Diabetic cardiomyopathy: Importance of direct evidence to support the roles of NOD-like receptor protein 3 inflammasome and pyroptosis

Abstract

Recently, the roles of pyroptosis, a form of cell death induced by activated NOD-like receptor protein 3 (NLRP3) inflammasome, in the pathogenesis of diabetic cardiomyopathy (DCM) have been extensively investigated. However, most studies have focused mainly on whether diabetes increases the NLRP3 inflammasome and associated pyroptosis in the heart of type 1 or type 2 diabetic rodent models, and whether various medications and natural products prevent the development of DCM, associated with decreased levels of cardiac NLRP3 inflammasome and pyroptosis. The direct link of NLRP3 inflammasome and associated pyroptosis to the pathogenesis of DCM remains unclear based on the limited evidence derived from the available studies, with the approaches of NLRP3 gene silencing or pharmaceutical application of NLRP3 specific inhibitors. We thus emphasize the requirement for more systematic studies that are designed to provide direct evidence to support the link, given that several studies have provided both direct and indirect evidence under specific conditions. This editorial emphasizes that the current investigation should be circumspect in its conclusion, i.e., not overemphasizing its role in the pathogenesis of DCM with the fact of only significantly increased expression or activation of NLRP3 inflammasome and pyroptosis in the heart of diabetic rodent models. Only clear-cut evidence-based causative roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM can help to develop effective and safe medications for the clinical management of DCM, targeting these biomarkers.

Key Words: Diabetic cardiomyopathy; Nucleotide oligomerization domain; NOD-like receptor protein 3 inflammasome; Cardiac cell death; Pyroptosis

Core Tip: The involvement of the NOD-like receptor protein 3 (NLRP3) inflammasome and pyroptosis in the pathogenesis of diabetic cardiomyopathy (DCM) has been extensively explored. However, most studies focused on whether diabetes causes NLRP3 inflammasome activation and pyroptosis in the diabetic heart, as well as the potential of medications and natural products to mitigate DCM progression along with reducing NLRP3 inflammasome expression and pyroptosis. Few studies directly investigated the roles of NLRP3 inflammasome and pyroptosis in the development of DCM, utilizing appropriate approaches, such as NLRP3 gene silencing or pharmaceutical NLRP3 inhibitors. Therefore, this aspect of investigation is an urgent need, as stated in this editorial.

INTRODUCTION

Deaths attributable to cardiovascular diseases (CVDs) in the United States remain on the rise in the last decade[1]. One of the CVDs, diabetic cardiomyopathy (DCM), defined as a specific diabetes-induced cardiac disease, was reported more than 50 years ago[2]. However, its underlying mechanisms are not fully understood, despite numerous studies that have been conducted[2,3]. Cardiomyopathy refers to a group of diseases that affect the heart muscle, leading to cardiac structural remodeling and dysfunction. Emerging evidence suggests that cardiomyocyte death is key to the pathogeneses of cardiomyopathy[4,5]. Cardiac cell death, particularly cardiomyocyte death, leads to a reduction in contractile units and impairment of cardiac function, while also triggering cardiac inflammation and remodeling, all of which are critical to the development and progression of cardiomyopathy[4,5]. In a recent issue of this journal, Zhang et al[6] reported an important study, showing the preventive effects on the development of DCM by teneligliptin through the inhibition of NPRLP3, one of the inflammasomes (macromolecular protein complexes) that plays an important role in the immune system. In this study, mice were induced with type 1 diabetes mellitus (T1DM) by multiple small dose i.p. injections of streptozotocin (STZ) and were treated with teneligliptin. The authors observed a significant prevention of cardiac remodeling and dysfunction in T1DM mice with teneligliptin compared to those without treatment. Moreover, NOD-like receptor protein 3 (NLRP3) inflammasome activation and increased release of interleukin-1β (IL-1β) in the heart of diabetic mice were also inhibited by teneligliptin treatment. Accordingly, this study establishes the association of the prevention of DCM with the inhibition of NLRP3, but does not provide additional insights into the causative role of the NLRP3 inflammasome in the pathogenesis of DCM.

EVIDENCE NEEDED TO SUPPORT THE CAUSATIVE ROLE OF THE NLRP3 INFLAMMASOME AND PYROPSOSIS IN THE PATHOGENESIS OF DCM

As discussed by the authors[6], the NLRP3 inflammasome, consisting of NLRP3 (pyrin domain-containing NOD-like receptor protein 3), CARD-containing apoptosis-associated speck-like protein (ASC), and effector protein Caspase-1, has been extensively studied[6,7]. NLRP3 is expressed predominantly in macrophages. As a component of the inflammasome, activated NLRP3 undergoes self-oligomerization and assembles with ASC and the protease caspase-1 to form the NLRP3 inflammasome to trigger the immune response. This process results in the production of the active form of the caspase-1 p10/p20 splicer and induces the conversion of the proinflammatory cytokines IL-1β and IL-18 from their immature to their active forms, inducing so-called pyroptosis, a rapid, inflammatory form of lytic programmed cell death[6,7]. It is now well-accepted that the NLRP3 inflammasome plays important roles in the pathogenesis of both T1DM and type 2 diabetes mellitus (T2DM) and is linked to a number of the complications associated with diabetes[8]. As a key feature of diabetes, hyperglycemia was found to be able to activate NLRP3 inflammasome-mediated pyroptosis in multi-organs, including the heart[9,10]. These findings suggest that the NLRP3 inflammasome may be involved in the development of DCM.

Zhang et al[6], did not provide direct evidence to confirm the capacity of specific inhibition of NLRP3 to prevent DCM, but instead cited several publications that discuss the crucial roles of NLRP3 (see their citations). However, these citations are of studies showing associations; in another words, not providing definitive evidence as to whether NLRP3 plays a causative role in the development of DCM or not. This is a general phenomenon in the current literature. Many publications have only cited studies that do not provide clear evidence to support the role of NLRP3 inflammasome and pyroptosis in the induction of DCM. For example, such studies demonstrated protective or preventive effects of different medications or natural compounds on the pathogenesis of DCM, accompanied by inhibition of cardiac inflammatory responses, including markers of the NLRP3 inflammasome and pyroptosis. Accordingly, they concluded that the protective or preventive effects of DCM are mediated by the inhibition of NLRP3 and/or pyroptosis (references are not cited due to page limitations). Therefore, it is appropriate to summarize the direct evidence to support the roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM as described below.

In 2014, an important study was reported by Luo et al[11], in which the silencing of NLRP3 gene ameliorated the development of DCM in a T2DM rat model. In their study, rats induced with T2DM by HFD/STZ exhibited severe metabolic disorder, cardiac inflammation, remodeling along with activated NLRP3, ASC, pro-caspase-1, cleaved caspase-1, mature IL-1β, and cell death (pyroptosis). These diabetic effects were markedly attenuated by NLRP3 gene silencing therapy, supporting the potential role of activation of NLRP3 inflammasome in the pathogenesis of DCM. Following this study, the pharmaceutical application of NLRP3 specific inhibitors to prevent diabetic complications was explored. For example, administration of the NLRP3 specific inhibitor MCC950, the most widely used NLRP3 specific inhibitor in preclinical and clinical studies[12], in STZ-induced diabetes in ApoE knockout mice resulted in the prevention of diabetes-induced atherosclerosis[13].

In terms of the heart, Yin et al[14] treated cultured cardiac H9C2 cells with palmitate as a commonly used fatty acid to mimic in vivo lipotoxicity in the diabetic condition. They showed the increased cardiac cell death by palmitate, when it was attenuated by the direct application of MCC950 into the medium, along with decreased gene and protein expression levels of NLRP3, ASC, Caspase-1, and GSDMD. Furthermore, in a mouse model of stroke-induced systemic inflammation with cardiac dysfunction, ischemic stroke-induced cardiac remodeling and dysfunction were more severe in mice with T2DM induced by HFD/STZ compared to mice without T2DM. M1-polarized macrophage infiltration and NLRP3 inflammasome activation in the heart following diabetic stroke are also more severe compared to stroke in non-diabetic mice. However, these effects are attenuated by the NLRP3 inflammasome inhibitor CY-09[15]. It is known that obesity increases the risk of cardiac fibrosis. Deng et al[16] recently reported that mice with HFD-induced obesity had more severe myocardial fibrosis than control mice, both in normal and ischemia/reperfusion conditions. However, the cardiac fibrosis could be attenuated by a NLRP3 inhibitor MCC950, supporting the concept that the heart is susceptible to fibrosis with obesity through activation of the NLRP3 inflammasome[16].

CONCLUSION

These pieces of evidence summarized above support, to a certain extent, a critical role of the NLRP3 inflammasome and associated pyroptosis in the development of DCM[11,13-16]. However, the relevant studies remain limited in terms of the experimental models of diabetes used, and DCM’s typical pathological changes and dysfunction examined, gene silencing efficiency, and the NLRP3 inflammasome specific inhibitor types, doses, and times of administration at early or late stage of diabetes. Accordingly, more systematic studies with direct evidence are urgently needed to support the direct link between NLRP3 inflammasome, associated pyroptosis and DCM. At this time, it is appropriate to be circumspect in relation to its role in the pathogenesis of DCM despite the fact that several medications and natural products that are able to inhibit NLRP3 inflammasome and associated pyroptosis, including the teneligliptin used in the study of Zhang et al[6], also prevent the pathogenesis of DCM[10,17-20]. We need to determine that these medications and natural products do not protect the heart from diabetes-associated damage via other mechanisms and that the increased expression/activation of NLRP3 inflammasome and pyroptosis-related markers that were attenuated do not represent non-specific responses. It is only when an unequivocal confirmation of their causative roles in the pathogenesis of DCM is provided, then these can be specifically targeted to develop effective and safe medications for clinical use. Zhang et al[6] have performed an important study and this editorial serves as a timely reminder in relation to the pivotal importance of direct, rather than indirect, evidence in this regard.