Costunolide alleviates hyperglycaemia-induced diabetic cardiomyopathy via inhibiting inflammatory responses and oxidative stress

Hyperglycaemia-induced myocardial injury promotes the induction of heart failure in diabetic patients. Impaired antioxidant capability and sustained chronic inflammation play a vital role in the progression of diabetic cardiomyopathy (DCM). Costunolide (Cos), a natural compound with anti-inflammatory and antioxidant properties, has exhibited therapeutic effects in various inflammatory diseases. However, the role of Cos in diabetes-induced myocardial injury remains poorly understood. In this study, we investigated the effect of Cos on DCM and explored the potential mechanisms. C57BL/6 mice were administered intraperitoneal streptozotocin for DCM induction. Cos-mediated anti-inflammatory and antioxidation activities were examined in heart tissues of diabetic mice and high glucose (HG)-stimulated cardiomyocytes. Cos markedly inhibited HG-induced fibrotic responses in diabetic mice and H9c2 cells, respectively. The cardioprotective effects of Cos could be correlated to the reduced expression of inflammatory cytokines and decreased oxidative stress. Further investigations demonstrated Cos reversed diabetes-induced nuclear factor-κB (NF-κB) activation and alleviated impaired antioxidant defence system, principally via activation of nuclear factor-erythroid 2 p45-related factor-2 (Nrf-2). Cos alleviated cardiac damage and improved cardiac function in diabetic mice by inhibiting NF-κB-mediated inflammatory responses and activating the Nrf-2-mediated antioxidant effects. Therefore, Cos could be a potential candidate for the treatment of DCM.     

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.     

Resveratrol inhibits ferroptosis and decelerates heart failure progression via Sirt1/p53 pathway activation

Resveratrol is an organic compound widely studied for its therapeutic uses. We investigated whether resveratrol exerts cardioprotective effects by inhibiting ferroptosis via the Sirt1/p53 pathway. A heart failure model was established by aortic coarctation in Sirt1 knockout mice. The superoxide dismutase (SOD), glutathione (GSH) levels and mitochondrial morphology in murine heart tissues were assessed at different time points to determine the role of ferroptosis in heart failure progression. The cardiac function of mice with heart failure was evaluated by determining the brain natriuretic peptide (BNP) and sST2 concentration and conducting echocardiography. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transfected with the p53 K382R mutant and Sirt1 interference lentiviral vectors. Immunoprecipitation (IP) experiments were performed to investigate whether Sirt1 influences ferroptosis via p53 K382 acetylation and SLC7A11 expression modulation. Resveratrol improved cardiac function in mice and decelerated ferroptosis and fibrosis progression in heart failure. However, the ability of resveratrol to prevent ferroptosis and treat heart failure was lost after silencing Sirt1. Sirt1 reduced ferroptosis by diminishing the levels of p53 K382 acetylation, reducing the degradation of SLC7A11, and increasing the levels of GSH and glutathione peroxidase 4 (GPX4) in cells. In conclusion, by activating the Sirt1/p53 pathway in heart failure, resveratrol decreased the depletion of SLC7A11, inhibited ferroptosis, and improved cardiac function.     

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H₂O₂)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.     

Dilated cardiomyopathy alters the expression patterns of CAR and other adenoviral receptors in human heart

Gene therapy trials for heart failure have demonstrated the key role of efficient gene transfer in achieving therapeutic efficacy. An attractive approach to improve adenoviral gene transfer is to use alternative virus serotypes with modified tropism. We performed a detailed analysis of cardiac expression of receptors for several adenovirus serotypes with a focus on differential expression of CAR and CD46, as adenoviruses targeting these receptors have been used in various applications. Explanted hearts from patients with DCM and healthy donors were analyzed using Q-RT-PCR, western blot and immunohistochemistry. Q-RT-PCR and Western analyses revealed robust expression of all receptors except CD80 in normal hearts with lower expression levels in DCM. Immunohistochemical analyses demonstrated that CD46 expression was somewhat higher than CAR both in normal and DCM hearts with highest levels of expression in intramyocardial coronary vessels. Total CAR expression was upregulated in DCM. Triple staining on these vessels demonstrated that both CAR and CD46 were confined to the subendothelial layer in normal hearts. The situation was clearly different in DCM, where both CAR and CD46 were expressed by endothelial cells. The induction of expression of CAR and CD46 by endothelial cells in DCM suggests that viruses targeting these receptors could more easily gain entry to heart cells after intravascular administration. This finding thus has potential implications for the development of targeted gene therapy for heart failure.     

Cardiac regenerative potential of cardiosphere‐derived cells from adult dog hearts

The regenerative potential of cardiosphere‐derived cells (CDCs) for ischaemic heart disease has been demonstrated in mice, rats, pigs and a recently completed clinical trial. The regenerative potential of CDCs from dog hearts has yet to be tested. Here, we show that canine CDCs can be produced from adult dog hearts. These cells display similar phenotypes in comparison to previously studied CDCs derived from rodents and human beings. Canine CDCs can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vitro. In addition, conditioned media from canine CDCs promote angiogenesis but inhibit cardiomyocyte death. In a doxorubicin‐induced mouse model of dilated cardiomyopathy (DCM), intravenous infusion of canine CDCs improves cardiac function and decreases cardiac fibrosis. Histology revealed that injected canine CDCs engraft in the mouse heart and increase capillary density. Out study demonstrates the regenerative potential of canine CDCs in a mouse model of DCM.     

Deletion of Smad3 protects against diabetic myocardiopathy in db/db mice

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF-β/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT-db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO-db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2-mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF-kB-driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3-dependent miRNAs by up-regulating cardiac miR-29b while suppressing miR-21 to exhibit the cardioprotective effect on Smad3KO-db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM.     

BEX1 is a critical determinant of viral myocarditis

Viral infection of the heart is a common but underappreciated cause of heart failure. Viruses can cause direct cardiac damage by lysing infected cardiomyocytes. Inflammatory immune responses that limit viral replication can also indirectly cause damage during infection, making regulatory factors that fine-tune these responses particularly important. Identifying and understanding these factors that regulate cardiac immune responses during infection will be essential for developing targeted treatments for virus-associated heart failure. Our laboratory has discovered Brain Expressed X-linked protein 1 (BEX1) as a novel stress-regulated pro-inflammatory factor in the heart. Here we report that BEX1 plays a cardioprotective role in the heart during viral infection. Specifically, we adopted genetic gain- and loss-of-function strategies to modulate BEX1 expression in the heart in the context of coxsackievirus B3 (CVB3)-induced cardiomyopathy and found that BEX1 limits viral replication in cardiomyocytes. Interestingly, despite the greater viral load observed in mice lacking BEX1, inflammatory immune cell recruitment in the mouse heart was profoundly impaired in the absence of BEX1. Overall, the absence of BEX1 accelerated CVB3-driven heart failure and pathologic heart remodeling. This result suggests that limiting inflammatory cell recruitment has detrimental consequences for the heart during viral infections. Conversely, transgenic mice overexpressing BEX1 in cardiomyocytes revealed the efficacy of BEX1 for counteracting viral replication in the heart in vivo. We also found that BEX1 retains its antiviral role in isolated cells. Indeed, BEX1 was necessary and sufficient to counteract viral replication in both isolated primary cardiomyocytes and mouse embryonic fibroblasts suggesting a broader applicability of BEX1 as antiviral agent that extended to viruses other than CVB3, including Influenza A and Sendai virus. Mechanistically, BEX1 regulated interferon beta (IFN-β) expression in infected cells. Overall, our study suggests a multifaceted role of BEX1 in the cardiac antiviral immune response.     

Identification of noncytotoxic and IL-10-producing CD8+AT2R+ T cell population in response to ischemic heart injury

Emerging evidence suggests a cardioprotective role of the angiotensin AT2R, albeit the underlying cellular mechanisms are not well understood. We aimed in this article to elucidate a potential role of cardiac angiotensin AT2R in regulating cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, double-immunofluorescence staining showed that AT2R was detected in a fraction of CD8(+) T cells infiltrating in the peri-infarct myocardium. We developed a method that allowed the isolation of myocardial infiltrating CD8(+)AT2R(+) T cells using modified MACS, and further characterization and purification with flow cytometry. Although the CD8(+)AT2R(-) T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes (CMs), the CD8(+)AT2R(+) T cells were noncytotoxic to these CMs. The CD8(+)AT2R(+) T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-γ expression when compared with CD8(+)AT2R(-) T cells. We further showed that IL-10 gene expression was enhanced in CD8(+) T cells on in vitro AT2R stimulation. Importantly, in vivo AT2R activation engendered an increment of CD8(+)AT2R(+) T cells and IL-10 production in the ischemic myocardium. In addition, intramyocardial transplantation of CD8(+)AT2R(+) T cells (versus CD8(+)AT2R(-)) led to reduced ischemic heart injury. Moreover, the CD8(+)AT2R(+) T cell population was also demonstrated in human peripheral blood. Thus, we have defined the cardioprotective CD8(+)AT2R(+) T cell population, which increases during ischemic heart injury and contributes to maintaining CM viability and providing IL-10, hence revealing an AT2R-mediated cellular mechanism in modulating adaptive immune response in the heart.     

Nrf2 prevents diabetic cardiomyopathy via antioxidant effect and normalization of glucose and lipid metabolism in the heart

Metabolic disorders and oxidative stress are the main causes of diabetic cardiomyopathy. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a powerful antioxidant effect and prevents the progression of diabetic cardiomyopathy. However, the mechanism of its cardiac protection and direct action on cardiomyocytes are not well understood. Here, we investigated in a cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) the direct effect of Nrf2 on cardiomyocytes in DCM and its mechanism. In this study, cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) were used to directly observe whether cardiomyocyte-specific overexpression of Nrf2 can prevent diabetic cardiomyopathy and correct glucose and lipid metabolism disorders in the heart. Compared to wild-type mice, Nrf2-TG mice showed resistance to diabetic cardiomyopathy in a streptozotocin-induced type 1 diabetes mouse model. This was primarily manifested as improved echocardiography results as well as reduced myocardial fibrosis, cardiac inflammation, and oxidative stress. These results showed that Nrf2 can directly act on cardiomyocytes to exert a cardioprotective role. Mechanistically, the cardioprotective effects of Nrf2 depend on its antioxidation activity, partially through improving glucose and lipid metabolism by directly targeting lipid metabolic pathway of AMPK/Sirt1/PGC-1α activation via upstream genes of sestrin2 and LKB1, and indirectly enabling AKT/GSK-3β/HK-Ⅱ activity via AMPK mediated p70S6K inhibition.     

Ghrelin,a novel growth hormone-releasing peptide,in the treatment of chronic heart failure

Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, which has been identified as an endogenous ligand for growth-hormone secretagogues receptor (GHS-R). This peptide also causes a positive energy balance by stimulating food intake and inducing adiposity through growth hormone-independent mechanisms. In addition, ghrelin has some cardiovascular effects, as indicated by the presence of its receptor in blood vessels and the cardiac ventricles. In vitro, ghrelin inhibits apoptosis of cardiomyocytes and endothelial cells. In humans, infusion of ghrelin decreases systemic vascular resistance and increases cardiac output in patients with heart failure. Repeated administration of ghrelin improves cardiac structure and function and attenuates the development of cardiac cachexia in rats with heart failure. These results suggest that ghrelin has cardiovascular effects and regulates energy metabolism through GH-dependent and -independent mechanisms. Thus, administration of ghrelin may be a new therapeutic strategy for the treatment of severe chronic heart failure (CHF).     

Testosterone and interleukin-1β increase cardiac remodeling during coxsackievirus B3 myocarditis via serpin A 3n

Myocarditis and dilated cardiomyopathy (DCM) are often caused by viral infections and occur more frequently in men than in women, but the reasons for the sex difference remain unclear. The aim of this study was to assess whether gene changes in the heart during coxsackievirus B3 (CVB3) myocarditis in male and female BALB/c mice predicted worse DCM in males. Although myocarditis (P = 4.2 × 10(-5)) and cardiac dilation (P = 0.008) were worse in males, there was no difference in viral replication in the heart. Fibrotic remodeling genes, such as tissue inhibitor of metalloproteinase (TIMP)-1 and serpin A 3n, were upregulated in males during myocarditis rather than during DCM. Using gonadectomy and testosterone replacement, we showed that testosterone increased cardiac TIMP-1 (P = 0.04), serpin A 3n (P = 0.007), and matrix metalloproteinase (MMP)-8 (P = 0.04) during myocarditis. Testosterone increased IL-1β levels in the heart (P = 0.02), a cytokine known to regulate cardiovascular remodeling, and IL-1β in turn increased cardiac serpin A 3n mRNA (P = 0.005). We found that 39 of 118 (33%) genes identified in acute DCM patients were significantly altered in the heart during CVB3 myocarditis in mice, including serpin A 3n (3.3-fold change, P = 0.0001). Recombinant serpin A 3n treatment induced cardiac fibrosis during CVB3 myocarditis (P = 0.0008) while decreasing MMP-3 (P = 0.04) and MMP-9 (P = 0.03) levels in the heart. Thus, serpin A 3n was identified as a gene associated with fibrotic cardiac remodeling and progression to DCM in male myocarditis patients and mice.     

Cell type-specific functions of the lysosomal protease cathepsin L in the heart

Deficiency of the lysosomal cysteine protease cathepsin L (Ctsl) in mice results in a phenotype affecting multiple tissues, including thymus, epidermis, and hair follicles, and in the heart develops as a progressive dilated cardiomyopathy (DCM). To understand the role of Ctsl in the maintenance of regular heart morphology and function, it is critical to determine whether the DCM in Ctsl-/- mice is primarily because of the lack of Ctsl expression and activity in the cardiomyocytes or is caused by the additional extracardiac pathologies. Cardiomyocyte-specific expression of Ctsl in Ctsl-/- mice, using an alpha-myosin heavy chain promoter-Ctsl transgene, results in improved cardiac contraction, normal mRNA expression of atrionatriuretic peptide, normal heart weight, and regular ultrastructure of cardiomyocytes. Epithelial expression of cathepsin L2 (CTSL2) by a K14 promoter-CTSL2-transgene resulted in rescue of the Ctsl-/- hair loss phenotype. In these mice, cardiac atrionatriuretic peptide expression and end systolic heart dimensions were also significantly attenuated. However, cardiac contraction was not improved, and increased heart weight as well as the typical changes in lysosomal ultrastructure of Ctsl-/- hearts persisted. Myocardial fibrosis was detected in all Ctsl-/- mice irrespective of transgene-mediated cardiac Ctsl expression or extracardiac CTSL2 expression. Expression of collagen 1 was not enhanced in Ctsl-/- hearts, but a reduced collagenolytic activity suggests a role for Ctsl in collagen turnover by cardiac fibroblasts. We conclude that the DCM of Ctsl-/- mice is primarily caused by absence of the protease in cardiomyocytes, whereas the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress to the heart.     

Kir6.1 improves cardiac dysfunction in diabetic cardiomyopathy via the AKT-FoxO1 signalling pathway

Previous studies have shown that the expression of inwardly rectifying potassium channel 6.1 (Kir6.1) in heart mitochondria is significantly reduced in type 1 diabetes. However, whether its expression and function are changed and what role it plays in type 2 diabetic cardiomyopathy (DCM) have not been reported. This study investigated the role and mechanism of Kir6.1 in DCM. We found that the cardiac function and the Kir6.1 expression in DCM mice were decreased. We generated mice overexpressing or lacking Kir6.1 gene specifically in the heart. Kir6.1 overexpression improved cardiac dysfunction in DCM. Cardiac-specific Kir6.1 knockout aggravated cardiac dysfunction. Kir6.1 regulated the phosphorylation of AKT and Foxo1 in DCM. We further found that Kir6.1 overexpression also improved cardiomyocyte dysfunction and up-regulated the phosphorylation of AKT and FoxO1 in neonatal rat ventricular cardiomyocytes with insulin resistance. Furthermore, FoxO1 activation down-regulated the expression of Kir6.1 and decreased the mitochondrial membrane potential (ΔΨm) in cardiomyocytes. FoxO1 inactivation up-regulated the expression of Kir6.1 and increased the ΔΨm in cardiomyocytes. Chromatin immunoprecipitation assay demonstrated that the Kir6.1 promoter region contains a functional FoxO1-binding site. In conclusion, Kir6.1 improves cardiac dysfunction in DCM, probably through the AKT-FoxO1 signalling pathway.     

应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病

本研究旨在应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病（dilated cardiomyopathy,DCM）小鼠（DCM小鼠）进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR（quantitative real-time PCR,Q-PCR）检测人源TNNT2 mRNA的表达水平。结果显示,gRNA 2引导Cas13b敲低目标基因的效率最高,达到80%（P<0.0001）。把gRNA2表达质粒包装到慢病毒载体中转导出生后1天的DCM小鼠原代心肌细胞,Q-PCR检测结果表明CRISPR/Cas13b系统对人源TNNT2 mRNA的敲低效率达到55%（P<0.01）。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,然后将200 μL 约1×1012 AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时,Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%（P<0.01）。对5月龄野生型（WT）、DCM（未注射病毒组）和AAV9+DCM（基因组编辑工具注射组）三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示：DCM小鼠的心脏形态异常,而AAV9+DCM小鼠心脏形态趋于正常;对三组小鼠的心脏进行超声心动图并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数（left ventricular percent ejection fraction,LV EF%）、左心室短轴缩短率（left ventricular percent fractional shortening,LV FS%）分别下降了50.4%（P<0.0001）,55.1%（P<0.0001）,而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升了66.5%（P<0.01）,77.0%（P<0.01）;通过Q-PCR和天狼星红染色检测三组小鼠的心脏纤维化程度,结果显示DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍（P<0.001）、4.5倍（P<0.01）,而AAV9+DCM组较DCM组小鼠两种基因表达分别下降了2.0倍（P<0.05）、1.4倍（NS）,天狼星红染色结果显示纤维化区域明显下降;通过Q-PCR和蛋白质免疫印迹分别检测三组小鼠的心脏心力衰竭基因Nppb mRNA和Nppa蛋白质的表达水平,结果表明DCM组较WT组小鼠Nppb mRNA表达上升14.2倍（P<0.01）,而AAV9+DCM组较DCM组小鼠Nppb mRNA表达明显下降下降2.8倍（P<0.05）,Nppa蛋白质表达趋势与Nppb相同。把gRNA 5和含有R141W突变（gRNA 5T）和正常的TNNT2 mRNA（gRNA 5V）序列分别组合转染到293T细胞中,通过Q-PCR检测两种序列mRNA的表达水平。结果显示,gRNA 5T序列表达效率为30%（P<0.0001）,而并未检测到gRNA 5V mRNA的敲低。本研究通过设计靶向TNNT2R141W mRNA的gRNA,特异性敲低TNNT2R141W转基因小鼠体内突变的mRNA,有效改善了转基因小鼠的心功能,为临床进一步探索扩张型心肌病的治疗奠定了实验室基础。     

Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells (r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.     

Small molecule regulators of postnatal Nkx2.5 cardiomyoblast proliferation and differentiation

While recent data have supported the capacity for a neonatal heart to undergo cardiomyogenesis, it is unclear whether these new cardiomyocytes arise from an immature cardiomyoblast population or from the division of mature cardiomyocytes. By following the expression of enhanced Green Fluorescent Protein (eGFP) in an Nkx2.5 enhancer-eGFP transgenic mice, we have identified a population of immature cells that can undergo cardiomyogenic as well as smooth muscle cell differentiation in the neonatal heart. Here, we examined growth factors and small molecule regulators that potentially regulate the proliferation and cardiomyogenic versus smooth muscle cell differentiation of neonatal Nkx2.5-GFP (+) cells in vitro. We found that A83-01 (A83), an inhibitor of TGF-βRI, was able to induce an expansion of neonatal Nkx2.5-eGFP (+) cells. In addition, the ability of A83 to expand eGFP (+) cells in culture was dependent on signalling from the mitogen-activated protein kinase kinase (MEK) as treatment with a MEK inhibitor, PD0325901, abolished this effect. On the other hand, activation of neonatal Nkx2.5-eGFP (+) cells with TGF-β1, but not activin A nor BMP2, led to smooth muscle cell differentiation, an effect that can be reversed by treatment with A83. In summary, small molecule inhibition of TGF-β signalling may be a promising strategy to induce the expansion of a rare population of postnatal cardiomyoblasts.     

Effect of human umbilical cord blood cells on Ang-II-induced hypertrophy in mice

We have assessed the capacity of human umbilical cord blood (hUCB)-derived stem cells to differentiate into cardiomyocytes and repair angiotensin II induced insult in culture and in mouse hearts when injected. hUCB were able to differentiate into cardiomyocyte-like cells, when induced with 5-azacytidine or co-cultured with rat neonatal cardiomyocytes (NRCM). When co-cultured, hUCB reversed the pathological effects induced by angiotensin II (Ang-II) in NRCM and in mice injected after Ang-II infusion. As assessed by increased heart weight to body mass ratio and Ang-II-induced fibrosis, cardiac hypertrophy was also reduced after hUCB were injected. hUCB also reversed the pathological heart failure markers induced by Ang-II in mice. Further, we observed a shift from pathological hypertrophy towards physiological hypertrophy by hUCB in Ang-II-challenged mice. Our findings support hUCB as a feasible model for experimentation in stem cell therapy and emphasize the relevance of the hUCB in reversing heart failure conditions.     

Klotho protects the heart from hyperglycemia-induced injury by inactivating ROS and NF-κB-mediated inflammation both in vitro and in vivo

Cardiac inflammation and oxidative stress play a key role in the pathogenesis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho has been found to protect cells from inflammation and oxidative stress. The current study aimed to explore the cardioprotective effects of Klotho on DCM and the underlying mechanisms. H9c2 cells and neonatal cardiomyocytes were incubated with 33 mM glucose in the presence or absence of Klotho. Klotho pretreatment effectively inhibited high glucose-induced inflammation, ROS generation, apoptosis, mitochondrial dysfunction, fibrosis and hypertrophy in both H9c2 cells and neonatal cardiomyocytes. In STZ-induced type 1 diabetic mice, intraperitoneal injection of Klotho at 0.01 mg/kg per 48 h for 3 months completely suppressed cardiac inflammatory cytokines and oxidative stress and prevented cardiac cell death and remodeling, which subsequently improved cardiac dysfunction without affecting hyperglycemia. This study revealed that Klotho may exert its protective effects by augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression and inactivating nuclear factor κB (NF-κB) activation both in vitro and in vivo. Thus, this work demonstrated for the first time that the anti-aging protein Klotho may be a potential therapeutic agent to treat DCM by inhibiting oxidative stress and inflammation. We also demonstrated the critical roles of the Nrf2 and NF-κB pathways in diabetes-stimulated cardiac injuries and indicated that they may be key therapeutic targets for diabetic complications.