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Fibro-adipogenic progenitor cells (FAPs) are a population of stem cells in skeletal muscle that play multiple roles in muscle repair and regeneration through their complex secretome; however, it is not well understood how the FAP secretome is altered with muscle disuse atrophy. Previous work suggests that the inflammatory cytokine IL-1β is increased in FAPs with disuse and denervation. Inflammasome activation and IL-1β secretion are also known to stimulate the release of extracellular vesicles (EVs). Here, we examined the microRNA (miRNA) cargo of FAP-derived, platelet-derived growth factor receptor A (PDGFRα+) EVs from hindlimb muscles of wild-type and IL-1β KO mice after 14 days of single-hindlimb immobilization. Hindlimb muscles were isolated from mice following the immobilization period, and PDGFRα+ extracellular vesicles were isolated using size-exclusion chromatography and immunoprecipitation. Microarrays were performed to detect changes in miRNAs with unloading and IL-1β deficiency. Results indicate that the PDGFRα+, FAP-derived EVs show a significant increase in miRNAs, such as miR-let-7c, miR-let-7b, miR-181a, and miR-124. These miRNAs have previously been demonstrated to play important roles in cellular senescence and muscle atrophy. Furthermore, the expression of these same miRNAs was not significantly altered in FAP-derived EVs isolated from the immobilized IL-1β KO. These data suggest that disuse-related activation of IL-1β can mediate the miRNA cargo of FAP-derived EVs, contributing directly to the release of senescence- and atrophy-related miRNAs. Therapies targeting FAPs in settings associated with muscle disuse atrophy may therefore have the potential to preserve muscle function and enhance muscle recovery.

Elderly patients are more susceptible to ischemic injury. N6-methyladenosine (m6A) modification is the most abundant reversible epitranscriptomic modification in mammalian RNA and plays a vital role in many biological processes. However, it is unclear whether age difference impacts m6A RNA methylation in hearts and their response to acute myocardial ischemia/reperfusion (I/R) injury. In this study, we measured the global level of m6A RNA methylation as well as the expression of m6A RNA “writers” (methylation enzymes) and “erasers” (demethylation enzymes) in the hearts of young and elderly female mice undergone sham surgery or acute MI/R injury. We found that m6A RNA level and associate modifier gene expression was similar in intact young and old female hearts. However, young hearts show a significant reduction in m6A RNA while elderly hearts showed only a slight reduction in m6A RNA in response to acute I/R injury. To explore the mechanism of differential level of m6A RNA modification, we use qRT-PCR and Western blotting to compare the mRNA and protein expression of major m6A-related “writers” (Mettl3, Mettl14, and WTAP) and ‘erasers” (ALKBH5 and FTO). Mettl3 mRNA and protein expression were significantly reduced in both young and elderly hearts. However, the levels of FTO’s mRNA and protein were only significantly reduced in ischemic elderly hearts, and age-related downregulation of FTO may offset the effect of reduced Mettl3 on reduced m6A RNA level in the hearts of aging mice hearts with acute I/R injury, indicating aging-related differences in epitranscriptomic m6A regulation in hearts in response to acute I/R injury. To further investigate specific I/R related targets of Mettl3, we overexpressed Mettl3 in cardiomyocyte line (HL1) using lentiviral vector, and the m6A enrichment of Bcl2, Bax and PTEN were quantified with m6A RIP-qPCR, we found that m6A modification of PTEN mRNA decreased after in vitro hypoxia/reperfusion injury (iH/R) while Mettl3 augments m6A levels of both Bax and PTEN after iH/R, indicating that Bax and PTEN are target genes of Mettl3 under iH/R stress.

Duchenne muscular dystrophy (DMD) is a progressive myopathic disease caused by mutations in the gene encoding dystrophin protein that eventually leads to the exhaustion of myogenic progenitor cells (MPC). Autologous induced pluripotent stem cells (iPSCs) provide an endless source of MPC, which can potentially replenish the progenitor cell pool, repair muscle damage, and prevent DMD progression. Deletion of mutant exon 23 (ΔEx23) with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify ΔEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify ΔEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables ΔEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, ΔEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted ΔEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice). In conclusion, the use of HDR donor vector increased the efficiency of ΔEx23 gene correction by CRISPR/Cas9, and facilitate the identification of successfully edited iPSC clones for cell therapy of DMD.

Myocardial ischemia/reperfusion (MI/R) causes loss of cardiomyocytes via oxidative stress-induced cardiomyocyte apoptosis. miR322, orthologous to human miR-424, was identified as an ischemia-induced angiogenic miRNA, but its cellular source and function in the setting of acute MI/R remains largely unknown. Using LacZ-tagged miR322 cluster reporter mice, we observed that vascular endothelial cells are the major cellular source of the miR322 cluster in adult hearts. Moreover, miR322 levels were significantly reduced in the heart at 24 h after MI/R injury. Intramyocardial injection of mimic-miR322 significantly diminished cardiac apoptosis (as determined by expression levels of active caspase 3 by Western blot analysis and immunostaining for TUNEL) and reduced infarct size by about 40%, in association with reduced FBXW7 and increased active Notch 1 levels in the ischemic hearts. FBXW7, which is an ubiquitin ligase that is crucial for activated Notch1 turnover, was identified as a direct target of miR322 via FBXW7 3’UTR reporter assay. Co-injection of FBXW7 plasmid with mimic-miR322 in ischemic hearts abolished the effect of mimic-miR322 to reduce apoptosis and infarct size in MI/R hearts. These data identify FBXW7 as a direct target of miR322 and suggest that miR322 could have potential therapeutic application for cardioprotection against ischemia/reperfusion-induced injury.

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.

MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by β-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte-specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline-rich protein 1a (Sprr1a) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a. Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.

Diabetes causes hyperglycemia, which can create a stressful environment for cardiac microvascular endothelial cells (CMECs). To investigate the impact of diabetes on the cellular metabolism of CMECs, we assessed glycolysis by quantifying the extracellular acidification rate (ECAR), and mitochondrial oxidative phosphorylation (OXPHOS) by measuring cellular oxygen consumption rate (OCR), in isolated CMECs from wild-type (WT) hearts and diabetic hearts (db/db) using an extracellular flux analyzer. Diabetic CMECs exhibited a higher level of intracellular reactive oxygen species (ROS), and significantly reduced glycolytic reserve and non-glycolytic acidification, as compared to WT CMECs. In addition, OCR assay showed that diabetic CMECs had increased maximal respiration, and significantly reduced non-mitochondrial oxygen consumption and proton leak. Quantitative PCR (qPCR) showed no difference in copy number of mitochondrial DNA (mtDNA) between diabetic and WT CMECs. In addition, gene expression profiling analysis showed an overall decrease in the expression of essential genes related to β-oxidation (Sirt1, Acox1, Acox3, Hadha, and Hadhb), tricarboxylic acid cycle (TCA) (Idh-3a and Ogdh), and electron transport chain (ETC) (Sdhd and Uqcrq) in diabetic CMECs compared to WT CMECs. Western blot confirmed that the protein expression of Hadha, Acox1, and Uqcrq was decreased in diabetic CMECs. Although lectin staining demonstrated no significant difference in capillary density between the hearts of WT mice and db/db mice, diabetic CMECs showed a lower percentage of cell proliferation by Ki67 staining, and a higher percentage of cellular apoptosis by TUNEL staining, compared with WT CMECs. In conclusion, excessive ROS caused by hyperglycemia is associated with impaired glycolysis and mitochondrial function in diabetic CMECs, which in turn may reduce proliferation and promote CMEC apoptosis.

Background: Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) have detrimental effects. Exercise not only improves endothelial dysfunction and angiogenesis in T2DM but also induces secretion of exosomes into circulation. Extracellular superoxide dismutase (ecSOD) is a major secretory Cu containing antioxidant enzyme that catalyzes dismutation of O 2 •- to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ecSOD-derived H 2 O 2 in endothelial cells (ECs) enhances angiogenesis while impaired ATP7A-ecSOD axis in diabetes induces endothelial dysfunction. Here we examined whether exercise-derived exosomes (Exe-Exo) may have pro-angiogenic effects via regulating ATP7A-ecSOD axis in T2DM. Results: Two weeks of voluntary wheel exercise of control C57Bl6 mice increased plasma exosome levels (6.2-fold) characterized by Nanosight, TEM and exosome markers (CD63, CD81, Tsg101). Treatment of HUVECs with equal number of exosomes revealed that angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold) were significantly enhanced by Exe-Exo compared to sedentary-derived exosomes (Sed-Exo). This was associated with increased ATP7A (2.9-fold) and ecSOD (1.4-fold) expression in Exe-Exo. Sed-Exo from high fat-induced T2DM mice significantly decreased EC migration (40%) and tube formation (10%) as well as ATP7A expression (28%) compared to Sed-Exo from control mice, which were restored by T2DM Exe-Exo, but not by T2DM/ecSOD KO Exe-Exo. Furthermore, exosomes overexpressing ecSOD (ecSOD-Exo) which mimic exercise increased angiogenesis and H2O2 levels in ECs, which were inhibited by overexpression of catalase. In vivo, skin wound healing model showed that direct application of T2DM Sed-Exo delayed while T2DM Exe-Exo enhanced wound healing of control mice. Furthermore, defective wound healing in T2DM mice or ecSOD KO mice were rescued by ecSOD-Exo application. Conclusion: Exercise training improves pro-angiogenic function of circulating exosomes in T2DM via increasing ATP7A-ecSOD axis, which may provide an effective therapy for promoting angiogenesis and wound repair in metabolic and cardiovascular diseases.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium (PCTC) and Cardiovascular Bioengineering Symposium (CVBE) was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present for more than 40 scientists presenting their latest works on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers and physicians in the field of cardiovascular sciences, met and discussed on the most recent advances in their works and propose future strategies in overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical and mechanical cues to cells in order to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here we present some important perspectives that emerged from this meeting.

Cardiac mesenchymal stem cells (C-MSCs) are endogenous cardiac stromal cells that play a crucial role in maintaining normal cardiac function. Rab27b is a member of the small GTPase Rab family that controls membrane trafficking and the secretion of exosomes. However, its role in regulating energy metabolism in C-MSC is unclear. In this study, we analyzed mitochondrial oxidative phosphorylation by quantifying cellular oxygen consumption rate (OCR) and quantified the extracellular acidification rate (ECAR) in C-MSC with/without Rab27b knockdown. Knockdown of Rab27b increased glycolysis, but significantly reduced mitochondrial oxidative phosphorylation (OXPHOS) with loss of mitochondrial membrane potential in C-MSC. Furthermore, knockdown of Rab27b reduced H3k4me3 expression in C-MSC and selectively decreased the expression of the essential genes involved in β-oxidation, tricarboxylic acid cycle (TCA), and electron transport chain (ETC). Taken together, our findings highlight a novel role for Rab27b in maintaining fatty acid oxidation in C-MSCs.

Cardiac progenitor cells (CPCs) being multipotent offer a promising source for cardiac repair due to their ability to proliferate and multiply into cardiac lineage cells. Here, we explored a novel strategy for human CPCs generation from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule, isoxazole (ISX-9) and their ability to grow in the scar tissue for functional improvement in the infarcted myocardium. CPCs were induced from hiPSCs with ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. The CPC survival and differentiation in the infarcted hearts were determined by in vivo transplantation in immunodeficient mice following left anterior descending artery ligation and their effects were determined on fibrosis and functional improvement. ISX-9 simultaneously induced expression of cardiac transcription factors, NK2 homeobox 5, islet-1, GATA binding protein 4, myocyte enhancer factor-2 in hiPSCs within 3 days of treatment and successfully differentiated into three cardiac lineages in vitro. Messenger RNA and microRNA-sequencing results showed that ISX-9 targeted multiple cardiac differentiation, proliferation signaling pathways and upregulated myogenesis and cardiac hypertrophy related-microRNA. ISX-9 activated multiple pathways including transforming growth factor β induced epithelial-mesenchymal transition signaling, canonical, and non-canonical Wnt signaling at different stages of cardiac differentiation. CPCs transplantation promoted myoangiogenesis, attenuated fibrosis, and led to functional improvement in treated mice.

Objective Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. Methods We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). Results Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. Conclusions These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.

Background. Ischemic heart disease (IHD) is the major cause of death in patients with cardiovascular disease. Cardiac remodeling is a common pathological change following myocardial infarction (MI), and cardiomyocyte apoptosis plays a key role in this change. Transcription factor recombination signal-binding protein-J (RBP-J)-mediated Notch signaling pathway has been implicated in several inherited cardiovascular diseases, including aortic valve diseases, but whether the RBP-J-mediated Notch signaling pathway plays a role in cardiomyocyte apoptosis after MI is unclear. Method. We crossed RBP-Jfl/fl mice and Myh6-Cre/Esr1 transgenic mice to delete RBP-J in vivo and to partly inhibit the canonical Notch signaling pathway. MI was induced in mice by permanent ligation of the left anterior descending coronary artery followed by the knockout of RBP-J. Cardiac function and morphology were assessed by echocardiography and histological analysis 4 weeks after infarction. In addition, the expression and regulation of apoptosis-related molecules were examined by real time PCR and western blot. Results. RBP-J knockout decreased the survival rate and deteriorated post-MI remodeling and function in mice, and this effect was associated with increased cardiomyocyte apoptosis. The potential mechanisms might be related to the downregulated expression of bcl-2, upregulated expression of bax, and cleaved-caspase 3 to exacerbate cardiomyocyte apoptosis. Conclusion. These findings show that the RBP-J-mediated Notch signaling pathway in cardiomyocytes limits ventricular remodeling and improves cardiac function after MI. The RBP-J-mediated Notch signaling pathway has a protective role in cardiomyocyte apoptosis following cardiac injury.

Research into the biology of extracellular vesicles (EVs), including exosomes and microvesicles, has expanded significantly with advances in EV isolation techniques, a better understanding of the surface markers that characterize exosomes and microvesicles, and greater information derived from –omics approaches on the proteins, lipids, mRNAs, and microRNAs (miRNAs) transported by EVs. We have recently discovered a role for exosome-derived miRNAs in age-related bone loss and osteoarthritis, two conditions that impose a significant public health burden on the aging global population. Previous work has also revealed multiple roles for EVs and their miRNAs in muscle regeneration and congenital myopathies. Thus, EVs appear to be involved in a number of degenerative conditions that impact the musculoskeletal system, indicating that the musculoskeletal system is an excellent model for investigating the role of EVs in tissue maintenance and repair. This review highlights the role of EVs in bone, skeletal muscle, and joint health, including both normal tissue metabolism as well as tissue injury repair and regeneration. A consistent theme that emerges from study of musculoskeletal EVs is that various miRNAs appear to mediate a number of key pathological processes. These findings point to a potential therapeutic opportunity to target EV-derived miRNAs as a strategy for improving musculoskeletal function.

Recent advancements in genome-wide analyses and RNA-sequencing technologies led to the discovery of small noncoding RNAs, such as microRNAs (miRs), as well as both linear long noncoding RNAs (lncRNAs) and circular long noncoding RNAs (circRNAs). The importance of miRs and lncRNAs in the treatment, prognosis and diagnosis of cardiovascular diseases (CVDs) has been extensively reported. We also previously reviewed their implications in therapies and as biomarkers for CVDs. More recently, circRNAs have also emerged as important regulators in CVDs. CircRNAs are circular genome products that are generated by back splicing of specific regions of pre-messenger RNAs (pre-mRNAs). Growing interest in circRNAs led to the discovery of a wide array of their pathophysiological functions. CircRNAs have been shown to be key regulators of CVDs such as myocardial infarction, atherosclerosis, cardiomyopathy and cardiac fibrosis. Accordingly, circRNAs have been recently proposed as potential therapeutic targets and biomarkers for CVDs. In this review, we summarize the current state of the literature on circRNAs, starting with their biogenesis and global mechanisms of actions. We then provide a synopsis of their involvement in various CVDs. Lastly, we emphasize the great potential of circRNAs as biomarkers for the early detection of CVDs, and discuss several patents and recent papers that highlight the utilization of circRNAs as promising biomarkers.

Suxiao Jiuxin pill (SJP) is a traditional Chinese medicine for the treatment of acute coronary syndrome in China, which contains two principal components, tetramethylpyrazine (TMP) and borneol (BOR). Thus far, however, the molecular mechanisms underlying the beneficial effects of SJP on the cardiac microenvironment are unknown. Cardiac mesenchymal stem cells (C-MSCs) communicate with cardiomyocytes (CMs) through the release of microvesicles (exosomes) to restore cardiac homeostasis and elicit repair, in part through epigenetic regulatory mechanisms. In this study, we examined whether SJP treatment altered C-MSC-derived exosomes (SJP-Exos) to cause epigenetic chromatic remodeling in recipient CMs. C-MSC isolated from mouse hearts were pretreated with SJP (SJP-Exos), TMP (TMP-Exos) or BOR (BOR-Exos). Then, HL-1 cells, a mouse cardiomyocyte line, were treated with exosomes from control C-MSCs (Ctrl-Exos), SJP-Exos, TMP-Exos or BOR-Exos. Treatment with SJP-Exos significantly increased the protein levels of histone 3 lysine 27 trimethylation (H3K27me3), a key epigenetic chromatin marker for cardiac transcriptional suppression, in the HL-1 cells. To further explore the mechanisms of SJP-Exo-mediated H3K27me3 upregulation, we assessed the mRNA expression levels of key histone methylases (EZH1, EZH2 and EED) and demethylases (JMJD3 and UTX) in the exosome-treated HL-1 cells. Treatment with SJP-Exo selectively suppressed UTX expression in the recipient HL-1 cells. Furthermore, PCNA, an endogenous marker of cell replication, was significantly higher in SJP-Exo-treated HL-1 cells than in Ctrl-Exo-treated HL-1 cells. These results show that SJP-Exos increase cardiomyocyte proliferation and demonstrate that SJP can modulate C-MSC-derived exosomes to cause epigenetic chromatin remodeling in recipient cardiomyocytes; consequently, SJP-Exos might be used to promote cardiomyocyte proliferation.

Stem cell transplantation is one of most valuable methods in the treatment of myocardial infarction, and adipose-derived stem cells (ASCs) are becoming a hot topic in medical research. Previous studies have shown that ASCs can be differentiated into cardiomyocyte-like cells, but the efficiency and survival rates are low. We investigated the role and mechanism of microRNA-1 (miR-1) in the differentiation of ASCs into cardiomyocyte-like cells. ASCs and cardiomyocytes were isolated from neonatal rats. We constructed lentivirus for overexpressing miR-1 and used DAPT, an antagonist of the Notch1 pathway, for in vitro analyses. We performed cocultures with ASCs and cardiomyocytes. The differentiation efficiency of ASCs was detected by cell-specific surface antigens. Our results showed that miR-1 can promote the expression of Notch1 and reduce the expression of Hes1, a Notch pathway factor, and overexpression of miR-1 can promote the differentiation of ASCs into cardiomyocyte-like cells, which may occur by regulating Notch1 and Hes1.

Background Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. MiR-125b-5p is downregulated in patients with end-stage dilated and ischemic cardiomyopathy, and has been proposed as a biomarker of heart failure. We previously reported that the β-blocker carvedilol promotes cardioprotection via β-arrestin-biased agonism of β1-adrenergic receptor while stimulating miR-125b-5p processing in the mouse heart. We hypothesize that β1-adrenergic receptor/β-arrestin1-responsive miR-125b-5p confers the improvement of cardiac function and structure after acute myocardial infarction. Methods and results Using cultured cardiomyocyte (CM) and in vivo approaches, we show that miR-125b-5p is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125b-5p exhibit increased susceptibility to stress-induced apoptosis, while CMs overexpressing miR-125b-5p have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b-5p in the mouse heart causes abnormalities in cardiac structure and function after acute myocardial infarction. Mechanistically, the improvement of cardiac function and structure elicited by miR-125b-5p is in part attributed to repression of the pro-apoptotic genes Bak1 and Klf13 in CMs. Conclusions In conclusion, these findings reveal a pivotal role for miR-125b-5p in regulating CM survival during acute myocardial infarction.

Tumorigenesis and attendant safety risks are significant concerns of induced pluripotent stem cell (iPSC)-based therapies. Thus, it is crucial to evaluate iPSC proliferation, differentiation, and tumor formation after transplantation. Several approaches have been employed for tracking the donor cells, including fluorescent protein and luciferase, but both have limitations. Here, we introduce a protocol using iRFP genetic labeling technology to track tumor formation of iPSCs in skeletal muscle after CRISPR/Cas9 gene editing.

Duchenne muscular dystrophy (DMD) is a progressive disease characterized by skeletal muscle atrophy, respiratory failure, and cardiomyopathy. Our previous studies have shown that transplantation with allogeneic myogenic progenitor-derived exosomes (MPC-Exo) can improve cardiac function in X-linked muscular dystrophy (Mdx) mice. In the present study we explored the molecular mechanisms underlying this beneficial effect. We quantified gene expression in the hearts of two strains of Mdx mice (D2.B10-Dmd(Mdx)/J and Utrn(tm1Ked)-Dmd(Mdx)/J). Two days after MPC-Exo or control treatment, we performed unbiased next-generation RNA-sequencing to identify differentially expressed genes (DEGs) in treated Mdx hearts. Venn diagrams show a set of 780 genes that were ≥2-fold upregulated, and a set of 878 genes that were ≥2-fold downregulated, in both Mdx strains following MPC-Exo treatment as compared with control. Gene ontology (GO) and protein-protein interaction (PPI) network analysis showed that these DEGs were involved in a variety of physiological processes and pathways with a complex connection. qRT-PCR was performed to verify the upregulated ATP2B4 and Bcl-2 expression, and downregulated IL-6, MAPK8 and Wnt5a expression in MPC-Exo-treated Mdx hearts. Western blot analysis verified the increased level of Bcl-2 and decreased level of IL-6 protein in MPC-Exo-treated Mdx hearts compared with control treatment, suggesting that anti-apoptotic and anti-inflammatory effects might be responsible for heart function improvement by MPC-Exo. Based on these findings, we believed that these DEGs might be therapeutic targets that can be explored to develop new strategies for treating DMD.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmd(mdx) mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Ischemic cardiac preconditioning protects the heart during myocardial infarction by activating critical cardioprotective genes such as eNOS, SOD, and HO-1. Clinical trials only show marginal effects of conventional preconditioning strategies, however, in part due to transient activation of cardioprotective genes. Recent studies have shown that N6-methyladenosine (m6A) mRNA methylation is the most abundant RNA modification in eukaryotes, and governs mRNA stability and, in turn, the level of protein expression. We hypothesize that regulation of m6A mRNA methylation levels of cardioprotective mRNAs will result in stable expression of the cardioprotective proteins, rendering ischemic cardiac preconditioning more robust and reducing infarct size. To test this hypothesis, we will test the effects of introducing m6A methylases/demethylases into ischemic preconditioned/post conditioned hearts and subjecting them to myocardial infarction. We will assess the half-life of key cardioprotective mRNAs (e.g., eNOS, SOD, and HO-1) and cardiac apoptosis to determine which m6A methylases/demethylases have a synergistic effect on cardiac preconditioning.

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMD(MDX) mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMD(MDX) mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMD(MDX) mutant mice.

G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation.

The nonselective β-adrenergic receptor antagonist (β-blocker) carvedilol has been shown to protect against myocardial injury, but the detailed underlying mechanisms are unclear. We recently reported that carvedilol stimulates the processing of microRNA (miR)-199a-3p and miR-214 in the heart via β-arrestin1-biased β1-adrenergic receptor (β1AR) cardioprotective signaling. Here, we investigate whether these β-arrestin1/β1AR-responsive miRs mediate the beneficial effects of carvedilol against simulated ischemia/reperfusion (sI/R). Using cultured cardiomyocyte cell lines and primary cardiomyocytes, we demonstrate that carvedilol upregulates miR-199a-3p and miR-214 in both ventricular and atrial cardiomyocytes subjected to sI/R. Overexpression of the two miRs in cardiomyocytes mimics the effects of carvedilol to activate p-AKT survival signaling and the expression of a downstream pluripotency marker Sox2 in response to sI/R. Moreover, carvedilol-mediated p-AKT activation is abolished by knockdown of either miR-199a-3p or miR-214. Along with previous studies to directly link the cardioprotective actions of carvedilol to upregulation of p-AKT/stem cell markers, our findings suggest that the protective roles of carvedilol during ischemic injury are in part attributed to activation of these two protective miRs. Loss of function of miR-199a-3p and miR-214 also increases cardiomyocyte apoptosis after sI/R. Mechanistically, we demonstrate that miR-199a-3p and miR-214 repress the predictive or known apoptotic target genes ddit4 and ing4, respectively, in cardiomyocytes. These findings suggest pivotal roles for miR-199a-3p and miR-214 as regulators of cardiomyocyte survival and contributors to the functional benefits of carvedilol therapy.

Myocardial infarction (MI) is the primary cause of cardiovascular mortality, and therapeutic strategies to prevent or mitigate the consequences of MI are a high priority. Cardiac progenitor cells (CPCs) have been used to treat cardiac injury post-MI, and despite poor engraftment, they have been shown to inhibit apoptosis and to promote angiogenesis through poorly understood paracrine effects. We previously reported that the direct injection of exosomes derived from CPCs (CPCexo) into mouse hearts provides protection against apoptosis in a model of acute ischemia/reperfusion injury. Moreover, we and others have reported that reactive oxygen species (ROS) derived from NADPH oxidase (NOX) can enhance angiogenesis in endothelial cells (ECs). Here we examined whether bioengineered CPCexo transfected with a pro-angiogenic miR-322 (CPCexo-322) can improve therapeutic efficacy in a mouse model of MI as compared to CPCexo. Systemic administration of CPCexo-322 in mice after ischemic injury provided greater protection post-MI than control CPCexo, in part, through enhanced angiogenesis in the border zones of infarcted hearts. Mechanistically, the treatment of cultured human ECs with CPCexo-322 resulted in a greater angiogenic response, as determined by increased EC migration and capillary tube formation via increased Nox2-derived ROS. Our study reveals that the engineering of CPCexo via microRNA (miR) programing can enhance angiogenesis, and this may be an effective therapeutic strategy for the treatment of ischemic cardiovascular diseases.

Stem cell-based therapies hold great promise as alternative therapeutic strategies for various chronic diseases, including ischemic cardiomyopathy. Tracking the engraftment of transplanted stem cells is critical to the assessment of donor cell survival in the host environment. Fluorescent proteins, such as green fluorescent protein (GFP), have been widely used to track the fate of donor cells; however, GFP labeling has limitations with regard to noninvasively measuring cell engraftment in vivo. Our research indicates that near-infrared fluorescent protein (iRFP) labeling offers advantages for noninvasive in vivo imaging and histological assessment. Here, we present a protocol for using the lentiviral vector-mediated iRFP vector system to label and track donor stem cells in ischemic mouse hearts.

Progressive cardiomyocyte loss in Duchenne muscular dystrophy (DMD) leads to cardiac fibrosis, cardiomyopathy, and eventually heart failure. In the present study, we observed that myogenic progenitor cells (MPC) carry mRNA for the dystrophin gene. We tested whether cardiac function can be improved in DMD by allograft transplantation of MPC-derived exosomes (MPC-Exo) into the heart to restore dystrophin protein expression. Exo from C2C12 cells (an MPC cell line) or vehicle were delivered locally into the hearts of MDX mice. After 2 days of treatment, we observed that MPC-Exo restored dystrophin expression in the hearts of MDX mice, which correlated with improved myocardial function in dystrophin-deficient MDX mouse hearts. In conclusion, this study demonstrated that allogeneic WT-MPC-Exo transplantation transiently restored dystrophin gene expression and improved cardiac function in MDX mice, suggesting that allogenic exosomal delivery may serve as an alternative treatment for cardiomyopathy of DMD.

The death rate from cancer in the USA has declined steadily over the past 2 decades (1). Improved cancer survival associated with anticancer agents has led to a greater recognition of the late effects of cardiotoxicity including left ventricular dysfunction, heart failure and coronary heart disease.

Our previous study demonstrated the beneficial effects of exosomes secreted by cardiac mesenchymal stem cells (C-MSC-Exo) in protecting acute ischemic myocardium from reperfusion injury. Here, we investigated the effect of exosomes from C-MSC on angiogenesis in ischemic myocardium. We intramyocardially injected C-MSC-Exo or PBS into the infarct border zone after induction of acute mouse myocardial infarction (MI). We observed that hearts treated with C-MSC-Exo exhibit improved cardiac function compared to control hearts treated with PBS at one month after MI. Capillary density and Ki67-postive cells were significantly higher following treatment with C-MSC-Exo as compared with PBS. Moreover, C-MSC-Exo treatment increased cardiomyocyte proliferation in infarcted hearts. In conclusion, intramyocardial delivery of C-MSC-Exo after myocardial infarction enhances cardiac angiogenesis, promotes cardiomyocyte proliferation, and preserves heart function. C-MSC-Exo constitute a novel form of cell-free therapy for cardiac repair.

The aryl hydrocarbon receptor (AhR) is a DNA binding protein that acts as a nuclear receptor mediating xenobiotic metabolism and environmental responses. Owing to the evolutionary conservation of this gene and its widespread expression in the immune and circulatory systems, AhR has for many years been almost exclusively studied by the pharmacological/toxicological field for its role in contaminant toxicity. More recently, the functions of AhR in environmental adaption have been examined in the context of the occurrence, development, and therapy of cardiovascular diseases. Increasing evidence suggests that AhR is involved in maintaining homeostasis or in triggering pathogenesis by modulating the biological responses of critical cell types in the cardiovascular system. Here, we describe the structure, distribution, and ligands of AhR and the AhR signaling pathway and review the impact of AhR on cardiovascular physiology. We also discuss the potential contribution of AhR as a new potential factor in the targeted treatment of cardiovascular diseases.

We demonstrated the effects of exosomes secreted by cardiac mesenchymal stem cells (C-MSC-Exo) in protecting acute ischemic myocardium from reperfusion injury. To investigate the effect of exosomes from C-MSC on angiogenesis, we injected C-MSC-Exo or PBS intramuscularly into ischemic hind limb. Blood perfusion of limb was evaluated by laser Doppler Imaging. We observed that ischemic limb treated with C-MSC-Exo exhibits improved blood perfusion compared to ischemic limb treated with PBS at 2 weeks and 1 month after induction of limb ischemia. To explore the potential mechanisms underlying C-MSC-Exo’s angiogenetic effect, we performed microRNA array analysis and identify mmu-miR-7116-5p as the most abundant enriched miRNA detected in C-MSC-Exo. Bioinformatics’ analysis shows that miR-7116-5p negatively regulates protein polyubiquitination. In conclusion, our study demonstrated that intramuscular delivery of C-MSC-Exo after limb ischemia improves blood perfusion, and we identified the most abundant miRNAs that are preferentially enriched in C-MSC-Exo.

Despite substantial advances in the development of medical and interventional strategies in ischemic and non-ischemic heart diseases, cardiovascular diseases (CVDs) remain the leading cause of mortality and morbidity worldwide. Stem cell therapy for heart disease has gained traction over the past two decades and is an emerging option for the treatment of myocardial dysfunction. In this review, we summarize the current literature on different types of stem cells and their potential usage in ischemic and non-ischemic heart diseases. We emphasize the clinical utility of stem cells to improve myocardial structural and function, promote microvascular angiogenesis, and diminish scar size and major adverse cardiovascular events. We also discuss the therapeutic potential of microvesicles, such as exosomes, in the treatment of CVDs, which may open novel avenues for further clinical studies.

Rationale MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate target genes. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed by Drosha into premature miRs (pre-miRs) and further cleaved by Dicer into mature miRs. Initially discovered to desensitize β-adrenergic receptor (βAR) signaling, β-arrestins are now well-appreciated to modulate multiple pathways independent of G protein signaling, a concept known as biased signaling. Using the β-arrestin-biased βAR ligand carvedilol, we previously showed that β-arrestin1 (not β-arrestin2)-biased β1AR (not β2AR) cardioprotective signaling stimulates Drosha-mediated processing of six miRs by forming a multi-protein nuclear complex, which includes β-arrestin1, the Drosha microprocessor complex and a single-stranded RNA binding protein hnRNPA1. Objective Here, we investigate whether β-arrestin-mediated βAR signaling induced by carvedilol could regulate Dicer-mediated miR maturation in the cytoplasm and whether this novel mechanism promotes cardioprotective signaling. Methods and results In mouse hearts, carvedilol indeed upregulates three mature miRs, but not their pre-miRs and pri-miRs, in a β-arrestin 1- or 2-dependent manner. Interestingly, carvedilol-mediated activation of miR-466g or miR-532-5p, and miR-674 is dependent on β2ARs and β1ARs, respectively. Mechanistically, β-arrestin 1 or 2 regulates maturation of three newly identified βAR/β-arrestin-responsive miRs (β-miRs) by associating with the Dicer maturation RNase III enzyme on three pre-miRs of β-miRs. Myocardial cell approaches uncover that despite their distinct roles in different cell types, β-miRs act as gatekeepers of cardiac cell functions by repressing deleterious targets. Conclusions Our findings indicate a novel role for βAR-mediated β-arrestin signaling activated by carvedilol in Dicer-mediated miR maturation, which may be linked to its protective mechanisms.

Rationale: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. Objective: The objective of this study was to investigate the effect of growth hormone–releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. Methods and Results: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor–selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A–selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. Conclusions: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.

Objective:To verify the influence of 3 potential factors on the accuracy of non-invasive central BP measurement, namely the calibration peripheral BP (brachial or radial), calibration methods (systolic/diastolic BP or mean/diastolic BP), and devices.Design and method:50 participants (aged 62.4 ± 8.9

Cardiovascular disease is the leading cause of death in the United States, accounting for nearly one in every seven deaths. Over the last decade, various targeted therapeutics have been introduced, but there has been no corresponding improvement in patient survival. Since the mortality rate of cardiovascular disease has not been significantly decreased, efforts have been made to understand the link between heart disease and novel therapeutic targets such as non-coding RNAs. Among multiple non-coding RNAs, long non-coding RNA (lncRNA) has emerged as a novel therapeutic in cardiovascular medicine. LncRNAs are endogenous RNAs that contain over 200 nucleotides and regulate gene expression. Recent studies suggest critical roles of lncRNAs in modulating the initiation and progression of cardiovascular diseases. For example, aberrant lncRNA expression has been associated with the pathogenesis of ischemic heart failure. In this article, we present a synopsis of recent discoveries that link the roles and molecular interactions of lncRNAs to cardiovascular diseases. Moreover, we describe the prevalence of circulating lncRNAs and assess their potential utilities as biomarkers for diagnosis and prognosis of heart disease.

Obesity is associated with insulin resistance and type 2 diabetes; molecular mechanisms that promote energy expenditure can be utilized for effective therapy. Src-associated in mitosis of 68 kDa (Sam68) is potentially significant, because knockout (KO) of Sam68 leads to markedly reduced adiposity. In the present study, we sought to determine the mechanism by which Sam68 regulates adiposity and energy homeostasis. We first found that Sam68 KO mice have a significantly reduced body weight as compared to controls, and the difference is explained entirely by decreased adiposity. Interestingly, these effects were not mediated by a difference in food intake; rather, they were associated with enhanced physical activity. When they were fed a high-fat diet, Sam68 KO mice gained much less body weight and fat mass than their WT littermates did, and they displayed an improved glucose and insulin tolerance. In Sam68 KO mice, the brown adipose tissue (BAT), inguinal, and epididymal depots were smaller, and their adipocytes were less hypertrophied as compared to their WT littermates. The BAT of Sam68 KO mice exhibited reduced lipid stores and expressed higher levels of Ucp1 and key thermogenic and fatty acid oxidation genes. Similarly, depots of inguinal and epididymal white adipose tissue (WAT) in Sam68 KO mice appeared browner, their multilocular Ucp1-positive cells were much more abundant, and the expression of Ucp1, Cidea, Prdm16, and Ppargc1a genes was greater as compared to WT controls, which suggests that the loss of Sam68 also promotes WAT browning. Furthermore, in all of the fat depots of the Sam68 KO mice, the expression of M2 macrophage markers was up-regulated, and that of M1 markers was down-regulated. Thus, Sam68 plays a crucial role in controlling thermogenesis and may be targeted to combat obesity and associated disorders.

Although stem cell therapy is promising for repairing damaged cardiac tissue and improving heart function, there are safety concerns, especially regarding the risk of arrhythmias, which can be life threatening. To address this issue, we propose to develop a novel screening system to evaluate arrhythmic risk associated with stem cell therapy using a high-throughput multielectrode array system that can measure conduction velocity and action potential duration in cardiomyocytes co-cultured with different types of stem cells, such as mesenchymal stem cells, skeletal myoblasts, and resident cardiac stem cells. We will assess the arrhythmic potential of each of these types of stem cells under normoxic and hypoxic conditions, with/without application of oxidative stress or catecholamines. We hypothesize that these methods will prove to be an effective way to screen for arrhythmic risk of cardiac stem cell therapy. Ultimately, our approach can potentially be personalized to develop a robust screening protocol in order to identify which stem cell type carries the least amount of risk for arrhythmia. This system will have great clinical benefit to improve the risk/benefit ratio of human stem cell therapy for heart disease.