peptide (ANP) and Mfn2 by real-time PCR. Phosphorylated-ERK1/2 (phospho-ERK) was measured by Western blot. Mfn2 was downregulated in phenylephrine treated NRCMs (by approximately 40%), hypertrophied hearts from SHR (by approximately 80%), mice with TAC (at 1 and 3 weeks, by approximately 50%), and beta(2)-TG mice (by approximately 20%). However, Mfn2 was not downregulated in hypertrophied hearts with 15 weeks of TAC, nor in hypertrophied non-infarcted myocardium following MI. phospho-ERK1/2 was increased in hypertrophied myocardium at 1 week post-TAC, but not in non-infarcted myocardium after MI, indicating that downregulated Mfn2 may be accompanied by an increase of phospho-ERK1/2. This study shows, for the first time, downregulated Mfn2 expression in hypertrophied hearts, which depends on the etiology and time course of hypertrophy. Further study is required to examine the causal relationship between Mfn2 and cardiac hypertrophy.     

A new regulation of IL-6 production in adult cardiomyocytes by β-adrenergic and IL-1β receptors and induction of cellular hypertrophy by IL-6 trans-signalling

The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic β-adrenergic receptor (β-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between β-AR stimulation and pro-inflammatory cytokines, we studied the effects of the β-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G_s protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3^Tyr705 in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that β-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.     

Neural regulation of the proinflammatory cytokine response to acute myocardial infarction

Within minutes of acute myocardial infarction (MI), proinflammatory cytokines increase in the brain, heart, and plasma. We hypothesized that cardiac afferent nerves stimulated by myocardial injury signal the brain to increase central cytokines. Urethane-anesthetized male Sprague-Dawley rats underwent ligation of the left anterior descending coronary artery (LAD) or sham LAD ligation after bilateral cervical vagotomy, sham vagotomy, or application of a 10% phenol solution to the epicardial surface of the myocardium at risk. MI caused a significant increase in tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the plasma and heart, which was blunted by vagotomy. MI also caused a significant increase in hypothalamic TNF-alpha and IL-1beta, which was not affected by vagotomy. In contrast, epicardial phenol blocked MI-induced increases in hypothalamic TNF-alpha and IL-1beta without affecting increases in the plasma and heart. These findings demonstrate that the appearance of proinflammatory cytokines in the brain after MI is independent of blood-borne cytokines and suggest that cardiac sympathetic afferent nerves activated by myocardial ischemia signal the brain to increase cytokine production. In addition, an intact vagus nerve is required for the full expression of proinflammatory cytokines in the injured myocardium and in the circulation. We conclude that the sympathetic and parasympathetic innervation of the heart both contribute to the acute proinflammatory response to MI.     

Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure

Chronic heart failure (CHF) is a state of immune activation, and pro-inflammatory cytokines play an important role in its development and progression. Macrophages (Mphis), when activated, are the main source of pro-inflammatory cytokines. We measured interleukin-6 (IL-6), interleukin (IL-1beta) and tumor necrosis factor - alpha (TNF-alpha) production after lipopolysaccharide (LPS)-stimulation, as well as peritoneal Mphis migration, phagocytic capacity, chemotaxis index, and hydrogen peroxide production, in an attempt to clarify the role of this cell in an animal model of CHF. Ligature of the left coronary artery or sham operation was performed in adult Wistar rats. After 12 weeks, resident and total cell number, phagocytic capacity, chemotaxis index, and hydrogen peroxide production in Mphis were significantly higher in CHF than in control rats. The production of IL-6 and TNF- alpha was similarly significantly enhanced in CHF as compared with controls. Mphis obtained from CHF rats were more responsive to LPS, suggesting the existence, in vivo, of possible factor(s) modulating the production of pro-inflammatory cytokines. The results demonstrated that there is modification of peritoneal Mphis function along CHF development, possibly contributing to the pathophysiological process in the establishment of CHF.     

Expression of macrophage migration inhibitory factor in acute ischemic myocardial injury.

Macrophage migration inhibitory factor (MIF) is a key mediator in inflammatory or immune-mediated diseases, although its role in heart diseases is unknown. This study investigated the expression of MIF in the myocardium in the development of acute myocardial infarction (AMI). By use of immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization, the gene and protein expression of MIF in the heart at 6 hr, 1 day, 3 days, 1 week, and 2 weeks after AMI was studied. In both normal and sham-operated rats, MIF mRNA and protein were expressed constitutively at low levels by the myocytes. By contrast, MIF mRNA was rapidly upregulated by the surviving myocytes in the infarcted region and, to a lesser extent, the non-infarcted region, accounting for a sevenfold increase at 6 hr after AMI (p<0.001). This was followed by a fourfold increase in MIF protein expression at day 1 after AMI (p<0.05). Macrophages were found accumulated in the infarcted region, being significant at day 1 (p<0.01) and progressive increased over the 2-week time course (p<0.01) in which MIF was found expressed in these cells. The results indicated that the infiltrating macrophages and myocytes were sources of MIF in the infarcted region. The latter cells became activated and involved in the amplification of inflammatory response in AMI. Therefore, upregulation of myocardial MIF may contribute to macrophage accumulation in the infarcted region and their pro-inflammatory role may participate in the myocyte damage seen in AMI.