Inhibition of Hmbox1 Promotes Cardiomyocyte Survival and Glucose Metabolism Through Gck Activation in Ischemia/Reperfusion Injury.

BACKGROUND: Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. METHODS: We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. RESULTS: We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. CONCLUSIONS: Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury.

Exercise-Induced miR-210 Promotes Cardiomyocyte Proliferation and Survival and Mediates Exercise-Induced Cardiac Protection against Ischemia/Reperfusion Injury.

Exercise can stimulate physiological cardiac growth and provide cardioprotection effect in ischemia/reperfusion (I/R) injury. MiR-210 is regulated in the adaptation process induced by exercise; however, its impact on exercise-induced physiological cardiac growth and its contribution to exercise-driven cardioprotection remain unclear. We investigated the role and mechanism of miR-210 in exercise-induced physiological cardiac growth and explored whether miR-210 contributes to exercise-induced protection in alleviating I/R injury. Here, we first observed that regular swimming exercise can markedly increase miR-210 levels in the heart and blood samples of rats and mice. Circulating miR-210 levels were also elevated after a programmed cardiac rehabilitation in patients that were diagnosed of coronary heart diseases. In 8-week swimming model in wild-type (WT) and miR-210 knockout (KO) rats, we demonstrated that miR-210 was not integral for exercise-induced cardiac hypertrophy but it did influence cardiomyocyte proliferative activity. In neonatal rat cardiomyocytes, miR-210 promoted cell proliferation and suppressed apoptosis while not altering cell size. Additionally, miR-210 promoted cardiomyocyte proliferation and survival in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and AC16 cell line, indicating its functional roles in human cardiomyocytes. We further identified miR-210 target genes, cyclin-dependent kinase 10 (CDK10) and ephrin-A3 (EFNA3), that regulate cardiomyocyte proliferation and apoptosis. Finally, miR-210 KO and WT rats were subjected to swimming exercise followed by I/R injury. We demonstrated that miR-210 crucially contributed to exercise-driven cardioprotection against I/R injury. In summary, this study elucidates the role of miR-210, an exercise-responsive miRNA, in promoting the proliferative activity of cardiomyocytes during physiological cardiac growth. Furthermore, miR-210 plays an essential role in mediating the protective effects of exercise against cardiac I/R injury. Our findings suggest exercise as a potent nonpharmaceutical intervention for inducing miR-210, which can alleviate I/R injury and promote cardioprotection.

Exercise Inhibits Doxorubicin-Induced Cardiotoxicity via Regulating B Cells.

BACKGROUND: Doxorubicin is an effective chemotherapeutic agent, but its use is limited by acute and chronic cardiotoxicity. Exercise training has been shown to protect against doxorubicin-induced cardiotoxicity, but the involvement of immune cells remains unclear. This study aimed to investigate the role of exercise-derived B cells in protecting against doxorubicin-induced cardiotoxicity and to further determine whether B cell activation and antibody secretion play a role in this protection. METHODS: Mice that were administered with doxorubicin (5 mg/kg per week, 20 mg/kg cumulative dose) received treadmill running exercise. The adoptive transfer of exercise-derived splenic B cells to µMT-/- (B cell-deficient) mice was performed to elucidate the mechanism of B cell regulation that mediated the effect of exercise. RESULTS: Doxorubicin-administered mice that had undergone exercise training showed improved cardiac function, and low levels of cardiac apoptosis, atrophy, and fibrosis, and had reduced cardiac antibody deposition and proinflammatory responses. Similarly, B cell pharmacological and genetic depletion alleviated doxorubicin-induced cardiotoxicity, which phenocopied the protection of exercise. In vitro performed coculture experiments confirmed that exercise-derived B cells reduced cardiomyocyte apoptosis and fibroblast activation compared with control B cells. Importantly, the protective effect of exercise on B cells was confirmed by the adoptive transfer of splenic B cells from exercised donor mice to µMT-/- recipient mice. However, blockage of Fc gamma receptor IIB function using B cell transplants from exercised Fc gamma receptor IIB-/- mice abolished the protection of exercise-derived B cells against doxorubicin-induced cardiotoxicity. Mechanistically, we found that Fc gamma receptor IIB, an important B cell inhibitory receptor, responded to exercise and increased B cell activation threshold, which participated in exercise-induced protection against doxorubicin-induced cardiotoxicity. CONCLUSIONS: Our results demonstrate that exercise training protects against doxorubicin-induced cardiotoxicity by upregulating Fc gamma receptor IIB expression in B cells, which plays an important anti-inflammatory role and participates in the protective effect of exercise against doxorubicin-induced cardiotoxicity.

Danlou Tablet Protects Against Myocardial Infarction Through Promoting eNOS-Dependent Endothelial Protection and Angiogenesis.

Myocardial infarction (MI) is one of the leading causes of death worldwide. Danlou tablet (Dan) is an effective traditional Chinese medicine for cardiac protection, although the underlying mechanism was not fully understood. In this study, we used a murine MI model and demonstrated that Dan administration effectively attenuated myocardial apoptosis, cardiac remodeling, and heart failure post MI. Dan increased CD31-positive capillaries in MI hearts, and reduced the apoptosis and oxidative stress in human umbilical vein endothelial cells after oxygen-glucose deprivation stress, simultaneously with the activated HIF-1α/VEGFA/eNOS signaling. Moreover, inhibition of eNOS by L-NAME attenuated Dan-induced protection against MI, and abolished its effect in promoting angiogenesis and reducing endothelial apoptosis and oxidative stress. Collectively, Dan is beneficial to promote eNOS-dependent endothelial protection and angiogenesis thus protecting against MI. A deep understanding of Dan-induced protection might help promote clinical usage of Dan in MI treatment.

Innovative Animal Models Of Cardiac Remodeling: Development And Evaluation.

ARTICLES DISCUSSED: Liao, R. et al. Generation and characterization of right ventricular myocardial infarction induced by permanent ligation of the right coronary artery in mice. Journal of Visualized Experiments. (180), e63508 (2022). Li, X. et al. Establishment and evaluation of a porcine vein graft disease model. Journal of Visualized Experiments. (185), e63896 (2022). Lu, A. et al. Viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk. Journal of Visualized Experiments. (185), e64073 (2022). Wang, Y., Gao, H., Li, Y., Sun, H., Liu, L. Estimating bilateral atrial function by cardiovascular magnetic resonance feature tracking in patients with paroxysmal atrial fibrillation. Journal of Visualized Experiments. (185), e63598 (2022). Wu, J. et al. Surgically induced cardiac volume overload by aortic regurgitation in mouse. Journal of Visualized Experiments. (186), e63579 (2022). Li, X. et al. A surgical model of heart failure with preserved ejection fraction in Tibetan minipigs. Journal of Visualized Experiments. (180), e63526 (2022). Wang, M. et al. Improved renal denervation mitigated hypertension induced by angiotensin II infusion. Journal of Visualized Experiments. (183), e63719 (2022). Xia, Y. et al. Investigating the pathogenesis of MYH7 mutation Gly823Glu in familial hypertrophic cardiomyopathy using a mouse model. Journal of Visualized Experiments. (186), e63949 (2022).

Danlou Tablet Protects Against Cardiac Remodeling and Dysfunction after Myocardial Ischemia/Reperfusion Injury through Activating AKT/FoxO3a Pathway.

Myocardial ischemia/reperfusion injury (I/RI) and ventricular remodeling are the critical pathological basis of heart failure. Danlou tablet (Dan) is a kind of Chinese patent medicine used in angina pectoris treatment in China. However, it remains unclear whether and how Dan could protect against cardiac remodeling after myocardial I/RI. In this study, both preventive and therapeutic administration of Dan attenuated ventricular remodeling and cardiac dysfunction at 3 weeks after myocardial I/RI. Dan inhibited Bax/Bcl2 ratio and Caspase3 cleavage in heart tissues and also inhibited apoptosis of human AC16 cells and neonatal rat cardiomyocytes stressed by oxygen and glucose deprivation/reperfusion. Mechanistically, Dan inhibited myocardial apoptosis through phosphorylating AKT and FoxO3a, thereby inhibiting downstream BIM and PUMA expressions. Collectively, these results demonstrate that Dan treatment is effective to protect against cardiac remodeling and dysfunction after myocardial I/RI and provide theoretical basis for its cardioprotection and clinical application in treating ischemic cardiac diseases.

Platelet Membrane-Fused Circulating Extracellular Vesicles Protect the Heart from Ischemia/Reperfusion Injury.

Myocardial ischemia/reperfusion injury (I/RI) may potentiate cardiac remodeling and heart failure, while effective therapies for I/RI remain lacking. Circulating human plasma-derived extracellular vesicles (hEV) have great potential to protect against I/RI. However, the effective delivery of hEV in vivo remains a limiting factor for clinical application. The present study constructs a biomimetic delivery system of platelet membrane-fused hEV (P-hEV), utilizing the natural affinity of platelets for hEV delivery to the injured vascular and myocardial sites. The results show that platelet membrane and hEV membrane fusion can be achieved through repeated extrusion. Compared to non-modified hEV, P-hEV uptake is greatly enhanced in human umbilical vein endothelial cells (HUVECs) stressed by oxygen-glucose deprivation/reperfusion (OGD/R). Functionally, P-hEV inhibits HUVEC and neonatal rat cardiomyocyte (NRCM) apoptosis and promotes HUVECs migration and tube formation under OGD/R stress in vitro. Intravenous delivery of P-hEV more effectively targets and accumulates at injury sites in the heart. Furthermore, P-hEV significantly enhances protection against acute I/RI and attenuates cardiac remodeling at three weeks post-I/RI. In conclusion, the platelet membrane-fused hEV delivery system enhances the target delivery of EV to protect against myocardial I/RI, presenting a novel drug delivery system for ischemic heart diseases.

HIPK1 Inhibition Protects against Pathological Cardiac Hypertrophy by Inhibiting the CREB-C/EBPß Axis.

Inhibition of pathological cardiac hypertrophy is recognized as an important therapeutic strategy for heart failure, although effective targets are still lacking in clinical practice. Homeodomain interacting protein kinase 1 (HIPK1) is a conserved serine/threonine kinase that can respond to different stress signals, however, whether and how HIPK1 regulates myocardial function is not reported. Here, it is observed that HIPK1 is increased during pathological cardiac hypertrophy. Both genetic ablation and gene therapy targeting HIPK1 are protective against pathological hypertrophy and heart failure in vivo. Hypertrophic stress-induced HIPK1 is present in the nucleus of cardiomyocytes, while HIPK1 inhibition prevents phenylephrine-induced cardiomyocyte hypertrophy through inhibiting cAMP-response element binding protein (CREB) phosphorylation at Ser271 and inactivating CCAAT/enhancer-binding protein ß (C/EBPß)-mediated transcription of pathological response genes. Inhibition of HIPK1 and CREB forms a synergistic pathway in preventing pathological cardiac hypertrophy. In conclusion, HIPK1 inhibition may serve as a promising novel therapeutic strategy to attenuate pathological cardiac hypertrophy and heart failure.

Danlou tablet inhibits high-glucose-induced cardiomyocyte apoptosis via the miR-34a-SIRT1 axis.

Diabetic cardiomyopathy (DCM) is highly prevalent and increases the risk of heart failure and sudden death. Therefore, proper and effective treatments for DCM are in urgent demand. Danlou tablet (Dan) is reported to confer protective effects on several heart diseases. However, to our knowledge, whether Dan provides protection against DCM is unclear. In this study, we explored the effect of Dan on DCM with the in vitro DCM model using AC16 cardiomyocytes. We found that Dan treatment significantly reduced cardiomyocyte apoptosis and oxidative stress in high-glucose (HG)-treated cardiomyocytes, as evidenced by decreased Annexin V-FITC+ cardiomyocytes, intracellular reactive oxygen species (ROS) levels, Bax/Bcl2 ratio, and cleaved-Caspase3/Caspase3 ratio. Interestingly, Dan treatment caused a decreased level of microRNA-34a (miR-34a), which could enhance cardiomyocyte apoptosis. Furthermore, miR-34a mimic blocked Dan's effect in apoptosis prevention. Finally, we observed that the miR-34a mimic effectively decreased the level of sirtuin 1 (SIRT1), while the miR-34a inhibitor increased the level of SIRT1. And downregulation of SIRT1 effectively reversed the effect of miR-34a inhibitor on cardiomyocyte apoptosis. Taken together, our study showed that Dan prevented HG-induced cardiomyocyte apoptosis through downregulating miR-34a and upregulating SIRT1. Our study has provided experimental support for the potential use of Dan in treating DCM. Further detailed study of Dan and the underlying mechanisms may shed light on the prevention and treatment of DCM.

A modified apical resection model with high accuracy and reproducibility in neonatal mouse and rat hearts.

Neonatal mouse heart can regenerate after left ventricle (LV) apical resection (AR). Since current AR rodent method is accomplished by resecting LV apex until exposure of LV chamber, it is relatively difficult to operate reproducibly. We aimed to develop a modified AR method with high accuracy and reproducibility and to investigate whether cardiac regenerative capacity could be replicated in neonatal rats. For 15% AR of whole heart weight in 1-day-old (P1) neonatal mice, a modified 10 µL pipette tip cut to 0.48 mm in internal diameter was connected to a vacuum pump working at 0.06 ± 0.005 MPa and gently kept in touch with LV apex for nearly but no more than 12 s. LV apex was resected by a single incision adjacent to the pipette tip. The modified AR method in P1 mice achieved cardiac structural and functional recovery at 21 days post resection (dpr). Data from different operators showed smaller variation of resected LV apex and higher reproducibility using the modified AR method. Furthermore, we showed that 5% AR of whole heart weight in P1 neonatal rats using a modified 200 µL pipette tip cut to 0.63 mm in internal diameter led to complete regeneration of LV apex and full preservation of cardiac function at 42 dpr. In conclusion, the modified AR rodent model leads to accurate resection of LV apex with high homogeneity and reproducibility and it is practically convenient for the study of structural, functional, and molecular mechanisms of cardiac regeneration in both neonatal mice and rats.

A Versatile Design-Enabled Analysis of Circulating Extracellular Vesicles in Disease Diagnosis.

Circulating extracellular vesicles (EVs) are considered as potential biomarkers for treatment and diagnosis of many diseases. Most of the existing methods for the EV analysis only have a single function and thus reveal limited information carried by EVs. Herein, a phosphatidylserine-targeting peptide-facilitated design that enables the versatile analysis of circulating EVs for varying requirement is proposed. In the design, DNA probes are inserted into the EV membrane through hydrophobic interactions, and accelerate the removal of protective shielding from DNA-gated metal-organic framework, thereby releasing a large number of methylene blue molecules to amplify the electrochemical signal. Electrochemical results demonstrate equally high sensitivities toward the quantification of EVs derived from different cell sources using an indiscriminative DNA probe. More importantly, the probe can be endowed with extended function for more accurate classification of cell-specific features through the identification of specific EV biomarkers, and demonstrates the potential use in the diagnosis of cardiovascular in a principle-of-proof study for clinical application. Therefore, the method provides a versatile design for the identification of EV features, and may address the needs of clinical diagnosis in the future.

Lymphatic Regulation in Tissue Repair and Regeneration: Recent Advances and Future Perspective.

Lymphatic vasculature plays essential role in interstitial tissue uptake, immune cell transport and dietary lipid absorption. Increasing evidence has demonstrated the contribution of lymphangiogenesis to tissue repair and regeneration, which is associated with multiple factors such as improved tissue homeostasis, inflammation resolution, and immunomodulation effects. Meanwhile, lymphangiogenesis has the potential to regulate cell growth and proliferation through paracrine effects. Lymphatic vessels can also be important components of the stem cell niche and participate in regulating stem cell quiescency or activity. In perspective, the functions and mechanisms of lymphatic vessels in tissue repair and regeneration deserve further investigation. Novel strategies to stimulate lymphangiogenesis by using pharmacological, genetic, and lymphatic tissue engineering will be prospective to promote tissue repair and regeneration.

Exercise training maintains cardiovascular health: signaling pathways involved and potential therapeutics.

Exercise training has been widely recognized as a healthy lifestyle as well as an effective non-drug therapeutic strategy for cardiovascular diseases (CVD). Functional and mechanistic studies that employ animal exercise models as well as observational and interventional cohort studies with human participants, have contributed considerably in delineating the essential signaling pathways by which exercise promotes cardiovascular fitness and health. First, this review summarizes the beneficial impact of exercise on multiple aspects of cardiovascular health. We then discuss in detail the signaling pathways mediating exercise's benefits for cardiovascular health. The exercise-regulated signaling cascades have been shown to confer myocardial protection and drive systemic adaptations. The signaling molecules that are necessary for exercise-induced physiological cardiac hypertrophy have the potential to attenuate myocardial injury and reverse cardiac remodeling. Exercise-regulated noncoding RNAs and their associated signaling pathways are also discussed in detail for their roles and mechanisms in exercise-induced cardioprotective effects. Moreover, we address the exercise-mediated signaling pathways and molecules that can serve as potential therapeutic targets ranging from pharmacological approaches to gene therapies in CVD. We also discuss multiple factors that influence exercise's effect and highlight the importance and need for further investigations regarding the exercise-regulated molecules as therapeutic targets and biomarkers for CVD as well as the cross talk between the heart and other tissues or organs during exercise. We conclude that a deep understanding of the signaling pathways involved in exercise's benefits for cardiovascular health will undoubtedly contribute to the identification and development of novel therapeutic targets and strategies for CVD.

PPARγ Mediates the Cardioprotective Roles of Danlou Tablet After Acute Myocardial Ischemia-Reperfusion Injury.

Ischemic heart disease is one of the biggest threats to human life in the world. Reperfusion therapy is an effective strategy to reduce infarct size and ischemic injury. However, reperfusion process may cause secondary myocardial injury which is defined as ischemia-reperfusion injury (IRI). Exploring potential therapeutic strategy to attenuate IRI is extremely important. Danlou tablet (Dan), a Chinese herbal compound consisting of ten herbs, has been identified to be protective for the heart. However, the mechanism of Dan-induced cardioprotection after acute reperfusion was unelucidated. In this study, to investigate the role and mechanism of Dan in myocardial IRI, we performed acute IRI modeling in mice and oxygen-glucose deprivation-reperfusion (OGD/R)-induced apoptosis in primary neonatal rat cardiomyocytes (NRCMs). We found that Dan had protective effect against acute IRI in mice, as evidenced by reduced infarct size, TUNEL-positive cardiomyocytes (CMs), and Bax/Bcl2 ratio and cleaved-caspase 3/caspase 3 ratio in vivo. Meanwhile, Dan inhibited OGD/R-induced apoptosis of NRCMs in vitro. Mechanistically, Dan could activate proliferator-activated receptor gamma (PPARγ) in both IRI hearts and OGD/R-stressed NRCMs, while inhibition of PPARγ attenuated the protective effect of Dan against IRI in vivo and OGD/R-induced CM apoptosis in vitro. These data reveal that Dan attenuates acute myocardial IRI and CM apoptosis through activating PPARγ. Our findings may extend the knowledge of Chinese medicine and provide potential strategy for the precise treatment of ischemic heart diseases.

Downregulation of circ-ZNF609 Promotes Heart Repair by Modulating RNA N6-Methyladenosine-Modified Yap Expression.

Circular RNAs take crucial roles in several pathophysiological processes. The regulatory role and its underlying mechanisms of circ-ZNF609 in the heart remains largely unknown. Here, we report that circ-ZNF609 is upregulated during myocardial ischemia/reperfusion (I/R) remodeling. Knockdown of circ-ZNF609 protects against acute I/R injury and attenuates left ventricle dysfunction after I/R remodeling in vivo. In vitro, circ-ZNF609 regulates cardiomyocyte survival and proliferation via modulating the crosstalk between Hippo-YAP and Akt signaling. Mechanically, N6-methyladenosine-modification is involved in the regulatory role of circ-ZNF609 on YAP. An in-depth study indicates that knockdown of circ-ZNF609 decreases the expression of YTHDF3 and further fine-tuned the accessibility of Yap mRNA to YTHDF1 and YTHDF2 to regulate YAP expression. circ-ZNF609 knockdown represents a promising therapeutic strategy to combat the pathological process of myocardial I/R injury.