Cardiovascular pharmacologyMoxonidine modulates cytokine signalling and effects on cardiac cell viability
Introduction
Left ventricular hypertrophy (LVH) and remodelling in hypertension and heart failure have been associated with increased circulating and cardiac expression of pro-inflammatory cytokines, tumor necrosis alpha (TNF-α) and interleukin (IL)-1β (Duerrschmid et al., 2013, Gullestad et al., 2012). These cytokines lead to cardiomyocyte hypertrophy and apoptosis in vivo and in vitro (Haudek et al., 2007, Hiraoka et al., 2001, Hu et al., 2009), and stimulation of cardiac fibroblast proliferation and differentiation into activated myofibroblasts, which produce large amounts of collagens. The ensuing structural changes, LVH and fibrosis, impede cardiac contractility and compromise cardiac performance. The intracellular signalling pathways include generation of reactive oxygen species (Khurana et al., 2014, Kohler et al., 2014), activation of phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) (Chu et al., 2012, Tullio et al., 2013) and mitogen activated protein kinases (MAPKs): extracellular signal-regulated protein kinase (ERK), p38, and c-Jun NH2-terminal protein kinase (JNK) (Chae et al., 2006, Haudek et al., 2007, Hiraoka et al., 2001, Sugden and Clerk, 1998, Yndestad et al., 2010).
Moxonidine is a centrally-acting sympatholytic imidazoline compound that shows higher affinity to non-adrenergic imidazoline I1-receptors than α2-adrenergic receptors. Moxonidine is used in hypertension treatment, including hypertension complicated with LVH (reviewed in (Mukaddam-Daher, 2012)). Our studies have shown that treatment of spontaneously hypertensive rats (SHR) with moxonidine improves cardiac performance, attenuates LVH and collagen accumulation, in association with a transient apoptotic effect in SHR ventricles. The effects also include a substantial reduction in left ventricular IL-1β and circulating TNF-α and IL-6 levels, as well as attenuated ventricular Akt and p38 phosphorylation and inducible nitric oxide synthase (iNOS) expression (Aceros et al., 2011, Mukaddam-Daher et al., 2009, Paquette et al., 2008). On the other hand, moxonidine improves cardiac performance in cardiomyopathic hamsters in association with differential inflammatory/anti-inflammatory responses that culminate in reduced cardiac apoptosis (Stabile et al., 2011). The cardioprotective effects of in vivo moxonidine may be secondary to inhibition of sympathetic nerve activity, reduced noradrenaline release and cytokine levels, and subsequently, their downstream actions. Moxonidine may also directly act on cardiac cells to modulate the actions of cytokines. This hypothesis is supported by data showing that cardiac cells express cytokine receptors and imidazoline I1-receptors (also known as nischarin), and in vitro moxonidine attenuates noradrenaline-induced cardiomyocyte apoptosis and fibroblast proliferation (Aceros et al., 2011). Accordingly, we sought to examine in primary cultures of neonatal rat cardiomyocytes and fibroblasts: i) the direct effect of moxonidine on cardiac cell viability and mechanisms involved, including the expression of imidazoline I1-receptor/nischarin; and ii) to identify at what level moxonidine may regulate cytokine-induced cellular effects, focussing on their common signalling pathways, MAPKs, iNOS, and Akt (Aceros et al., 2011, Akira and Takeda, 2004, Edwards et al., 2001, Peng et al., 2009, Tesfai et al., 2011). The results demonstrate for the first time, the direct effect of moxonidine on cardiac cell viability. The mechanisms involve I1-receptor activation and subsequent ERK and Akt activation in cardiomyocytes, and ERK, p38 and JNK, and Akt in fibroblasts. Furthermore, moxonidine modulates cytokine-induced effects on cardiomyocyte and fibroblast viability, through differential effects on cytokine-induced p38, JNK and iNOS.
Section snippets
Cell cultures
Ventricular cardiomyocytes and fibroblasts were isolated, enzymatically digested, and purified from 1 to 2 day old Sprague Dawley rat pups, using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA) following manufacturer instructions, as we previously described (Aceros et al., 2011). Recovered cells were pre-plated on plastic flasks twice, for 30 min each, to reduce non-myocyte cell numbers. The adherent cells are mostly fibroblasts while cardiomyocytes
Expression of nischarin in neonatal cardiomyocytes and fibroblasts
Nischarin mRNA expression was detected in mouse brain and heart at 706 bp, confirming previous results (Alahari et al., 2000). Nischarin mRNA was also detected in neonatal rat cardiomyocytes and second passage fibroblasts. Nischarin mRNA was reduced after 24 h incubation with moxonidine in cardiomyocytes, but not altered in fibroblasts (Fig. 1). Imidazoline I1-receptor/nischarin expression and regulation were further explored by Western blot. Fig. 2A shows that nischarin protein expression is
Discussion
Our results provide evidence that moxonidine protects cultured neonatal cardiomyocytes against serum starvation-induced apoptosis as well as IL-1β- and TNF-α-reduced viability, mainly by maintaining membrane integrity, and in part, by improving mitochondrial activity. The protection primarily involves PI3K/Akt, ERK1/2, p38, and iNOS, in IL-1β-incubated cells, but activation of ERK1/2 in TNF-α-incubated cells. In contrast, moxonidine stimulates fibroblast mortality by mechanisms that include JNK
Conclusion
Studies on isolated neonatal cardiac cells in culture cannot be extrapolated to whole animals, especially in those with cardiac disease, where myriad of diverse mediators are activated. However, the finding that moxonidine/imidazoline I1-receptor protects cardiomyocytes and favors elimination of myofibroblasts in inflammatory conditions, is consistent with regression of LVH and fibrosis and improved cardiac function in SHR (Aceros et al., 2011) and heart failure hamsters (Stabile et al., 2011).
Funding
This work was supported by grants from the Canadian Institutes of Health Research (MOP-82708) and the Heart and Stroke Foundation of Canada (to SMD).
Henry Aceros received a doctoral scholarship award from Quebec Health Research Fund (FRSQ), Quebec, Canada.
Funding agencies had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.
Acknowledgments
Moxonidine was kindly provided by Solvay Pharmaceuticals, GMBH.
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