SAH is a major metabolic sensor mediating worsening metabolic crosstalk in metabolic syndrome.

In this study, we observed worsening metabolic crosstalk in mouse models with concomitant metabolic disorders such as hyperhomocysteinemia (HHcy), hyperlipidemia, and hyperglycemia and in human coronary artery disease by analyzing metabolic profiles. We found that HHcy worsening is most sensitive to other metabolic disorders. To identify metabolic genes and metabolites responsible for the worsening metabolic crosstalk, we examined mRNA levels of 324 metabolic genes in Hcy, glucose-related and lipid metabolic systems. We examined Hcy-metabolites (Hcy, SAH and SAM) by LS-ESI-MS/MS in 6 organs (heart, liver, brain, lung, spleen, and kidney) from C57BL/6J mice. Through linear regression analysis of Hcy-metabolites and metabolic gene mRNA levels, we discovered that SAH-responsive genes were responsible for most metabolic changes and all metabolic crosstalk mediated by Serine, Taurine, and G3P. SAH-responsive genes worsen glucose metabolism and cause upper glycolysis activation and lower glycolysis suppression, indicative of the accumulation of glucose/glycogen and G3P, Serine synthesis inhibition, and ATP depletion. Insufficient Serine due to negative correlation of PHGDH with SAH concentration may inhibit the folate cycle and transsulfurarion pathway and consequential reduced antioxidant power, including glutathione, taurine, NADPH, and NAD+. Additionally, we identified SAH-activated pathological TG loop as the consequence of increased fatty acid (FA) uptake, FA ß-oxidation and Ac-CoA production along with lysosomal damage. We concluded that HHcy is most responsive to other metabolic changes in concomitant metabolic disorders and mediates worsening metabolic crosstalk mainly via SAH-responsive genes, that organ-specific Hcy metabolism determines organ-specific worsening metabolic reprogramming, and that SAH, acetyl-CoA, Serine and Taurine are critical metabolites mediating worsening metabolic crosstalk, redox disturbance, hypomethylation and hyperacetylation linking worsening metabolic reprogramming in metabolic syndrome.

miR-182/183-Rasa1 axis induced macrophage polarization and redox regulation promotes repair after ischemic cardiac injury.

Few therapies have produced significant improvement in cardiac structure and function after ischemic cardiac injury (ICI). Our possible explanation is activation of local inflammatory responses negatively impact the cardiac repair process following ischemic injury. Factors that can alter immune response, including significantly altered cytokine levels in plasma and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI is a valid strategy for reducing infarct size and damage after myocardial injury. Our previous studies showed that cortical bone stem cells (CBSCs) possess reparative effects after ICI. In our current study, we have identified that the beneficial effects of CBSCs appear to be mediated by miRNA in their extracellular vesicles (CBSC-EV). Our studies showed that CBSC-EV treated animals demonstrated reduced scar size, attenuated structural remodeling, and improved cardiac function versus saline treated animals. These effects were linked to the alteration of immune response, with significantly altered cytokine levels in plasma, and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI. Our detailed in vitro studies demonstrated that CBSC-EV are enriched in miR-182/183 that mediates the pro-reparative polarization and metabolic reprogramming in macrophages, including enhanced OXPHOS rate and reduced ROS, via Ras p21 protein activator 1 (RASA1) axis under Lipopolysaccharides (LPS) stimulation. In summary, CBSC-EV deliver unique molecular cargoes, such as enriched miR-182/183, that modulate the immune response after ICI by regulating macrophage polarization and metabolic reprogramming to enhance repair.

Cardiomyocyte Ploidy, Metabolic Reprogramming and Heart Repair.

The adult heart is made up of cardiomyocytes (CMs) that maintain pump function but are unable to divide and form new myocytes in response to myocardial injury. In contrast, the developmental cardiac tissue is made up of proliferative CMs that regenerate injured myocardium. In mammals, CMs during development are diploid and mononucleated. In response to cardiac maturation, CMs undergo polyploidization and binucleation associated with CM functional changes. The transition from mononucleation to binucleation coincides with unique metabolic changes and shift in energy generation. Recent studies provide evidence that metabolic reprogramming promotes CM cell cycle reentry and changes in ploidy and nucleation state in the heart that together enhances cardiac structure and function after injury. This review summarizes current literature regarding changes in CM ploidy and nucleation during development, maturation and in response to cardiac injury. Importantly, how metabolism affects CM fate transition between mononucleation and binucleation and its impact on cell cycle progression, proliferation and ability to regenerate the heart will be discussed.

Systemic Hypoxemia Induces Cardiomyocyte Hypertrophy and Right Ventricular Specific Induction of Proliferation.

BACKGROUND: A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce adult cardiomyocyte cell cycle reentry, and to determine if hypoxemia also induces cardiomyocyte proliferation in female mice. METHODS: EdU-containing mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were placed in a hypoxia chamber, and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% oxygen for 2 weeks before terminal studies. Myocyte proliferation was also studied with a mosaic analysis with double markers mouse model. RESULTS: Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced an increase (0.01±0.01% in normoxia to 0.11±0.09% in hypoxia) in the number of EdU+ RV cardiomyocytes, with no effect on LV myocytes in male C57BL/6 mice. Similar results were observed in female mice. Furthermore, in mosaic analysis with double markers mice, hypoxia induced a significant increase in RV myocyte proliferation (0.03±0.03% in normoxia to 0.32±0.15% in hypoxia of RFP+ myocytes), with no significant change in LV myocyte proliferation. RNA sequencing showed upregulation of mitotic cell cycle genes and a downregulation of Cullin genes, which promote the G1 to S phase transition in hypoxic mice. There was significant proliferation of nonmyocytes and mild cardiac fibrosis in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression following hypoxia. CONCLUSIONS: Systemic hypoxia induces a global hypertrophic stress response that was associated with increased RV proliferation, and while LV myocytes did not show increased proliferation, our results minimally confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in vivo.

RNA-Binding Protein LIN28a Regulates New Myocyte Formation in the Heart Through Long Noncoding RNA-H19.

BACKGROUND: Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear diploid cardiomyocytes. On maturation, mononuclear diploid cardiomyocytes become binucleated or polyploid and exit the cell cycle. Cardiomyocyte metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative mononuclear diploid cardiomyocytes enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism in cardiac repair after injury. METHODS: LIN28a overexpression was tested using mouse transgenesis on postnatal cardiomyocyte numbers, cell cycle, and response to apical resection injury. With the use of neonatal and adult cell culture systems and adult and Mosaic Analysis with Double Markers myocardial injury models in mice, the effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Last, isolated adult cardiomyocytes from LIN28a and wild-type mice 4 days after myocardial injury were used for RNA-immunoprecipitation sequencing. RESULTS: LIN28a was found to be active primarily during cardiac development and rapidly decreases after birth. LIN28a reintroduction at postnatal day (P) 1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing cardiomyocyte cell cycle activity in LIN28a transgenic pups compared with wild-type littermates. Moreover, LIN28a overexpression extended cardiomyocyte cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated cardiomyocyte apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared with wild-type littermate controls. Instead, LIN28a small molecule inhibitor attenuated the proreparative effects of LIN28a on the heart. Neonatal rat ventricular myocytes overexpressing LIN28a mechanistically showed increased glycolysis, ATP production, and levels of metabolic enzymes compared with control. LIN28a immunoprecipitation followed by RNA-immunoprecipitation sequencing in cardiomyocytes isolated from LIN28a-overexpressing hearts after injury identified long noncoding RNA-H19 as its most significantly altered target. Ablation of long noncoding RNA-H19 blunted LIN28a-induced enhancement on cardiomyocyte metabolism and cell cycle activity. CONCLUSIONS: Collectively, LIN28a reprograms cardiomyocyte metabolism and promotes persistence of mononuclear diploid cardiomyocytes in the injured heart, enhancing proreparative processes, thereby linking cardiomyocyte metabolism to regulation of ploidy/nucleation and repair in the heart.

Combination of mesenchymal stem cells and three-dimensional collagen scaffold preserves ventricular remodeling in rat myocardial infarction model.

BACKGROUND: Cardiovascular diseases are the major cause of mortality worldwide. Regeneration of the damaged myocardium remains a challenge due to mechanical constraints and limited healing ability of the adult heart tissue. Cardiac tissue engineering using biomaterial scaffolds combined with stem cells and bioactive molecules could be a highly promising approach for cardiac repair. Use of biomaterials can provide suitable microenvironment to the cells and can solve cell engraftment problems associated with cell transplantation alone. Mesenchymal stem cells (MSCs) are potential candidates in cardiac tissue engineering because of their multilineage differentiation potential and ease of isolation. Use of DNA methyl transferase inhibitor, such as zebularine, in combination with three-dimensional (3D) scaffold can promote efficient MSC differentiation into cardiac lineage, as epigenetic modifications play a fundamental role in determining cell fate and lineage specific gene expression. AIM: To investigate the role of collagen scaffold and zebularine in the differentiation of rat bone marrow (BM)-MSCs and their subsequent in vivo effects. METHODS: MSCs were isolated from rat BM and characterized morphologically, immunophenotypically and by multilineage differentiation potential. MSCs were seeded in collagen scaffold and treated with 3 µmol/L zebularine in three different ways. Cytotoxicity analysis was done and cardiac differentiation was analyzed at the gene and protein levels. Treated and untreated MSC-seeded scaffolds were transplanted in the rat myocardial infarction (MI) model and cardiac function was assessed by echocardiography. Cell tracking was performed by DiI dye labeling, while regeneration and neovascularization were evaluated by histological and immunohistochemical analysis, res pectively. RESULTS: MSCs were successfully isolated and seeded in collagen scaffold. Cytotoxicity analysis revealed that zebularine was not cytotoxic in any of the treatment groups. Cardiac differentiation analysis showed more pronounced results in the type 3 treatment group which was subsequently chosen for the transplantation in the in vivo MI model. Significant improvement in cardiac function was observed in the zebularine treated MSC-seeded scaffold group as compared to the MI control. Histological analysis also showed reduction in fibrotic scar, improvement in left ventricular wall thickness and preservation of ventricular remodeling in the zebularine treated MSC-seeded scaffold group. Immunohistochemical analysis revealed significant expression of cardiac proteins in DiI labeled transplanted cells and a significant increase in the number of blood vessels in the zebularine treated MSC-seeded collagen scaffold transplanted group. CONCLUSION: Combination of 3D collagen scaffold and zebularine treatment enhances cardiac differentiation potential of MSCs, improves cell engraftment at the infarcted region, reduces infarct size and improves cardiac function.

UCP2 modulates cardiomyocyte cell cycle activity, acetyl-CoA, and histone acetylation in response to moderate hypoxia.

Developmental cardiac tissue is regenerative while operating under low oxygen. After birth, ambient oxygen is associated with cardiomyocyte cell cycle exit and regeneration. Likewise, cardiac metabolism undergoes a shift with cardiac maturation. Whether there are common regulators of cardiomyocyte cell cycle linking metabolism to oxygen tension remains unknown. The objective of the study is to determine whether mitochondrial UCP2 is a metabolic oxygen sensor regulating cardiomyocyte cell cycle. Neonatal rat ventricular myocytes (NRVMs) under moderate hypoxia showed increased cell cycle activity and UCP2 expression. NRVMs exhibited a metabolic shift toward glycolysis, reducing citrate synthase, mtDNA, mitochondrial membrane potential (ΔΨm), and DNA damage/oxidative stress, while loss of UCP2 reversed this phenotype. Next, WT and mice from a global UCP2-KO mouse line (UCP2KO) kept under hypoxia for 4 weeks showed significant decline in cardiac function that was more pronounced in UCP2KO animals. Cardiomyocyte cell cycle activity was reduced, while fibrosis and DNA damage was significantly increased in UCP2KO animals compared with WT under hypoxia. Mechanistically, UCP2 increased acetyl-CoA levels and histone acetylation, and it altered chromatin modifiers linking metabolism to cardiomyocyte cell cycle under hypoxia. Here, we show a potentially novel role for mitochondrial UCP2 as an oxygen sensor regulating cardiomyocyte cell cycle activity, acetyl-CoA levels, and histone acetylation in response to moderate hypoxia.

Cell Surface and Functional Features of Cortical Bone Stem Cells.

The newly established mouse cortical-bone-derived stem cells (mCBSCs) are unique stem cells compared to mouse mesenchymal stem cells (mMSCs). The mCBSC-treated hearts after myocardial infarction have been reported to have greater improvement in myocardial structure and functions. In this study, we examined the stemness features, cell surface glycan profiles, and paracrine functions of mCBSCs compared with mMSCs. The stemness analysis revealed that the self-renewing capacity of mCBSCs was greater than mMSCs; however, the differentiation capacity of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles by lectin array analysis revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. In contrast, the lactosamine (Galß1-4GlcNAc) structure, poly lactosamine- or poly N-acetylglucosamine structure, and α2-3sialic acid on both N- and O-glycans were more highly expressed in mCBSCs. Moreover, we found that mCBSCs secrete a greater amount of TGF-ß1 compared to mMSCs, and that the TGF-ß1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Together, these results suggest that unique characteristics in mCBSCs compared to mMSCs may lead to advanced utility of mCBSCs for cardiac and noncardiac repair.

LIN28a induced metabolic and redox regulation promotes cardiac cell survival in the heart after ischemic injury.

RATIONALE: Cell-based therapeutics have been extensively used for cardiac repair yet underperform due to inability of the donated cells to survive in near anoxia after cardiac injury. Cellular metabolism is linked to maintenance of cardiac stem cell (CSC) renewal, proliferation and survival. Ex vivo expansion alters (CSC) metabolism increasing reliance on oxygen dependent respiration. Whether promoting 'metabolic flexibility' in CSCs augments their ability to survive in near anoxia and repair the heart after injury remains untested. OBJECTIVE: Determine the effect of LIN28a induced metabolic flexibility on cardiac tissue derived stem like cell (CTSC) survival and repair after cardiac injury. METHODS AND RESULTS: LIN28a expression coincides during heart development but is lost in adult CTSCs. Reintroduction of LIN28a in adult CTSC (CTSC-LIN) increased proliferation, survival, expression of pluripotency genes and reduced senescence compared to control (CTSC-GFP). Metabolomic analysis show glycolytic intermediates upregulated in CTSC-LIN together with increased lactate production, pyruvate kinase activity, glucose uptake, ECAR and expression of glycolytic enzymes compared to CTSC-GFP. Additionally, CTSC-LIN showed significantly reduced ROS generation and increase antioxidant markers. In response to H2O2 induced oxidative stress, CTSC-LIN showed increased survival and expression of glycolytic genes. LIN28a salutary effects on CTSCs were linked to PDK1/let-7 signaling pathway with loss of PDK1 or alteration of let-7 abrogating LIN28a effects. Following transplantation in the heart after myocardial infarction (MI), CTSC-LIN showed 6% survival rate at day 7 after injection compared to control cells together with increased proliferation and significant increase in cardiac structure and function 8 weeks after MI. Finally, CSTC-LIN showed enhanced ability to secrete paracrine factors under hypoxic conditions and ability to promote cardiomyocyte proliferation following ischemic cardiac injury. CONCLUSIONS: LIN28a modification promotes metabolic flexibility in CTSCs enhancing proliferation and survival post transplantation including ability to repair the heart after myocardial injury.

Cortical bone stem cell-derived exosomes' therapeutic effect on myocardial ischemia-reperfusion and cardiac remodeling.

Heart failure is the one of the leading causes of death in the United States. Heart failure is a complex syndrome caused by numerous diseases, including severe myocardial infarction (MI). MI occurs after an occlusion of a cardiac artery causing downstream ischemia. MI is followed by cardiac remodeling involving extensive remodeling and fibrosis, which, if the original insult is severe or prolonged, can ultimately progress into heart failure. There is no "cure" for heart failure because therapies to regenerate dead tissue are not yet available. Previous studies have shown that in both post-MI and post-ischemia-reperfusion (I/R) models of heart failure, administration of cortical bone stem cell (CBSC) treatment leads to a reduction in scar size and improved cardiac function. Our first study investigated the ability of mouse CBSC-derived exosomes (mCBSC-dEXO) to recapitulate mouse CBSCs (mCBSC) therapeutic effects in a 24-h post-I/R model. This study showed that injection of mCBSCs and mCBSC-dEXOs into the ischemic region of an infarct had a protective effect against I/R injury. mCBSC-dEXOs recapitulated the effects of CBSC treatment post-I/R, indicating exosomes are partly responsible for CBSC's beneficial effects. To examine if exosomes decrease fibrotic activation, adult rat ventricular fibroblasts (ARVFs) and adult human cardiac fibroblasts (NHCFs) were treated with transforming growth factor ß (TGFß) to activate fibrotic signaling before treatment with mCBSC- and human CBSC (hCBSC)-dEXOs. hCBSC-dEXOs caused a 100-fold decrease in human fibroblast activation. To further understand the signaling mechanisms regulating the protective decrease in fibrosis, we performed RNA sequencing on the NHCFs after hCBSC-dEXO treatment. The group treated with both TGFß and exosomes showed a decrease in small nucleolar RNA (snoRNA), known to be involved with ribosome stability.NEW & NOTEWORTHY Our work is noteworthy due to the identification of factors within stem cell-derived exosomes (dEXOs) that alter fibroblast activation through the hereto-unknown mechanism of decreasing small nucleolar RNA (snoRNA) signaling within cardiac fibroblasts. The study also shows that the injection of stem cells or a stem-cell-derived exosome therapy at the onset of reperfusion elicits cardioprotection, emphasizing the importance of early treatment in the post-ischemia-reperfusion (I/R) wounded heart.

Cardiomyocyte Proliferation as a Source of New Myocyte Development in the Adult Heart.

Cardiac diseases such as myocardial infarction (MI) can lead to adverse remodeling and impaired contractility of the heart due to widespread cardiomyocyte death in the damaged area. Current therapies focus on improving heart contractility and minimizing fibrosis with modest cardiac regeneration, but MI patients can still progress to heart failure (HF). There is a dire need for clinical therapies that can replace the lost myocardium, specifically by the induction of new myocyte formation from pre-existing cardiomyocytes. Many studies have shown terminally differentiated myocytes can re-enter the cell cycle and divide through manipulations of the cardiomyocyte cell cycle, signaling pathways, endogenous genes, and environmental factors. However, these approaches result in minimal myocyte renewal or cardiomegaly due to hyperactivation of cardiomyocyte proliferation. Finding the optimal treatment that will replenish cardiomyocyte numbers without causing tumorigenesis is a major challenge in the field. Another controversy is the inability to clearly define cardiomyocyte division versus myocyte DNA synthesis due to limited methods. In this review, we discuss several studies that induced cardiomyocyte cell cycle re-entry after cardiac injury, highlight whether cardiomyocytes completed cytokinesis, and address both limitations and methodological advances made to identify new myocyte formation.

Bmi1 Augments Proliferation and Survival of Cortical Bone-Derived Stem Cells after Injury through Novel Epigenetic Signaling via Histone 3 Regulation.

Ischemic heart disease can lead to myocardial infarction (MI), a major cause of morbidity and mortality worldwide. Multiple stem cell types have been safely transferred into failing human hearts, but the overall clinical cardiovascular benefits have been modest. Therefore, there is a dire need to understand the basic biology of stem cells to enhance therapeutic effects. Bmi1 is part of the polycomb repressive complex 1 (PRC1) that is involved in different processes including proliferation, survival and differentiation of stem cells. We isolated cortical bones stem cells (CBSCs) from bone stroma, and they express significantly high levels of Bmi1 compared to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs). Using lentiviral transduction, Bmi1 was knocked down in the CBSCs to determine the effect of loss of Bmi1 on proliferation and survival potential with or without Bmi1 in CBSCs. Our data show that with the loss of Bmi1, there is a decrease in CBSC ability to proliferate and survive during stress. This loss of functionality is attributed to changes in histone modification, specifically histone 3 lysine 27 (H3K27). Without the proper epigenetic regulation, due to the loss of the polycomb protein in CBSCs, there is a significant decrease in cell cycle proteins, including Cyclin B, E2F, and WEE as well as an increase in DNA damage genes, including ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). In conclusion, in the absence of Bmi1, CBSCs lose their proliferative potential, have increased DNA damage and apoptosis, and more cell cycle arrest due to changes in epigenetic modifications. Consequently, Bmi1 plays a critical role in stem cell proliferation and survival through cell cycle regulation, specifically in the CBSCs. This regulation is associated with the histone modification and regulation of Bmi1, therefore indicating a novel mechanism of Bmi1 and the epigenetic regulation of stem cells.

Cortical bone stem cells modify cardiac inflammation after myocardial infarction by inducing a novel macrophage phenotype.

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-ß, but not IL-4. CBSC therapy increased the number of galectin-3+ macrophages, CD4+ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-ß, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.

Transcriptional Profiling of Cardiac Cells Links Age-Dependent Changes in Acetyl-CoA Signaling to Chromatin Modifications.

Metabolism has emerged as a regulator of core stem cell properties such as proliferation, survival, self-renewal, and multilineage potential. Metabolites serve as secondary messengers, fine-tuning signaling pathways in response to microenvironment alterations. Studies show a role for central metabolite acetyl-CoA in the regulation of chromatin state through changes in histone acetylation. Nevertheless, metabolic regulators of chromatin remodeling in cardiac cells in response to increasing biological age remains unknown. Previously, we identified novel cardiac-derived stem-like cells (CTSCs) that exhibit increased functional properties in the neonatal heart (nCTSC). These cells are linked to a unique metabolism which is altered with CTSC aging (aCTSC). Here, we present an in-depth, RNA-sequencing-based (RNA-Seq) bioinformatic with cluster analysis that details a distinct epigenome present in nCTSCs but not in aCTSCs. Gene Ontology (GO) and pathway enrichment reveal biological processes, including metabolism, gene regulation enriched in nCTSCs, and STRING analysis that identifies a network of genes related to acetyl-CoA that can potentially influence chromatin remodeling. Additional validation by Western blot and qRT-PCR shows increased acetyl-CoA signaling and histone acetylation in nCTSCs compared to aCTSCs. In conclusion, our data reveal that the link between metabolism and histone acetylation in cardiac cells is altered with the aging of the cardiac tissue.

Cardiac Remodeling During Pregnancy With Metabolic Syndrome: Prologue of Pathological Remodeling.

BACKGROUND: The heart undergoes physiological hypertrophy during pregnancy in healthy individuals. Metabolic syndrome (MetS) is now prevalent in women of child-bearing age and might add risks of adverse cardiovascular events during pregnancy. The present study asks if cardiac remodeling during pregnancy in obese individuals with MetS is abnormal and whether this predisposes them to a higher risk for cardiovascular disorders. METHODS: The idea that MetS induces pathological cardiac remodeling during pregnancy was studied in a long-term (15 weeks) Western diet-feeding animal model that recapitulated features of human MetS. Pregnant female mice with Western diet (45% kcal fat)-induced MetS were compared with pregnant and nonpregnant females fed a control diet (10% kcal fat). RESULTS: Pregnant mice fed a Western diet had increased heart mass and exhibited key features of pathological hypertrophy, including fibrosis and upregulation of fetal genes associated with pathological hypertrophy. Hearts from pregnant animals with WD-induced MetS had a distinct gene expression profile that could underlie their pathological remodeling. Concurrently, pregnant female mice with MetS showed more severe cardiac hypertrophy and exacerbated cardiac dysfunction when challenged with angiotensin II/phenylephrine infusion after delivery. CONCLUSIONS: These results suggest that preexisting MetS could disrupt physiological hypertrophy during pregnancy to produce pathological cardiac remodeling that could predispose the heart to chronic disorders.

Uncoupling protein 2-mediated metabolic adaptations define cardiac cell function in the heart during transition from young to old age.

Cellular replacement in the heart is restricted to postnatal stages with the adult heart largely postmitotic. Studies show that loss of regenerative properties in cardiac cells seems to coincide with alterations in metabolism during postnatal development and maturation. Nevertheless, whether changes in cellular metabolism are linked to functional alternations in cardiac cells is not well studied. We report here a novel role for uncoupling protein 2 (UCP2) in regulation of functional properties in cardiac tissue derived stem-like cells (CTSCs). CTSC were isolated from C57BL/6 mice aged 2 days (nCTSC), 2 month (CTSC), and 2 years old (aCTSC), subjected to bulk-RNA sequencing that identifies unique transcriptome significantly different between CTSC populations from young and old heart. Moreover, results show that UCP2 is highly expressed in CTSCs from the neonatal heart and is linked to maintenance of glycolysis, proliferation, and survival. With age, UCP2 is reduced shifting energy metabolism to oxidative phosphorylation inversely affecting cellular proliferation and survival in aged CTSCs. Loss of UCP2 in neonatal CTSCs reduces extracellular acidification rate and glycolysis together with reduced cellular proliferation and survival. Mechanistically, UCP2 silencing is linked to significant alteration of mitochondrial genes together with cell cycle and survival signaling pathways as identified by RNA-sequencing and STRING bioinformatic analysis. Hence, our study shows UCP2-mediated metabolic profile regulates functional properties of cardiac cells during transition from neonatal to aging cardiac states.

Stem Cell Metabolism: Powering Cell-Based Therapeutics.

Cell-based therapeutics for cardiac repair have been extensively used during the last decade. Preclinical studies have demonstrated the effectiveness of adoptively transferred stem cells for enhancement of cardiac function. Nevertheless, several cell-based clinical trials have provided largely underwhelming outcomes. A major limitation is the lack of survival in the harsh cardiac milieu as only less than 1% donated cells survive. Recent efforts have focused on enhancing cell-based therapeutics and understanding the biology of stem cells and their response to environmental changes. Stem cell metabolism has recently emerged as a critical determinant of cellular processes and is uniquely adapted to support proliferation, stemness, and commitment. Metabolic signaling pathways are remarkably sensitive to different environmental signals with a profound effect on cell survival after adoptive transfer. Stem cells mainly generate energy through glycolysis while maintaining low oxidative phosphorylation (OxPhos), providing metabolites for biosynthesis of macromolecules. During commitment, there is a shift in cellular metabolism, which alters cell function. Reprogramming stem cell metabolism may represent an attractive strategy to enhance stem cell therapy for cardiac repair. This review summarizes the current literature on how metabolism drives stem cell function and how this knowledge can be applied to improve cell-based therapeutics for cardiac repair.

Role of Stem Cell-Derived Microvesicles in Cardiovascular Disease.

The role of stem cells in augmenting reparative processes in the heart after ischemic injury has been successfully demonstrated in small and large animal models. However, the outcomes of cell therapy in clinical trials have been somewhat variable, with overall effects of autologous stem cell therapies demonstrating a modest improvement in cardiac structure and function. How stem cells repair the heart after cardiac injury is still not well understood. Most recent studies suggest that adult derived stem cells act primarily through paracrine signaling to exert beneficial effects, including modulation of immune response, stimulation of new blood vessel formation, or by inducing mature myocytes to transiently reenter the cell cycle, rather than robust direct differentiation of the transplanted cells into myocytes. In addition, data from multiple laboratory results confirmed clearance of stem cells themselves within a few days still leading to functional benefits further confirming the role of paracrine signaling in augmenting cardiac reparative processes rather than direct differentiation of cells. These findings rapidly evolved the field of extracellular vesicles specifically microvesicles (MVs) as they are active hubs of autocrine, paracrine, and endocrine signaling targeting different biological processes. The beneficial effects seen after stem cell transplantation could be linked to the cardioprotective factors packaged in the MVs secreted from stem cells. Therefore, stem cell MVs provide a new avenue for the treatment of cardiovascular disease through a multitude of mechanisms including cellular communication within the stem cell niches, delivery of genetic information, regulation of the immune system in the heart, and stimulation of angiogenesis which will be discussed in this review.

Cortical Bone Derived Stem Cells Modulate Cardiac Fibroblast Response via miR-18a in the Heart After Injury.

The adult heart following injury such as a myocardial infarction forms a fibrotic scar associated with transformation of resident cardiac fibroblasts into myofibroblast, accelerating cardiac remodeling and dysfunction. Cell therapies provide a novel direction for the enhancement of cardiac structure and function but remain poorly described in terms of the effect on resident cardiac fibroblasts. We have shown cortical bone derived stem cells (CBSCs) exhibit an ability to repair the heart after myocardial injury together with reduced scar formation. Nevertheless, whether CBSCs possess ability to modulate resident fibroblast response after myocardial injury remains untested. OBJECTIVE: To determine the effect of secreted factors from CSBCs to attenuate myofibroblast formation in the heart after injury. METHODS AND RESULTS: CBSCs were injected in mice after myocardial infarction which demonstrated reduced fibrosis as determined by Masson's trichrome and Picro-Sirius red staining. In parallel, decreased expression of myofibroblast markers such as Acta2 was observed compared to PBS injected mice. To determine the effect of CBSCs on cardiac fibrosis, adult mouse cardiac fibroblasts were isolated from C57BL/6 mice, primed with CBSC pre-conditioned media for 12 h, and treated with 10ng TGF-ß for 48 h to mimic cardiac injury. Decreased expression of Acta2, periostin and CTGF was observed in adult cardiac fibroblasts cultured in CBSC medium compared to control cells. Additionally, analysis of myofibroblast markers such as vimentin and pSMAD/SMAD was also decreased compared to control cells. To determine the mechanism, we looked for enriched miRNA in CBSCs that can mediate anti fibrotic response after injury. Results showed significantly increased expression of miR-18a in CBSCs. The upregulation of miR-18a was also validated in adult fibroblasts treated with CBSCs compared to control cells. Adult fibroblasts treated with mimic for miR-18a followed by TGF-ß showed significant decrease in myofibroblast formation while miR-18a inhibitor completely inhibited the effect of CBSC medium. CONCLUSION: CBSCs reduce fibroblast to myofibroblast transition and differentiation in adult cardiac fibroblasts via miR-18a-5p. This finding reveals a new avenue for cell therapies to target myocardial scar modulation and provides a resolution for the cardiac repair response after injury in the adult myocardium.