In Vitro Studies Of Pathways Involved In IL-1β-Mediated Cardiomyocyte Apoptosis In Mice

IntroductionIL-1consists of two distinct ligands (IL-1α and IL-1β) with indistinguishable biological activities that signal through the IL-1type I receptor (IL-1RI). A naturally occurring IL-1receptor antagonist (IL-1Ra) binds to IL-1RI without initiating signal transduction and prevents IL-1signaling, competitively inhibiting IL-1-mediated responses. IL-1, the prototypic pro-inflammatory cytokine, was originally described as the first "endogenous pyrogen" because it exerts fever-inducing effects in both rabbits and humans. IL-1consists of two distinct ligands (IL-1α and IL-1β) with high sequence homology, but only IL-1β can be released from cytosol to exocellular space after activation. So the IL-1β, other than IL-1α, is known to play a leading role on the cardiovascular system. With further research, more evidence showed that IL-1β played a crucial role in a variety of cardiovascular diseases. In the pathogenesis of cardiovascular diseases, IL-1β signaling plays a role in atherothrombotic diseases by promoting the formation of atheromatous lesions, enhancing the vascular inflammation, and triggering the plaque destabilization. Following the myocardial infarction, IL-1β signaling critically regulates the inflammatory response and is involved in the development of adverse remodeling by enhancing the expression of matrix metalloproteinases. Further, emerging evidence suggests that IL-1β signaling still plays an essential role in heart failure by suppressing the cardiac function, promoting the myocardial hypertrophy and even inducing the cardiomyocyte apoptosis.IL-1β has been proved to induce rat cardiomyocyte apoptosis which is associated with the activation of Bcl-2family members. Although the activation of pro-apoptotic pathways mediated by IL-1stimulation during the cardiomyocyte injury has been confirmed, the mechanism involved in this process remains poorly defined. Coincidentally, the marked upregulation of IL-1has been found in ischemic heart definitely. So understanding the mechanism of IL-1β-mediated cardiomyocyte apoptosis is important for designing the optimal therapeutic strategies against heart failure.ObjectivesIn this study, we used the cardiomyocytes isolated from neonatal mice and explored the molecular pathways, including caspase-dependent, caspase-independent pathways and inhibitor of apoptosis proteins, involved in the IL-1β-mediated cardiomyocyte apoptosis in vitro. The aim of this study was to explore the molecular pathways involved in the IL-1β-mediated cardiomyocyte apoptosis.MethodsThe neonatal (1-3days after birth) C57BL/6J mouse were performed in accordance with the institutional guidelines for the care and use of animals. We referred to the methods for primary culture of neonatal rat cardiomyocytes and modified it to fit for neonatal mouse. The neonatal mouse cardiomyocytes were isolated by the digestion combined by the collagenase and trypsin. During the first3days culture we added the fibroblast growth inhibitor (0.1mmol/L bromodeoxyuridine) into the medium to purify the cardiac myocytes from the isolated population (finally, cardiac troponin I positive staining cells>90%).The purified cardiomyocytes were incubated with the addition of10ng/ml recombinant murine IL-1β for72hours in vitro. Then the cell apoptotic ratios were measured by flow cytometry under Annexin V staining and the expressions of relative molecules were analyzed by western blotting or real-time quantitative polymerase chain reaction.Results1. IL-1β induced the apoptosis of mouse cardiomyocyte in vitro. Caspase3were activated under the IL-1β treatment.Compared with controls (without IL-1β incubation) IL-1P incubated groups showed significantly higher cell apoptotic ratio (36.71±8.4%vs.5.5±2.3%, p=0.0034). Thus, IL-1β induced the apoptosis of mouse cardiomyocyte in vitro. Western blotting analysis revealed that the cardiomyocytes showed significantly higher active (cleaved) caspase3expressions after IL-1β incubation (10ng/ml,72hours) compared with controls (without IL-1β incubation). As a central executioner caspase, caspase3were activated under the IL-1β treatment. The results indicated that IL-1β signaling triggered the caspase-dependent apoptosis pathways in cardiomyocytes.2. Endonuclease G (Endo G) and high temperature requirement protein A2(HtrA2/Omi) were involved in the caspase-independent pathways during IL-1β-mediated cardiomyocyte apoptosis.About caspase-independent pathways the real-time quantitative PCR analysis demonstrated that IL-1β incubated cardiomyocytes had increased Endo G and HtrA2/Omi mRNA expressions compared with controls (Endo G:p=0.0084, HtrA2/Omi:p=0.0483). However, we didn’t find the significant difference on second mitochondria-derived activator of caspase (Smac/Diablo) and apoptosis inducing factor (AIF) mRNA expressions between the groups with or without IL-1β incubation. In cardiomyocytes IL-1β signaling also triggered the caspase-independent apoptosis pathways which were mediated by Endo G and HtrA2/Omi.3. IL-1β signaling mediated the inhibition of survivin and X-linked inhibitor of apoptosis protein (XIAP) in cardiomyocyte apoptosis.Except for pro-apoptotic molecules we still want to know whether there had any anti-apoptotic molecules (e.g. inhibitor of apoptosis proteins, IAPs) involved in IL-1β-mediated cardiomyocyte apoptosis. The real-time quantitative PCR analysis indicated that IL-1β incubated cardiomyocytes had decreased survivin and XIAP mRNA expressions compared with controls (survivin:p=0.0001, XIAP:p=0.0069). Unexpectedly, c-IAP1and c-IAP2, two other IAPs family members, have been found both increased on mRNA level after IL-1β incubation (c-IAP1:p=0.0227, c-IAP2: p=0.0003). Therefore IL-1β signaling simultaneously mediated the inhibition of IAPs family members including survivin and XIAP in cardiomyocyte apoptosis.ConclusionIL-1β induced the activation of both caspase-dependent and caspase-independent pathways as well as inhibition of IAPs during the cardiomyocyte apoptosis in vitro. This discovery has not been reported in the literature. Understanding the molecular mechanisms involved in the IL-1β-mediated cardiomyocyte apoptosis may benefit for designing the therapeutic strategies for protecting cardiomyocyte function and preventing heart failure.