Abstract
Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are critical regulators of programmed necrosis or necroptosis. However, the role of the RIPK1/RIPK3 signaling pathway in myocardial fibrosis and related diabetic cardiomyopathy is still unclear. We hypothesized that RIPK1/RIPK3 activation mediated myocardial fibrosis by impairing the autophagic flux. To this end, we established in vitro and in vivo models of type 2 diabetes mellitus with high glucose fat (HGF) medium and diet respectively. HGF induced myocardial fibrosis, and impaired cardiac diastolic and systolic function by activating the RIPK1/RIPK3 pathway, which increased the expression of autophagic related proteins such as LC3-II, P62 and active-cathepsin D. Inhibition of RIPK1 or RIPK3 alleviated HGF-induced death and fibrosis of cardiac fibroblasts by restoring the impaired autophagic flux. The autophagy blocker neutralized the effects of the RIPK1 inhibitor necrostatin-1 (Nec-1) and RIPK3 inhibitor GSK872 (GSK). RIPK1/RIPK3 inhibition respectively decreased the levels of RIPK3/p-RIPK3 and RIPK1/p-RIPK1. P62 forms a complex with RIPK1-RIPK3 and promotes the binding of RIPK1 and RIPK3, silencing of RIPK1 decreased the association of RIPK1 with P62 and the binding of P62 to LC3. Furthermore, inhibition of both kinases in combination with a low dose of Nec-1 and GSK in the HGF-treated fibroblasts significantly decreased cell death and fibrosis, and restored the autophagic flux. In the diabetic rat model, Nec-1 (1.65 mg/kg) treatment for 4 months markedly alleviated myocardial fibrosis, downregulated autophagic related proteins, and improved cardiac systolic and diastolic function. In conclusion, HGF induces myocardial fibrosis and cardiac dysfunction by activating the RIPK1-RIPK3 pathway and by impairing the autophagic flux, which is obviated by the pharmacological and genetic inhibition of RIPK1/RIPK3.
Highlights
Necroptosis is a newly discovered caspase-independent mode of programed cell death that mimics the characteristics of both apoptosis and necrosis [1]
The necroptotic pathway is triggered by the receptor-interacting protein kinase 1 (RIPK1), which binds to RIPK3 via interacting with their RIP homotypic interaction motif (RHIM) domains, recruits, and subsequently phosphorylates mixed lineage kinase domain-like protein (MLKL) in the necrosome [2, 3]
The aim of this study was to determine whether RIPK1/RIPK3 signaling is the mechanistic basis of HGFimpaired autophagic flux in diabetic myocardial fibrosis
Summary
Necroptosis is a newly discovered caspase-independent mode of programed cell death that mimics the characteristics of both apoptosis and necrosis [1]. Chronic hyperglycemia during type 2 diabetic mellitus (T2DM) induces cardiomyocyte death and myocardial fibrosis (MF) by accelerating proliferation of cardiac fibroblasts (CFs) and the secretion of extracellular matrix proteins. These pathological changes eventually lead to ventricular remodeling and dysfunction [4, 5]. High glucose and fat (HGF) conditions can impair the ubiquitin–protease complex system, leading to the accumulation of damaged proteins and senescent organelles. This in turn blocks the autophagic flux and accelerates cell aging and death [7]. Our findings indicate that the activation of RIPK1/RIPK3 pathway impairs the autophagic flux in CFs exposed to hyperglycemic conditions in vitro and in vivo, and inhibition of the kinases synergistically restored the autophagic flux and alleviated the HGF-induced pathological changes
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