Transcriptome analysis reveals organ-specific effects of 2-deoxyglucose treatment in healthy mice.

OBJECTIVE: Glycolytic inhibition via 2-deoxy-D-glucose (2DG) has potential therapeutic benefits for a range of diseases, including cancer, epilepsy, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and COVID-19, but the systemic effects of 2DG on gene function across different tissues are unclear. METHODS: This study analyzed the transcriptional profiles of nine tissues from C57BL/6J mice treated with 2DG to understand how it modulates pathways systemically. Principal component analysis (PCA), weighted gene co-network analysis (WGCNA), analysis of variance, and pathway analysis were all performed to identify modules altered by 2DG treatment. RESULTS: PCA revealed that samples clustered predominantly by tissue, suggesting that 2DG affects each tissue uniquely. Unsupervised clustering and WGCNA revealed six distinct tissue-specific modules significantly affected by 2DG, each with unique key pathways and genes. 2DG predominantly affected mitochondrial metabolism in the heart, while in the small intestine, it affected immunological pathways. CONCLUSIONS: These findings suggest that 2DG has a systemic impact that varies across organs, potentially affecting multiple pathways and functions. The study provides insights into the potential therapeutic benefits of 2DG across different diseases and highlights the importance of understanding its systemic effects for future research and clinical applications.

Transcriptome Analysis Reveals Organ-Specific Effects of 2-Deoxyglucose Treatment in Healthy Mice.

OBJECTIVE: Glycolytic inhibition via 2-deoxy-D-glucose (2DG) has potential therapeutic benefits for a range of diseases, including cancer, epilepsy, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and COVID-19, but the systemic effects of 2DG on gene function across different tissues are unclear. METHODS: This study analyzed the transcriptional profiles of nine tissues from C57BL/6J mice treated with 2DG to understand how it modulates pathways systemically. Principal component analysis (PCA), weighted gene co-network analysis (WGCNA), analysis of variance, and pathway analysis were all performed to identify modules altered by 2DG treatment. RESULTS: PCA revealed that samples clustered predominantly by tissue, suggesting that 2DG affects each tissue uniquely. Unsupervised clustering and WGCNA revealed six distinct tissue-specific modules significantly affected by 2DG, each with unique key pathways and genes. 2DG predominantly affected mitochondrial metabolism in the heart, while in the small intestine, it affected immunological pathways. CONCLUSIONS: These findings suggest that 2DG has a systemic impact that varies across organs, potentially affecting multiple pathways and functions. The study provides insights into the potential therapeutic benefits of 2DG across different diseases and highlights the importance of understanding its systemic effects for future research and clinical applications.

Upregulated Angiogenesis Is Incompetent to Rescue Dilated Cardiomyopathy Phenotype in Mice.

Dilated cardiomyopathy (DCM) is characterized by pathologic cardiac remodeling resulting in chambers enlargement and impaired heart contractility. Previous reports and our in-silico analysis support the association of DCM phenotype and impaired tissue angiogenesis. Here, we explored whether the modulation in cardiac angiogenesis partly intervenes or rescues the DCM phenotype in mice. Here, a DCM mouse model [α-tropomyosin 54 (α-TM54) mutant] was crossbred with microRNA-210 transgenic mice (210-TG) to develop microRNA-210 (miR-210) overexpressing α-TM54 mutant mice (TMx210). Contrary to wild-type (WT) and 210-TG mice, a significant increase in heart weight to body weight ratio in aged mixed-gender TMx210 and DCM mice was recorded. Histopathological analysis revealed signs of pathological cardiac remodeling such as myocardial disarray, myofibrillar loss, and interstitial fibrosis in DCM and TMx210 mice. Contrary to WT and DCM, a significant increase in angiogenic potential was observed in TMx210 and 210-TG mice hearts which is reflected by higher blood vessel density and upregulated proangiogenic vascular endothelial growth factor-A. The echocardiographic assessment showed comparable cardiac dysfunction in DCM and TMx210 mice as compared to WT and 210-TG. Overall, the present study concludes that miR-210 mediated upregulated angiogenesis is not sufficient to rescue the DCM phenotype in mice.

High-Resolution Transcriptomic Profiling of the Heart During Chronic Stress Reveals Cellular Drivers of Cardiac Fibrosis and Hypertrophy.

BACKGROUND: Cardiac fibrosis is a key antecedent to many types of cardiac dysfunction including heart failure. Physiological factors leading to cardiac fibrosis have been recognized for decades. However, the specific cellular and molecular mediators that drive cardiac fibrosis, and the relative effect of disparate cell populations on cardiac fibrosis, remain unclear. METHODS: We developed a novel cardiac single-cell transcriptomic strategy to characterize the cardiac cellulome, the network of cells that forms the heart. This method was used to profile the cardiac cellular ecosystem in response to 2 weeks of continuous administration of angiotensin II, a profibrotic stimulus that drives pathological cardiac remodeling. RESULTS: Our analysis provides a comprehensive map of the cardiac cellular landscape uncovering multiple cell populations that contribute to pathological remodeling of the extracellular matrix of the heart. Two phenotypically distinct fibroblast populations, Fibroblast-Cilp and Fibroblast-Thbs4, emerged after induction of tissue stress to promote fibrosis in the absence of smooth muscle actin-expressing myofibroblasts, a key profibrotic cell population. After angiotensin II treatment, Fibroblast-Cilp develops as the most abundant fibroblast subpopulation and the predominant fibrogenic cell type. Mapping intercellular communication networks within the heart, we identified key intercellular trophic relationships and shifts in cellular communication after angiotensin II treatment that promote the development of a profibrotic cellular microenvironment. Furthermore, the cellular responses to angiotensin II and the relative abundance of fibrogenic cells were sexually dimorphic. CONCLUSIONS: These results offer a valuable resource for exploring the cardiac cellular landscape in health and after chronic cardiovascular stress. These data provide insights into the cellular and molecular mechanisms that promote pathological remodeling of the mammalian heart, highlighting early transcriptional changes that precede chronic cardiac fibrosis.

Inhibition of Senescence-Associated Genes Rb1 and Meis2 in Adult Cardiomyocytes Results in Cell Cycle Reentry and Cardiac Repair Post-Myocardial Infarction.

Background Myocardial infarction results in a large-scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult mammalian cardiomyocytes are principally nonproliferative. Despite historical presumptions of stem cell-mediated cardiac regeneration, numerous recent studies using advanced lineage-tracing methods demonstrated that the only source of cardiomyocyte renewal originates from the extant myocardium; thus, the augmented proliferation of preexisting adult cardiomyocytes remains a leading therapeutic approach toward cardiac regeneration. In the present study we investigate the significance of suppressing cell cycle inhibitors Rb1 and Meis2 to promote adult cardiomyocyte reentry to the cell cycle. Methods and Results In vitro experiments with small interfering RNA-mediated simultaneous knockdown of Rb1 and Meis2 in both adult rat cardiomyocytes, isolated from 12-week-old Fischer rats, and human induced pluripotent stem cell-derived cardiomyocytes showed a significant increase in cell number, a decrease in cell size, and an increase in mononucleated cardiomyocytes. In vivo, a hydrogel-based delivery method for small interfering RNA-mediated silencing of Rb1 and Meis2 is utilized following myocardial infarction. Immunofluorescent imaging analysis revealed a significant increase in proliferation markers 5-ethynyl-2'-deoxyuridine, PH3, KI67, and Aurora B in adult cardiomyocytes as well as improved cell survivability with the additional benefit of enhanced peri-infarct angiogenesis. Together, this intervention resulted in a reduced infarct size and improved cardiac function post-myocardial infarction. Conclusions Silencing of senescence-inducing pathways in adult cardiomyocytes via inhibition of Rb1 and Meis2 results in marked cardiomyocyte proliferation and increased protection of cardiac function in the setting of ischemic injury.

Metformin intervention prevents cardiac dysfunction in a murine model of adult congenital heart disease.

OBJECTIVE: Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown. METHODS: We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition. Metformin intervention was used to further evaluate potential therapeutic amelioration of cardiac dysfunction in this model. RESULTS: ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence as main cardiac energy source. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin administration prevented the development of heart dysfunction in ACHD predisposed mice. CONCLUSIONS: This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our study suggests that interactions between genetic and metabolic disturbances ultimately lead to the clinical presentation of heart failure in patients with ACHD. Early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities. These interactions raise the prospect for a translational reassessment of ACHD presentation in the clinic.

MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents.

An innovative approach for cardiac regeneration following injury is to induce endogenous cardiomyocyte (CM) cell cycle re-entry. In the present study, CMs from adult rat hearts were isolated and transfected with cel-miR-67 (control) and rno-miR-210. A significant increase in CM proliferation and mono-nucleation were observed in miR-210 group, in addition to a reduction in CM size, multi-nucleation, and cell death. When compared to control, ß-catenin and Bcl-2 were upregulated while APC (adenomatous polyposis coli), p16, and caspase-3 were downregulated in miR-210 group. In silico analysis predicted cell cycle inhibitor, APC, as a direct target of miR-210 in rodents. Moreover, compared to control, a significant increase in CM survival and proliferation were observed with siRNA-mediated inhibition of APC. Furthermore, miR-210 overexpressing C57BL/6 mice (210-TG) were used for short-term ischemia/reperfusion study, revealing smaller cell size, increased mono-nucleation, decreased multi-nucleation, and increased CM proliferation in 210-TG hearts in contrast to wild-type (NTG). Likewise, myocardial infarction (MI) was created in adult mice, echocardiography was performed, and the hearts were harvested for immunohistochemistry and molecular studies. Compared to NTG, 210-TG hearts showed a significant increase in CM proliferation, reduced apoptosis, upregulated angiogenesis, reduced infarct size, and overall improvement in cardiac function following MI. ß-catenin, Bcl-2, and VEGF (vascular endothelial growth factor) were upregulated while APC, p16, and caspase-3 were downregulated in 210-TG hearts. Overall, constitutive overexpression of miR-210 rescues heart function following cardiac injury in adult mice via promoting CM proliferation, cell survival, and angiogenesis. KEY MESSAGES: MiRNA-210 transfected adult rat CMs show proliferation and reduced cell death in vitro. Cell cycle inhibitor APC is a target of miR-210. MiR-210 overexpressing (210-TG) mouse hearts show CMs cell cycle re-entry and survival post myocardial injury. 210-TG mice show significant neovascularization and angiogenic potential post myocardial infarction. 210-TG hearts show reduced infarct size following ischemic injury.

MicroRNA-1825 induces proliferation of adult cardiomyocytes and promotes cardiac regeneration post ischemic injury.

In mammals, proliferative capacity of cardiomyocytes is lost soon after birth, while zebrafish and other lower organisms like newts are known to regenerate injured hearts even at an adult age. Here, we show that miR-1825 can induce robust proliferation of adult rat cardiomyocytes and can improve cardiac function in-vivo post myocardial infarction. Rat adult cardiomyocytes transfected with miR-1825 showed a significant increase in DNA synthesis, mitosis, cytokinesis, and an increase in cell number when compared to cel-miR-67 transfected control. We also observed a reduction in mitochondrial number and a decrease in ROS and DNA-damage. RNA-sequencing data identified NDUFA10, a key gene involved in the mitochondrial electron transport chain to be a direct target of miR-1825. SiRNA mediated silencing of NDUFA10 showed a significant increase in cardiomyocyte proliferation indicating its role downstream of miRNA-1825. In addition, microRNA microarray results identified miR-1825 to regulate expression of a known proliferation inducing miRNA, miR-199a. We also identified the direct targets of miR-199a, namely p16, Rb1, and Meis2 to be downregulated following miR-1825 transfection. However, miR-199a alone did not have similar proliferation inducing effects as miR-1825, indicating that miR-1825 works through multiple pathways and is a master regulator of cardiomyocyte proliferation. In addition, our in-vivo analysis in animal models of LAD ligation and intra-cardiac miRNA delivery showed proliferation of endogenous cardiomyocytes in the peri-infarcted region and an improvement in heart function. These findings establish miR-1825 as a potential therapeutic agent for induction of cardiomyocyte proliferation and cardiac regeneration, with a significant translational potential.

MicroRNAs regulating meis1 expression and inducing cardiomyocyte proliferation.

Cardiovascular disease has been the biggest killer in the United States for decades, with almost a million new cases each year. Even though mammalian rodent neonatal cardiomyocytes show proliferative potential for up to 5 days, adult cardiomyocytes lose this ability. Insufficient cardiomyocyte proliferation is one of the major reasons for the lack of regeneration of myocardial tissue, post injury. Several studies have looked at the mechanisms responsible for the arrest in proliferation at an adult stage. Following up on a recent study by Eulalio et al's study on functional screening of 875 miRNAs for neonatal cardiomyocyte proliferation, we recently identified several miRNAs that induce proliferation in naturally senescent adult cardiomyocytes. Additional studies by Mahmood et al 2013 have identified Meis1 as the major regulator of cardiomyocyte cell cycle. In our present study we have identified three of the adult cardiomyocyte proliferation inducing miRNAs to have binding sites on the 3'UTR of Meis1 gene by in-silico analysis and luciferase assay. Additionally we found these miRNAs; miR-548c-3p, miR-509-3p, and miR-23b-3p to induce significant proliferation in adult cardiomyocytes through translational inhibition of Meis1. We found a significant increase in the number of ACMs with each miRNA, in combination, and with siRNA mediated inhibition of Meis1 gene. We confirmed that these microRNAs, through inhibition of Meis1, affect its downstream targets and thereby regulate cell-cycle progression. Further investigating of the mechanism of action of these miRNAs can identify other treatment options for abnormalities associated with the lack of cardiac regeneration post myocardial injury.

MicroRNAs Inducing Proliferation of Quiescent Adult Cardiomyocytes.

In the United States, each year over 700,000 people suffer from a heart attack and over 25% of deaths are related to heart disease, making it the leading cause of death. Following ischemic injury a part of the heart muscle is replaced by a scar tissue, reducing its functioning capacity. Recent advancements in surgical intervention and pharmacotherapy only provide symptomatic relief and do not address the root cause of the problem which is the massive loss of cardiomyocytes (CM). Therefore, the development of novel therapeutic intervention for the repair and regeneration of ischemic myocardium remains an area of intense research. While existing CM in zebra fish and neonatal mice are known to proliferate and replenish the infarcted heart, it has been shown that adult mammalian CM lose this ability, thus preventing regeneration of the scar tissue. There have been many attempts to facilitate regeneration of ischemic heart but have met with limited success. Micro-RNAs (miRNAs) are one of the promising candidates towards this goal as they are known to play important regulatory roles during differentiation and tissue regeneration, and regulate genetic information by post-transcriptional modification as well as regulation of other miRNAs. While previous work by Eulalio et al., showed miRNAs inducing proliferation in neonatal CM (NCM), we here identify miRNAs inducing proliferation of rat adult-CM (ACM). This commentary while analyses recent work by Eulalio et al[1] also shows some new data with microRNAs in rat adult-CMs. Further work into the mechanism of these miRNAs can determine their therapeutic potential towards regenerating cardiac tissue post ischemic injury.

Cyclosporin a disrupts notch signaling and vascular lumen maintenance.

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.

TRPM3 and miR-204 establish a regulatory circuit that controls oncogenic autophagy in clear cell renal cell carcinoma.

Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant-negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes the growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca(2+) influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca(2+) and Zn(2+) fluxes inhibit miR-214, which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 directly through miR-204 and indirectly through another miR-204 target, Caveolin 1 (CAV1).