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 HL-1 and H9c2 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from non-standardized 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 HL-1 and H9c2 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.

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.

Genome Editing and Diabetic Cardiomyopathy.

Differential gene expression is associated with diabetic cardiomyopathy (DMCM) and culminates in adverse remodeling in the diabetic heart. Genome editing is a technology utilized to alter endogenous genes. Genome editing also provides an option to induce cardioprotective genes or inhibit genes linked to adverse cardiac remodeling and thus has promise in ameliorating DMCM. Non-coding genes have emerged as novel regulators of cellular signaling and may serve as potential therapeutic targets for DMCM. Specifically, there is a widespread change in the gene expression of fetal cardiac genes and microRNAs, termed genetic reprogramming, that promotes pathological remodeling and contributes to heart failure in diabetes. This genetic reprogramming of both coding and non-coding genes varies with the progression and severity of DMCM. Thus, genetic editing provides a promising option to investigate the role of specific genes/non-coding RNAs in DMCM initiation and progression as well as developing therapeutics to mitigate cardiac remodeling and ameliorate DMCM. This chapter will summarize the research progress in genome editing and DMCM and provide future directions for utilizing genome editing as an approach to prevent and/or treat DMCM.

Guidelines on models of diabetic heart disease.

Diabetes is a major risk factor for cardiovascular diseases, including diabetic cardiomyopathy, atherosclerosis, myocardial infarction, and heart failure. As cardiovascular disease represents the number one cause of death in people with diabetes, there has been a major emphasis on understanding the mechanisms by which diabetes promotes cardiovascular disease, and how antidiabetic therapies impact diabetic heart disease. With a wide array of models to study diabetes (both type 1 and type 2), the field has made major progress in answering these questions. However, each model has its own inherent limitations. Therefore, the purpose of this guidelines document is to provide the field with information on which aspects of cardiovascular disease in the human diabetic population are most accurately reproduced by the available models. This review aims to emphasize the advantages and disadvantages of each model, and to highlight the practical challenges and technical considerations involved. We will review the preclinical animal models of diabetes (based on their method of induction), appraise models of diabetes-related atherosclerosis and heart failure, and discuss in vitro models of diabetic heart disease. These guidelines will allow researchers to select the appropriate model of diabetic heart disease, depending on the specific research question being addressed.

Impaired microcirculatory function, mitochondrial respiration, and oxygen utilization in skeletal muscle of claudicating patients with peripheral artery disease.

Peripheral artery disease (PAD) is an atherosclerotic disease that impairs blood flow and muscle function in the lower limbs. A skeletal muscle myopathy characterized by mitochondrial dysfunction and oxidative damage is present in PAD; however, the underlying mechanisms are not well established. We investigated the impact of chronic ischemia on skeletal muscle microcirculatory function and its association with leg skeletal muscle mitochondrial function and oxygen delivery and utilization capacity in PAD. Gastrocnemius samples and arterioles were harvested from patients with PAD (n = 10) and age-matched controls (Con, n = 11). Endothelium-dependent and independent vasodilation was assessed in response to flow (30 µL·min-1), acetylcholine, and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiration was quantified by high-resolution respirometry, microvascular oxygen delivery, and utilization capacity (tissue oxygenation index, TOI) were assessed by near-infrared spectroscopy. Vasodilation was attenuated in PAD (P < 0.05) in response to acetylcholine (Con: 71.1 ± 11.1%, PAD: 45.7 ± 18.1%) and flow (Con: 46.6 ± 20.1%, PAD: 29.3 ± 10.5%) but not SNP (P = 0.30). Complex I + II state 3 respiration (P < 0.01) and TOI recovery rate were impaired in PAD (P < 0.05). Both flow and acetylcholine-mediated vasodilation were positively associated with complex I + II state 3 respiration (r = 0.5 and r = 0.5, respectively, P < 0.05). Flow-mediated vasodilation and complex I + II state 3 respiration were positively associated with TOI recovery rate (r = 0.8 and r = 0.7, respectively, P < 0.05). These findings suggest that chronic ischemia attenuates skeletal muscle arteriole endothelial function, which may be a key mediator for mitochondrial and microcirculatory dysfunction in the PAD leg skeletal muscle. Targeting microvascular dysfunction may be an effective strategy to prevent and/or reverse disease progression in PAD.NEW & NOTEWORTHY Ex vivo skeletal muscle arteriole endothelial function is impaired in claudicating patients with PAD, and this is associated with attenuated skeletal muscle mitochondrial respiration. In vivo skeletal muscle oxygen delivery and utilization capacity is compromised in PAD, and this may be due to microcirculatory and mitochondrial dysfunction. These results suggest that targeting skeletal muscle arteriole function may lead to improvements in skeletal muscle mitochondrial respiration and oxygen delivery and utilization capacity in claudicating patients with PAD.

Metabolites and Genes behind Cardiac Metabolic Remodeling in Mice with Type 1 Diabetes Mellitus.

Metabolic remodeling is at the heart of diabetic cardiomyopathy. High glycemic fluctuations increase metabolic stress in the type 1 diabetes mellitus (T1DM) heart. There is a lack of understanding on how metabolites and genes affect metabolic remodeling in the T1DM heart. We hypothesize that differential expression of metabolic genes and metabolites synergistically influence metabolic remodeling preceding T1DM cardiomyopathy. To test our hypothesis, we conducted high throughput analysis of hearts from adult male hyperglycemic Ins2+/- (Akita) and littermate normoglycemic Ins2+/+ (WT) mice. The Akita mouse is a spontaneous, genetic model of T1DM that develops increased levels of consistent glycemic variability without the off-target cardiotoxic effects present in chemically- induced models of T1DM. After validating the presence of a T1DM phenotype, we conducted metabolomics via LC-MS analysis and genomics via next-generation sequencing in left ventricle tissue from the Akita heart. Ingenuity Pathway Analyses revealed that 108 and 30 metabolic pathways were disrupted within the metabolomics and genomics datasets, respectively. Notably, a comparison between the two analyses showed 15 commonly disrupted pathways, including ketogenesis, ketolysis, cholesterol biosynthesis, acetyl CoA hydrolysis, and fatty acid biosynthesis and beta-oxidation. These identified metabolic pathways predicted by the differential expression of metabolites and genes provide the foundation for understanding metabolic remodeling in the T1DM heart. By limited experiment, we revealed a predicted disruption in the metabolites and genes behind T1DM cardiac metabolic derangement. Future studies targeting these genes and metabolites will unravel novel therapies to prevent/improve metabolic remodeling in the T1DM heart.

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.

Infarct in the Heart: What's MMP-9 Got to Do with It?

Over the past three decades, numerous studies have shown a strong connection between matrix metalloproteinase 9 (MMP-9) levels and myocardial infarction (MI) mortality and left ventricle remodeling and dysfunction. Despite this fact, clinical trials using MMP-9 inhibitors have been disappointing. This review focuses on the roles of MMP-9 in MI wound healing. Infiltrating leukocytes, cardiomyocytes, fibroblasts, and endothelial cells secrete MMP-9 during all phases of cardiac repair. MMP-9 both exacerbates the inflammatory response and aids in inflammation resolution by stimulating the pro-inflammatory to reparative cell transition. In addition, MMP-9 has a dual effect on neovascularization and prevents an overly stiff scar. Here, we review the complex role of MMP-9 in cardiac wound healing, and highlight the importance of targeting MMP-9 only for its detrimental actions. Therefore, delineating signaling pathways downstream of MMP-9 is critical.

Generating Ins2+/-/miR-133aTg Mice to Model miRNA-Driven Cardioprotection of Human Diabetic Heart.

Diabetes mellitus (DM) is caused either due to insulin deficiency (T1DM) or insulin resistance (T2DM). DM increases the risk of heart failure by diabetic cardiomyopathy (DMCM), a cardiac muscle disorder that leads to a progressive decline in diastolic function, and ultimately systolic dysfunction. Mouse models of T1DM and T2DM exhibit clinical signs of DMCM. Growing evidence implicates microRNA (miRNA), an endogenous, non-coding, regulatory RNA, in the pathogenesis and signaling of DMCM. Therefore, inhibiting deleterious miRNAs and mimicking cardioprotective miRNAs could provide a potential therapeutic intervention for DMCM. miRNA-133a (miR-133a) is a highly abundant miRNA in the human heart. It is a cardioprotective miRNA, which is downregulated in the DM heart. It has anti-hypertrophic and anti-fibrotic effects. miR-133a mimic treatment after the onset of early DMCM can reverse histological and clinical signs of the disease in mice. We hypothesized that overexpression of cardiac-specific miR-133a in Ins2+/- Akita (T1DM) mice can prevent progression of DMCM. Here, we describe a method to create and validate cardiac-specific Ins2+/-/miR-133aTg mice to determine whether cardiac-specific miR-133a overexpression prevents development of DMCM. These strategies demonstrate the value of genetic modeling of human disease such as DMCM and evaluate the potential of miRNA as a therapeutic intervention.

Intracellular matrix metalloproteinase-9 mediates epigenetic modifications and autophagy to regulate differentiation in human cardiac stem cells.

Epigenetic reprogramming and autophagy have critical roles in differentiation of stem cells. However, very little is known about how epigenetic modifications are mediated and how they contribute to autophagy and differentiation in human cardiac stem cells (hCSCs). Previously, we have reported that intracellular matrix metalloproteinase-9 (MMP9), a collagenase, mediates cell death in hCSCs. Here, we investigated whether intracellular MMP9 mediates epigenetic modifications and autophagy in hCSCs. We created MMP9KO hCSCs and treated them with 5-azacytidine, an inhibitor of DNA methylation, and bafilomycin A1, an inhibitor of autophagosome degradation, and evaluated epigenetic modifications, autophagic flux, and differentiation. Our results showed compromised epigenetic modifications, reduced autophagy, and impaired differentiation in MMP9KO hCSCs. Remarkably, paracrine MMP9 supplementation restored epigenetic modifications but further reduced autophagy in MMP9KO hCSCs. We conclude that intracellular MMP9 is a critical mediator of epigenetic modifications and autophagy in hCSCs. Furthermore, the endocrine and paracrine effects of MMP9 vary for regulating autophagy in hCSCs. These novel roles of MMP9 are valuable for stem cell therapy.

Diabetes and COVID-19 risk: an miRNA perspective.

Coronavirus disease 2019 (COVID-19) and diabetes outcomes (CORONADO) trial revealed that 10.6% of patients with diabetes mellitus hospitalized for COVID-19 (COVID-19) die within 7 days. Several studies from New York, Italy, and China confirm that patients with diabetes are at a much higher risk for mortality due to COVID-19. Besides respiratory illness, COVID-19 increases cardiac injury and diabetic ketoacidosis. In the absence of specific guidelines for the prevention and treatment of COVID-19 for patients with diabetes, they remain at higher risk and are more susceptible to COVID-19. Furthermore, there is a scarcity of basic knowledge on how diabetes affects pathogenesis of severe acute respiratory coronavirus (SARS-CoV-2) infection. In patients with diabetes, impaired glucose use alters metabolic and consequently biological processes instigating pathological remodeling, which has detrimental effects on cardiovascular systems. A majority of biological processes are regulated by noncoding microRNAs (miRNAs), which have emerged as a promising therapeutic candidate for several diseases. In consideration of the higher risk of mortality in patients with diabetes and COVID-19, novel diagnostic test and treatment strategy are urgently warranted in post-COVID-19 era. Here, we describe potential roles of miRNA as a biomarker and therapeutic candidate, especially for heart failure, in patients with diabetes and COVID-19.

Acute mitochondrial antioxidant intake improves endothelial function, antioxidant enzyme activity, and exercise tolerance in patients with peripheral artery disease.

Peripheral artery disease (PAD) is a manifestation of atherosclerosis in the leg arteries, which causes claudication. This may be in part due to vascular mitochondrial dysfunction and excessive reactive oxygen species (ROS) production. A mitochondrial-targeted antioxidant (MitoQ) has been shown to improve vascular mitochondrial function that, in turn, led to improved vascular function in older adults and animal models. However, the roles of vascular mitochondria in vascular function including endothelial function and arterial stiffness in patients with PAD are unknown; therefore, with the use of acute MitoQ intake, this study examined the roles of vascular mitochondria in endothelial function, arterial stiffness, exercise tolerance, and skeletal muscle function in patients with PAD. Eleven patients with PAD received either MitoQ or placebo in a randomized crossover design. At each visit, blood samples, brachial and popliteal artery flow-mediated dilation (FMD), peripheral and central pulse-wave velocity (PWV), blood pressure (BP), maximal walking capacity, time to claudication (COT), and oxygen utility capacity were measured pre- and-post-MitoQ and placebo. There were significant group by time interactions (P < 0.05) for brachial and popliteal FMD that both increased by Δ2.6 and Δ3.3%, respectively, and increases superoxide dismutase (Δ0.03 U/mL), maximal walking time (Δ73.8 s), maximal walking distance (Δ49.3 m), and COT (Δ44.2 s). There were no changes in resting heart rate, BP, malondialdehyde, total antioxidant capacity, PWV, or oxygen utility capacity (P > 0.05). MitoQ intake may be an effective strategy for targeting the vascular mitochondrial environment, which may be useful for restoring endothelial function, leg pain, and walking time in patients with PAD.NEW & NOTEWORTHY The results of this study reveal for the first time that acute oral intake of mitochondrial-targeted antioxidant (MitoQ, 80 mg) is effective for improving vascular endothelial function and superoxide dismutase in patients with peripheral artery disease (PAD). Acute MitoQ intake is also effective for improving maximal walking capacity and delaying the onset of claudication in patients with PAD. These findings suggest that the acute oral intake of MitoQ-mediated improvements in vascular mitochondria play a pivotal role for improving endothelial function, the redox environment, and skeletal muscle performance in PAD.

MMP9 mediates acute hyperglycemia-induced human cardiac stem cell death by upregulating apoptosis and pyroptosis in vitro.

Providing a conducive microenvironment is critical to increase survival of transplanted stem cells in regenerative therapy. Hyperglycemia promotes stem cell death impairing cardiac regeneration in the diabetic heart. Understanding the molecular mechanisms of high glucose-induced stem cell death is important for improving cardiac regeneration in diabetic patients. Matrix metalloproteinase-9 (MMP9), a collagenase, is upregulated in the diabetic heart, and ablation of MMP9 decreases infarct size in the non-diabetic myocardial infarction heart. In the present study, we aim to investigate whether MMP9 is a mediator of hyperglycemia-induced cell death in human cardiac stem cells (hCSCs) in vitro. We created MMP9-/- hCSCs to test the hypothesis that MMP9 mediates hyperglycemia-induced oxidative stress and cell death via apoptosis and pyroptosis in hCSCs, which is attenuated by the lack of MMP9. We found that hyperglycemia induced oxidative stress and increased cell death by promoting pyroptosis and apoptosis in hCSCs, which was prevented in MMP9-/- hCSCs. These findings revealed a novel intracellular role of MMP9 in mediating stem cell death and provide a platform to assess whether MMP9 inhibition could improve hCSCs survival in stem cell therapy at least in acute hyperglycemic microenvironment.

Exercise Training Promotes Cardiac Hydrogen Sulfide Biosynthesis and Mitigates Pyroptosis to Prevent High-Fat Diet-Induced Diabetic Cardiomyopathy.

Obesity increases the risk of developing diabetes and subsequently, diabetic cardiomyopathy (DMCM). Reduced cardioprotective antioxidant hydrogen sulfide (H2S) and increased inflammatory cell death via pyroptosis contribute to adverse cardiac remodeling and DMCM. Although exercise training (EX) has cardioprotective effects, it is unclear whether EX mitigates obesity-induced DMCM by increasing H2S biosynthesis and mitigating pyroptosis in the heart. C57BL6 mice were fed a high-fat diet (HFD) while undergoing treadmill EX for 20 weeks. HFD mice developed obesity, hyperglycemia, and insulin resistance, which were reduced by EX. Left ventricle pressure-volume measurement revealed that obese mice developed reduced diastolic function with preserved ejection fraction, which was improved by EX. Cardiac dysfunction was accompanied by increased cardiac pyroptosis signaling, structural remodeling, and metabolic remodeling, indicated by accumulation of lipid droplets in the heart. Notably, EX increased cardiac H2S concentration and expression of H2S biosynthesis enzymes. HFD-induced obesity led to features of type 2 diabetes (T2DM), and subsequently DMCM. EX during the HFD regimen prevented the development of DMCM, possibly by promoting H2S-mediated cardioprotection and alleviating pyroptosis. This is the first report of EX modulating H2S and pyroptotic signaling in the heart.

Guidelines for evaluating myocardial cell death.

Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms.

Hydrogen Sulfide Ameliorates Homocysteine-Induced Cardiac Remodeling and Dysfunction.

Patients with diabetes, a methionine-rich meat diet, or certain genetic polymorphisms show elevated levels of homocysteine (Hcy), which is strongly associated with the development of cardiovascular disease including diabetic cardiomyopathy. However, reducing Hcy levels with folate shows no beneficial cardiac effects. We have previously shown that a hydrogen sulfide (H2S), a by-product of Hcy through transsulfuration by cystathionine beta synthase (CBS), donor mitigates Hcy-induced hypertrophy in cardiomyocytes. However, the in vivo cardiac effects of H2S in the context of hyperhomocysteinemia (HHcy) have not been studied. We tested the hypothesis that HHcy causes cardiac remodeling and dysfunction in vivo, which is ameliorated by H2S. Twelve-week-old male CBS+/- (a model of HHcy) and sibling CBS+/+ (WT) mice were treated with SG1002 (a slow release H2S donor) diet for 4 months. The left ventricle of CBS+/- mice showed increased expression of early remodeling signals c-Jun and c-Fos, increased interstitial collagen deposition, and increased cellular hypertrophy. Notably, SG1002 treatment slightly reduced c-Jun and c-Fos expression, decreased interstitial fibrosis, and reduced cellular hypertrophy. Pressure volume loop analyses in CBS+/- mice revealed increased end systolic pressure with no change in stroke volume (SV) suggesting increased afterload, which was abolished by SG1002 treatment. Additionally, SG1002 treatment increased end-diastolic volume and SV in CBS+/- mice, suggesting increased ventricular filling. These results demonstrate SG1002 treatment alleviates cardiac remodeling and afterload in HHcy mice. H2S may be cardioprotective in conditions where H2S is reduced and Hcy is elevated.

Transgenic Expression of miR-133a in the Diabetic Akita Heart Prevents Cardiac Remodeling and Cardiomyopathy.

Advanced diabetes mellitus (DM) may have both insulin resistance and deficiency (double DM) that accelerates diabetic cardiomyopathy (DMCM), a cardiac muscle disorder. Reduced cardiac miR-133a, a cardioprotective miRNA, is associated with DMCM. However, it is unclear whether increasing miR-133a levels in the double DM heart could prevent DMCM. We hypothesized that increasing cardiac levels of miR-133a could prevent DMCM in Akita, a mouse model of double DM. To test the hypothesis, we created Akita/miR-133aTg mice, a new strain of Akita where miR-133a is overexpressed in the heart, by crossbreeding male Akita with female cardiac-specific miR-133a transgenic mice. We validated Akita/miR-133aTg mice by genotyping and phenotyping (miR-133a levels in the heart). To determine whether miR-133a overexpression could prevent cardiac remodeling and cardiomyopathy, we evaluated cardiac fibrosis, hypertrophy, and dysfunction (P-V loop) in 13-15 week male WT, Akita, Akita/miR-133aTg, and miR-133aTg mice. Our results revealed that miR-133a overexpression in the Akita heart prevents DM-induced cardiac fibrosis (reduced collagen deposition), hypertrophy (decreased beta-myosin heavy chain), and impaired contractility (downregulated calcium handling protein sarco-endoplasmic reticulum-ATPase-2a). These results demonstrate that increased levels of miR-133a in the DM heart could prevent cardiac remodeling. Our P-V loop analysis showed a trend of decreased cardiac output, stroke volume, and ± dp/dt in Akita, which were blunted in Akita/miR-133aTg heart. These findings suggest that 13-15 week Akita heart undergoes adverse remodeling toward cardiomyopathy, which is prevented by miR-133a overexpression. In addition, increased cardiac miR-133a in the Akita heart did not change blood glucose levels but decreased lipid accumulation in the heart, suggesting inhibition of metabolic remodeling in the heart. Thus, miR-133a could be a promising therapeutic candidate to prevent DMCM.

Hydrogen sulfide-mediated regulation of cell death signaling ameliorates adverse cardiac remodeling and diabetic cardiomyopathy.

The death of cardiomyocytes is a precursor for the cascade of hypertrophic and fibrotic remodeling that leads to cardiomyopathy. In diabetes mellitus (DM), the metabolic environment of hyperglycemia, hyperlipidemia, and oxidative stress causes cardiomyocyte cell death, leading to diabetic cardiomyopathy (DMCM), an independent cause of heart failure. Understanding the roles of the cell death signaling pathways involved in the development of cardiomyopathies is crucial to the discovery of novel targeted therapeutics and biomarkers for DMCM. Recent evidence suggests that hydrogen sulfide (H2S), an endogenous gaseous molecule, has cardioprotective effects against cell death. However, very little is known about signaling by which H2S and its downstream targets regulate myocardial cell death in the DM heart. This review focuses on H2S in the signaling of apoptotic, autophagic, necroptotic, and pyroptotic cell death in DMCM and other cardiomyopathies, abnormalities in H2S synthesis in DM, and potential H2S-based therapeutic strategies to mitigate myocardial cell death to ameliorate DMCM.