MMP9 drives ferroptosis by regulating GPX4 and iron signaling.

Ferroptosis, defined by the suppression of glutathione peroxidase-4 (GPX4) and iron overload, is a distinctive form of regulated cell death. Our in-depth research identifies matrix metalloproteinase-9 (MMP9) as a critical modulator of ferroptosis through its influence on GPX4 and iron homeostasis. Employing an innovative MMP9 construct without collagenase activity, we reveal that active MMP9 interacts with GPX4 and glutathione reductase, reducing GPX4 expression and activity. Furthermore, MMP9 suppresses key transcription factors (SP1, CREB1, NRF2, FOXO3, and ATF4), alongside GPX1 and ferroptosis suppressor protein-1 (FSP1), thereby disrupting the cellular redox balance. MMP9 regulates iron metabolism by modulating iron import, storage, and export via a network of protein interactions. LC-MS/MS has identified 83 proteins that interact with MMP9 at subcellular levels, implicating them in ferroptosis regulation. Integrated pathway analysis (IPA) highlights MMP9's extensive influence on ferroptosis pathways, underscoring its potential as a therapeutic target in conditions with altered redox homeostasis and iron metabolism.

Regulation of cardiac ferroptosis in diabetic human heart failure: uncovering molecular pathways and key targets.

Diabetes significantly increases the risk of heart failure by inducing myocardial cell death, potentially through ferroptosis-an iron-dependent, non-apoptotic cell death pathway characterized by lipid peroxidation. The role of cardiac ferroptosis in human heart failure, however, remains poorly understood. In this study, we compared cardiac ferroptosis in humans with diabetic heart failure to that in healthy controls. Our findings reveal that diabetes not only intensifies myocardial cell death but also upregulates markers of ferroptosis in human hearts. This is linked to decreased transcription and activity of glutathione peroxidase-4 (GPX4), influenced by reduced levels of activating transcription factor-4 (ATF4) and nuclear factor erythroid-2-related factor-2 (NRF2), and downregulation of glutathione reductase (GSR). Additionally, diabetic hearts showed an increased labile iron pool due to enhanced heme metabolism by heme oxygenase-1 (HMOX1), elevated iron import via divalent metal transporter-1 (DMT1), reduced iron storage through ferritin light chain (FLC), and decreased iron export via ferroportin-1 (FPN1). The reduction in FPN1 levels likely results from decreased stabilization by amyloid precursor protein (APP) and diminished NRF2-mediated transcription. Furthermore, diabetes upregulates lysophosphatidylcholine acyltransferase-3 (LPCAT3), facilitating the integration of polyunsaturated fatty acids (PUFA) into phospholipid membranes, and downregulates acyl-CoA thioesterase-1 (ACOT1), which further promotes ferroptosis. LC-MS/MS analysis identified several novel proteins implicated in diabetes-induced cardiac ferroptosis, including upregulated ceruloplasmin, which enhances iron metabolism, and cytochrome b-245 heavy chain (CYBB), a key component of NADPH oxidase that aids in the production of reactive oxygen species (ROS), along with downregulated voltage-dependent anion-selective channel protein-2 (VDAC2), essential for maintaining mitochondrial membrane potential. In conclusion, our study not only confirms the presence and potentially predominant role of cardiac ferroptosis in humans with diabetic heart failure but also elucidates its molecular mechanisms, offering potential therapeutic targets to mitigate heart failure complications in diabetic patients.

Tailoring transfection for cardiomyocyte cell lines: balancing efficiency and toxicity in lipid versus polymer-based transfection methods in H9c2 and HL-1 cells.

Cardiovascular research relies heavily on the veracity of in vitro cardiomyocyte models, with H9c2 and HL-1 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from nonstandardized culturing and transfection methods poses a significant challenge to data uniformity and reliability. In this study, we introduce meticulously crafted protocols to enhance the culture and transfection of H9c2 and HL-1 cells, emphasizing the reduction of cytotoxic effects while improving transfection efficiency. Through the examination of polymer-based and lipid-based transfection methods, we offer a comparative analysis that underscores the heightened efficiency and reduced toxicity of these approaches. Our research provides an extensive array of step-by-step procedures designed to foster robust cell cultures and outlines troubleshooting practices to rectify issues of low transfection rates. We discuss the merits and drawbacks of both transfection techniques, equipping researchers with the knowledge to choose the most fitting method for their experimental goals. By offering a definitive guide to these cell lines' culturing and transfection, our work seeks to set a new standard in procedural consistency, ensuring that the cardiovascular research community can achieve more dependable and reproducible results, thereby pushing the boundaries of current methodologies toward impactful clinical applications.NEW & NOTEWORTHY We have developed standardized protocols that significantly reduce cytotoxicity and enhance transfection efficiency in H9c2 and HL-1 cardiomyocyte cell lines. Our detailed comparative analysis of polymer-based and lipid-based transfection methods has identified optimized approaches with superior performance. Accompanying these protocols are comprehensive troubleshooting strategies to address common issues related to low transfection rates. Implementing these protocols is expected to yield more consistent and reproducible results, driving the field of cardiovascular research toward impactful clinical breakthroughs.

Deciphering MMP9's dual role in regulating SOD3 through protein-protein interactions.

Although the collagenase enzyme activity of matrix metalloproteinase-9 (MMP9) is well-documented, its non-enzymatic functions remain less understood. The interaction between intracellular superoxide dismutase-1 (SOD1) and MMP9 is known, with SOD1 suppressing MMP9. However, the mechanism by which MMP9, a secretory protein, influences the extracellular antioxidant superoxide dismutase-3 (SOD3) is not yet clear. To explore MMP9's regulatory impact on SOD3, we employed human embryonic kidney-293 cells, transfecting them with MMP9 overexpresssion and catalytic-site mutant plasmids. Additionally, MMP9 overexpressing cells were treated with an MMP9 activator and inhibitor. Analyses of both cell lysates and culture medium provided insights into MMP9's intracellular and extracellular regulatory roles. In-silico analysis and experimental approaches like proximal ligation assay and co-immunoprecipitation were utilized to delineate the protein-protein interactions between MMP9 and SOD3. Our findings indicate that activated MMP9 enhances SOD3 levels, a regulation not hindered by MMP9 inhibitors. Intriguingly, catalytically inactive MMP9 appeared to reduce SOD3 levels, likely due to MMP9's binding with SOD3, leading to their proteolytic degradation. This MMP9 influence on SOD3 was consistent in both intracellular and extracellular environments, suggesting a parallel in MMP9-SOD3 interactions across these domains. Ultimately, this study unveils a novel interaction between MMP9 and SOD3, highlighting the unique regulatory role of catalytically inactive MMP9 in diminishing SOD3 levels, contrasting its usual upregulation by active MMP9.

Ironing out the details: ferroptosis and its relevance to diabetic cardiomyopathy.

Ferroptosis is a newly identified myocardial cell death mechanism driven by iron-dependent lipid peroxidation. The presence of elevated intramyocardial lipid levels and excessive iron in patients with diabetes suggest a predominant role of ferroptosis in diabetic cardiomyopathy. As myocardial cell death is a precursor of heart failure, and intensive glycemic control cannot abate the increased risk of heart failure in patients with diabetes, targeting myocardial cell death via ferroptosis is a promising therapeutic avenue to prevent and/or treat diabetic cardiomyopathy. This review provides updated and comprehensive molecular mechanisms underpinning ferroptosis, clarifies several misconceptions about ferroptosis, emphasizes the importance of ferroptosis in diabetes-induced myocardial cell death, and offers valuable approaches to evaluate and target ferroptosis in the diabetic heart. Furthermore, basic concepts and ideas presented in this review, including glutathione peroxidase-4-independent and mitochondrial mechanisms of ferroptosis, are also important for investigating ferroptosis in other diabetic organs, as well as nondiabetic and metabolically compromised hearts.

Differential effects of CMV infection on the viability of cardiac cells.

Cytomegalovirus (CMV) is a widely prevalent herpesvirus that reaches seroprevalence rates of up to 95% in several parts of the world. The majority of CMV infections are asymptomatic, albeit they have severe detrimental effects on immunocompromised individuals. Congenital CMV infection is a leading cause of developmental abnormalities in the USA. CMV infection is a significant risk factor for cardiovascular diseases in individuals of all ages. Like other herpesviruses, CMV regulates cell death for its replication and establishes and maintains a latent state in the host. Although CMV-mediated regulation of cell death is reported by several groups, it is unknown how CMV infection affects necroptosis and apoptosis in cardiac cells. Here, we infected primary cardiomyocytes, the contractile cells in the heart, and primary cardiac fibroblasts with wild-type and cell-death suppressor deficient mutant CMVs to determine how CMV regulates necroptosis and apoptosis in cardiac cells. Our results reveal that CMV infection prevents TNF-induced necroptosis in cardiomyocytes; however, the opposite phenotype is observed in cardiac fibroblasts. CMV infection also suppresses inflammation, reactive oxygen species (ROS) generation, and apoptosis in cardiomyocytes. Furthermore, CMV infection improves mitochondrial biogenesis and viability in cardiomyocytes. We conclude that CMV infection differentially affects the viability of cardiac cells.

Regulating Polyamine Metabolism by miRNAs in Diabetic Cardiomyopathy.

PURPOSE OF REVIEW: Insulin is at the heart of diabetes mellitus (DM). DM alters cardiac metabolism causing cardiomyopathy, ultimately leading to heart failure. Polyamines, organic compounds synthesized by cardiomyocytes, have an insulin-like activity and effect on glucose metabolism, making them metabolites of interest in the DM heart. This review sheds light on the disrupted microRNA network in the DM heart in relation to developing novel therapeutics targeting polyamine biosynthesis to prevent/mitigate diabetic cardiomyopathy. RECENT FINDINGS: Polyamines prevent DM-induced upregulation of glucose and ketone body levels similar to insulin. Polyamines also enhance mitochondrial respiration and thereby regulate all major metabolic pathways. Non-coding microRNAs regulate a majority of the biological pathways in our body by modulating gene expression via mRNA degradation or translational repression. However, the role of miRNA in polyamine biosynthesis in the DM heart remains unclear. This review discusses the regulation of polyamine synthesis and metabolism, and its impact on cardiac metabolism and circulating levels of glucose, insulin, and ketone bodies. We provide insights on potential roles of polyamines in diabetic cardiomyopathy and putative miRNAs that could regulate polyamine biosynthesis in the DM heart. Future studies will unravel the regulatory roles these miRNAs play in polyamine biosynthesis and will open new doors in the prevention/treatment of adverse cardiac remodeling in diabetic cardiomyopathy.