Wenfeng Cai

Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

Heat Shock Improves Sca‐1+ Stem Cell Survival and Directs Ischemic Cardiomyocytes Toward a Prosurvival Phenotype Via Exosomal Transfer: A Critical Role for HSF1/miR‐34a/HSP70 Pathway

Stem cell‐based therapy is a promising intervention for ischemic heart diseases. However, the functional integrity of stem cells is impaired in an ischemic environment. Here, we report a novel finding that heat shock significantly improves Sca‐1+stem cell survival in an ischemic environment by the regulation of the triangle: heat shock factor 1 (HSF1), HSF1/miR‐34a, and heat shock protein 70 (HSP70). Initially we prove that HSP70 is the key chaperone‐mediating cytoprotective effect of heat shock in Sca‐1+cells and then we establish miR‐34a as a direct repressor of HSP70. We found that HSP70 was downregulated in heat shocked Sca‐1+ stem cells (HSSca‐1+ cells). Intriguingly, we demonstrate that the downregulation of miR‐34a is attributed to HSF1‐mediated epigenetic repression through histone H3 Lys27 trimethylation (H3K27me3) on miR‐34a promoter. Moreover, we show that heat shock induces exosomal transfer of HSF1 from Sca‐1+ cells, which directs ischemic cardiomyocytes toward a prosurvival phenotype by epigenetic repression of miR‐34a. In addition, our in vivo study demonstrates that transplantation of HSSca‐1+ cells significantly reduces apoptosis, attenuates fibrosis, and improves global heart functions in ischemic myocardium. Hence, our study provides not only novel insights into the effects of heat shock on stem cell survival and paracrine behavior but also may have therapeutic values for stem cell therapy in ischemic heart diseases. Stem Cells 2014;32:462–472

Exosomes Secreted from CXCR4 Overexpressing Mesenchymal Stem Cells Promote Cardioprotection via Akt Signaling Pathway following Myocardial Infarction

Background and Objective. Exosomes secreted from mesenchymal stem cells (MSC) have demonstrated cardioprotective effects. This study examined the role of exosomes derived from MSC overexpressing CXCR4 for recovery of cardiac functions after myocardial infarction (MI). Methods. In vitro, exosomes from MSC transduced with lentiviral CXCR4 (ExoCR4) encoding a silencing sequence or null vector were isolated and characterized by transmission electron microscopy and dynamic light scattering. Gene expression was then analyzed by qPCR and Western blotting. Cytoprotective effects on cardiomyocytes were evaluated and effects of exosomes on angiogenesis analyzed. In vivo, an exosome-pretreated MSC-sheet was implanted into a region of scarred myocardium in a rat MI model. Angiogenesis, infarct size, and cardiac functions were then analyzed. Results. In vitro, ExoCR4 significantly upregulated IGF-1α and pAkt levels and downregulated active caspase 3 level in cardiomyocytes. ExoCR4 also enhanced VEGF expression and vessel formation. However, effects of ExoCR4 were abolished by an Akt inhibitor or CXCR4 knockdown. In vivo, ExoCR4 treated MSC-sheet implantation promoted cardiac functional restoration by increasing angiogenesis, reducing infarct size, and improving cardiac remodeling. Conclusions. This study reveals a novel role of exosomes derived from MSCCR4 and highlights a new mechanism of intercellular mediation of stem cells for MI treatment.

Small Heat Shock Protein 20 Interacts With Protein Phosphatase-1 and Enhances Sarcoplasmic Reticulum Calcium Cycling

Background: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. Methods and Results: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1–PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. Conclusions: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1–PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.

Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation.

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+).

Toll-Like Receptor (TLR) 2 and TLR4 Differentially Regulate Doxorubicin Induced Cardiomyopathy in Mice

Recent evidence indicates that toll-like receptor (TLR) 2 and 4 are involved in the pathogenesis of dilated cardiomyopathy (DCM), but the exact mechanisms of their actions have not been elucidated. We explored the therapeutic potential of blocking TLRs in mice with established cardiomyopathy. Cardiomyopathy was generated by a single intraperitoneal injection of doxorubicin (10 mg/kg). Two weeks later, the mice were treated with TLR2 or TLR4 neutralizing antibody. Blocking TLR2, but not TLR4, activity not only reduced mortality, but also attenuated doxorubicin-induced cardiac dysfunction by 20% and inhibited myocardial fibrosis. To determine the differential effects of blocking TLR2 and TLR4 in chronic cardiomyopathy, mice were injected with doxorubicin (3.5 mg/kg) once a week for 8 weeks, followed by treatment with TLR2 or TLR4 neutralizing antibody for 40 days. Blocking TLR2 activity blunted cardiac dysfunction by 13% and inhibited cardiac fibrosis, which was associated with a significant suppression of myocardial inflammation. The underlying mechanism involved interrupting the interaction of TLR2 with its endogenous ligands, resulting in attenuation of inflammation and fibrosis. In contrast, blocking TLR4 exacerbated cardiac dysfunction and fibrosis by amplifying inflammation and suppressing autophagy. Our studies demonstrate that TLR2 and TLR4 play distinct roles in the progression of doxorubicin-induced DCM. TLR4 activity is crucial for the resolution of inflammation and cardiac fibrosis, while blocking TLR2 activity has therapeutic potential for the treatment of DCM.

MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF

MicroRNAs have been appreciated in various cellular functions, including the regulation of angiogenesis. Mesenchymal-stem-cells (MSCs) transplanted to the MI heart improve cardiac function through paracrine-mediated angiogenesis. However, whether microRNAs regulate MSC induced angiogenesis remains to be clarified. Using microRNA microarray analysis, we identified a microRNA expression profile in hypoxia-treated MSCs and observed that among all dysregulated microRNAs, microRNA-377 was decreased the most significantly. We also validated that vascular endothelial growth factor (VEGF) is a target of microRNA-377 using dual-luciferase reporter assay and Western-blotting. Knockdown of endogenous microRNA-377 promoted tube formation in human umbilical vein endothelial cells. We then engineered rat MSCs with lentiviral vectors to either overexpress microRNA-377 (MSCmiR-377) or knockdown microRNA-377 (MSCAnti-377) to investigate whether microRNA-377 regulated MSC-induced myocardial angiogenesis, using MSCs infected with lentiviral empty vector to serve as controls (MSCNull). Four weeks after implantation of the microRNA-engineered MSCs into the infarcted rat hearts, the vessel density was significantly increased in MSCAnti-377-hearts, and this was accompanied by reduced fibrosis and improved myocardial function as compared to controls. Adverse effects were observed in MSCmiR-377-treated hearts, including reduced vessel density, impaired myocardial function, and increased fibrosis in comparison with MSCNull-group. These findings indicate that hypoxia-responsive microRNA-377 directly targets VEGF in MSCs, and knockdown of endogenous microRNA-377 promotes MSC-induced angiogenesis in the infarcted myocardium. Thus, microRNA-377 may serve as a novel therapeutic target for stem cell-based treatment of ischemic heart disease.

Expression of active protein phosphatase 1 inhibitor-1 attenuates chronic beta-agonist-induced cardiac apoptosis

Cardiac apoptosis has been considered an important contributing factor to heart failure. Several subcellular mechanisms, including increased protein phosphatase 1 activity, have been suggested to induce apoptosis. Protein phosphatase 1 is regulated by an endogenous inhibitor-1 (I-1) that is activated upon phosphorylation at threonine 35 via protein kinase A. Here, we tested whether cardiac-specific overexpression of a constitutively active (T35D, AA 1-65) inhibitor-1 (I-1c), could also affect cardiac apoptosis and heart failure progression induced by prolonged β-adrenergic stimulation. We found that either acute or chronic expression of I-1c reduced isoproterenol (ISO)-induced apoptosis assessed by nuclear condensation, TUNEL staining and DNA fragmentation. The beneficial effects of I-1c were associated with increased expression of the anti-apoptotic protein Bcl-2, decreased expression of the pro-apoptotic protein Bax and reduced levels of active caspases as well as increased activation of ERK. These findings suggest that mitochondrial signaling and ERK activation may be involved in the I-1c cardioprotective effects against apoptosis induced by prolonged β-adrenergic stimulation.

A novel human R25C-phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia

AIMS Depressed sarcoplasmic reticulum (SR) Ca(2+) cycling, a universal characteristic of human and experimental heart failure, may be associated with genetic alterations in key Ca(2+)-handling proteins. In this study, we identified a novel PLN mutation (R25C) in dilated cardiomyopathy (DCM) and investigated its functional significance in cardiomyocyte Ca(2+)-handling and contractility. METHODS AND RESULTS Exome sequencing identified a C73T substitution in the coding region of PLN in a family with DCM. The four heterozygous family members had implantable cardiac defibrillators, and three developed prominent ventricular arrhythmias. Overexpression of R25C-PLN in adult rat cardiomyocytes significantly suppressed the Ca(2+) affinity of SR Ca(2+)-ATPase (SERCA2a), resulting in decreased SR Ca(2+) content, Ca(2+) transients, and impaired contractile function, compared with WT-PLN. These inhibitory effects were associated with enhanced interaction of R25C-PLN with SERCA2, which was prevented by PKA phosphorylation. Accordingly, isoproterenol stimulation relieved the depressive effects of R25C-PLN in cardiomyocytes. However, R25C-PLN also elicited increases in the frequency of Ca(2+) sparks and waves as well as stress-induced aftercontractions. This was accompanied by increased Ca(2+)/calmodulin-dependent protein kinase II activity and hyper-phosphorylation of RyR2 at serine 2814. CONCLUSION The findings demonstrate that human R25C-PLN is associated with super-inhibition of SERCA2a and Ca(2+) transport as well as increased SR Ca(2+) leak, promoting arrhythmogenesis under stress conditions. This is the first mechanistic evidence that increased PLN inhibition may impact both SR Ca(2+) uptake and Ca(2+) release activities and suggests that the human R25C-PLN may be a prognostic factor for increased ventricular arrhythmia risk in DCM carriers.

HAX-1 regulates cyclophilin-D levels and mitochondria permeability transition pore in the heart

Significance The massive cell death, associated with a heart attack, is mainly due to disruption of mitochondrial membrane integrity upon activation of the mitochondrial permeability transition pore. Thus, it is important to understand how this pore is regulated to prevent cardiac cell death. In this study, we reported that hematopoietic-substrate-1 associated protein X-1 (HAX-1) is an inhibitor of the pore and promotes cell survival. HAX-1 works through recruitment of a chaperone protein called Hsp90 from cyclophilin-D, a major component of the pore. Displacement of Hsp90 from cyclophilin-D promotes cyclophilin-D degradation, resulting in inhibition of pore opening and cell death. Given that the opening of the mitochondrial permeability transition pore contributes to various diseases, our findings have broader applications reaching beyond the heart. The major underpinning of massive cell death associated with myocardial infarction involves opening of the mitochondrial permeability transition pore (mPTP), resulting in disruption of mitochondria membrane integrity and programmed necrosis. Studies in human lymphocytes suggested that the hematopoietic-substrate-1 associated protein X-1 (HAX-1) is linked to regulation of mitochondrial membrane function, but its role in controlling mPTP activity remains obscure. Herein we used models with altered HAX-1 expression levels in the heart and uncovered an unexpected role of HAX-1 in regulation of mPTP and cardiomyocyte survival. Cardiac-specific HAX-1 overexpression was associated with resistance against loss of mitochondrial membrane potential, induced by oxidative stress, whereas HAX-1 heterozygous deficiency exacerbated vulnerability. The protective effects of HAX-1 were attributed to specific down-regulation of cyclophilin-D levels leading to reduction in mPTP activation. Accordingly, cyclophilin-D and mPTP were increased in heterozygous hearts, but genetic ablation of cyclophilin-D in these hearts significantly alleviated their susceptibility to ischemia/reperfusion injury. Mechanistically, alterations in cyclophilin-D levels by HAX-1 were contributed by the ubiquitin-proteosomal degradation pathway. HAX-1 overexpression enhanced cyclophilin-D ubiquitination, whereas proteosomal inhibition restored cyclophilin-D levels. The regulatory effects of HAX-1 were mediated through interference of cyclophilin-D binding to heat shock protein-90 (Hsp90) in mitochondria, rendering it susceptible to degradation. Accordingly, enhanced Hsp90 expression in HAX-1 overexpressing cardiomyocytes increased cyclophilin-D levels, as well as mPTP activation upon oxidative stress. Taken together, our findings reveal the role of HAX-1 in regulating cyclophilin-D levels via an Hsp90-dependent mechanism, resulting in protection against activation of mPTP and subsequent cell death responses.