Pigment epithelium-derived factor (PEDF) is a multifunctional protein that exhibits anti-angiogenic, antitumor, anti-inflammatory, antioxidative, anti-atherogenic, and ca... More
Pigment epithelium-derived factor (PEDF) is a multifunctional protein that exhibits anti-angiogenic, antitumor, anti-inflammatory, antioxidative, anti-atherogenic, and cardioprotective properties. While it was recently shown that PEDF expression is inhibited under low oxygen conditions, the functional role of PEDF in response to hypoxia/reoxygenation (H/R) remains unclear. The goal of this study was to therefore investigate the influence of PEDF on myocardial H/R injury. For these analyses, PEDF-specific small interfering RNA-expressing and PEDF-expressing lentivirus (PEDF-LV) vectors were utilized to knockdown or stably overexpress PEDF, respectively, within human cardiomyocytes (HCM) in vitro. We noted that reactive oxygen species (ROS) play important roles in the induction of cell death pathways, including apoptosis and autophagy in ischemic hearts. Our findings demonstrate that overexpression of PEDF resulted in a significant reduction in ROS production and attenuation of mitochondrial membrane potential depletion under H/R conditions. Furthermore, PEDF inhibited the activation of a two-step apoptotic pathway in which caspase-dependent (caspase-9 and caspase-3) and caspase-independent (apoptosis inducing factor and endonuclease G), which in turn cleaves several crucial substrates including the DNA repair enzyme poly (ADP-ribose) polymerase. Meanwhile, overexpression of PEDF also promoted autophagy, a process that is typically activated in response to H/R. Therefore, these findings suggest that PEDF plays a critical role in preventing H/R injury by modulating anti-oxidant and anti-apoptotic factors and promoting autophagy. Less
Congenital heart disease (CHD) is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite it... More
Congenital heart disease (CHD) is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable. Less
Although it has been observed that aggregate size affects cardiac development, an incomplete understanding of the cellular mechanisms underlying human pluripotent stem ce... More
Although it has been observed that aggregate size affects cardiac development, an incomplete understanding of the cellular mechanisms underlying human pluripotent stem cell-derived cardiomyogenesis has limited the development of robust defined-condition cardiac cell generation protocols. Our objective was thus to elucidate cellular and molecular mechanisms underlying the endogenous control of human embryonic stem cell (hESC) cardiac tissue development, and to test the hypothesis that hESC aggregate size influences extraembryonic endoderm (ExE) commitment and cardiac inductive properties. hESC aggregates were generated with 100, 1000, or 4000 cells per aggregate using microwells. The frequency of endoderm marker (FoxA2 and GATA6)-expressing cells decreased with increasing aggregate size during early differentiation. Cardiogenesis was maximized in aggregates initiated from 1000 cells, with frequencies of 0.49±0.06 cells exhibiting a cardiac progenitor phenotype (KDR(low)/C-KIT(neg)) on day 5 and 0.24±0.06 expressing cardiac Troponin T on day 16. A direct relationship between ExE and cardiac differentiation efficiency was established by forming aggregates with varying ratios of SOX7 (a transcription factor required for ExE development) overexpressing or knockdown hESCs to unmanipulated hESCs. We demonstrate, in a defined, serum-free cardiac induction system, that robust and efficient cardiac differentiation is a function of endogenous ExE cell concentration, a parameter that can be directly modulated by controlling hESC aggregate size. Less
Aims: Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell ... More
Aims: Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX). Methods and results: Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure–volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1+/+), and these effects were markedly attenuated in CB1 knockouts (CB1−/−). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT–PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX ± AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation. Conclusion: CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular diseases. Keywords: Heart failure, CB1 receptor, Endocannabinoids, Rimonabant, SR141716, AM281 Less
The proteasome has been identified as a target of the humoral autoimmune response in different inflammatory disease entities including dilated cardiomyopathy (DCM). Howev... More
The proteasome has been identified as a target of the humoral autoimmune response in different inflammatory disease entities including dilated cardiomyopathy (DCM). However, the role of proteasome autoantibodies (ProtAb) remains to be studied. Here, we have isolated human ProtAb by affinity-purification from the IgG fractions obtained from DCM patients, which predominantly detected the outer ring subunits alpha3 of the 20S proteasome. In an attempt to study the cellular effects potentially exerted by these ProtAb, simultaneous calcium and cell contractility measurements were performed in rat cardiomyocytes revealing no short-term effects upon human ProtAb exposure. Immunofluorescence staining and FACS analysis pointed towards a failure of human ProtAb to bind to the intact cell membrane, whereas human ProtAb detected 20S proteasomes in the cytoplasm and nucleus. The lack of the cell surface interaction of human ProtAb was in agreement with the failure of these autoantibodies to interfere with the cellular viability. Further, we investigated whether the removal of ProtAb by immunoadsorption (IA) resulted in functional improvement in DCM patients. IA was performed in 90 DCM patients (left ventricular ejection fraction < or =45%, ProtAb detection at baseline in 30% of these DCM patients). Improvement of LVEF was not associated with the initial detection and removal of ProtAb in DCM patients. ProtAb were reconstituted to baseline levels as soon as after 3 months post-IA/IgG treatment despite the overall improvement of LVEF in this study group. In conclusion, our data argue against a direct impact of ProtAb in the pathogenesis of DCM. Less
Human amniotic fluid stem cells (hAFSCs) derived from second-trimester amniocentesis were evaluated for the therapeutic potential of cardiac repair. Whether hAFSCs can be... More
Human amniotic fluid stem cells (hAFSCs) derived from second-trimester amniocentesis were evaluated for the therapeutic potential of cardiac repair. Whether hAFSCs can be differentiated into cardiomyogenic cells and toward the maturation of endothelial cell lineage was investigated in vitro using mimicking differentiation milieu. Employing an immune-suppressed rat model with experimental myocardial infarction, an intramyocardial injection was conducted with a needle directly into the peri-infarct areas. There were three treatment groups: sham, saline, and hAFSCs (n > or = 10). When cultured with rat neonatal cardiomyocytes or in endothelial growth medium-2 enriched with vascular endothelial growth factor, hAFSCs were differentiated into cardiomyocyte-like cells and cells of endothelial lineage, respectively. After 4 weeks, hAFSC-treated animals showed a preservation of the infarcted thickness, an attenuation of left ventricle remodeling, a higher vascular density, and thus an improvement in cardiac function, when compared with the saline injection group. Survival and proliferation of the transplanted hAFSCs were revealed by immunohistochemical staining. Expressions of the cardiac-specific markers such as Nkx2.5, alpha-actinin, and cardiac Troponin T were observed in the transplanted hAFSCs. Additionally, Cx43 was clearly expressed at the borders of the transplanted/transplanted and host/transplanted cells, an indication of enhancement of cell connection. The results demonstrated that hAFSCs induce angiogenesis, have cardiomyogenic potential, and may be used as a new cell source for cellular cardiomyoplasty. Less
To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that m... More
To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. Keywords: Septic sera; gene expression modulation; human myocytes. Less
Coxsackievirus B3 (CVB3) generates 5'-terminally deleted genomes (TDs) during replication in murine hearts. We show here that CVB3 populations with TDs can also be genera... More
Coxsackievirus B3 (CVB3) generates 5'-terminally deleted genomes (TDs) during replication in murine hearts. We show here that CVB3 populations with TDs can also be generated within two to three passages of CVB3 in primary, but not immortalized, cell cultures. Deletions of less than 49 nucleotides increase in size during passage, while 5' TDs of 49 nucleotides appear to be the maximum deletion size. The cellular environment of contact-inhibited primary cell cultures or the myocardium in vivo is sufficient for the selection of 5' TDs over undeleted genomes. Less
Angiotensin II (Ang II) is a critical effector in the renin-angiotensin system (RAS), which modulates cardiovascular homeostasis, and the matrix metalloproteinases (MMPs)... More
Angiotensin II (Ang II) is a critical effector in the renin-angiotensin system (RAS), which modulates cardiovascular homeostasis, and the matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) related metabolism of extracellular matrix (ECM). Angiotensin(1-7) [Ang(1-7)] is another bioactive peptide in the RAS and is considered to have opposite effects to Ang II. However, the modulation of MMPs and TIMPs by Ang(1-7) is largely unclear in cardiocytes, and the antagonistic effects of Ang(1-7) on Ang II-mediated expression of MMPs and TIMPs have yet to be identified. In the present study, we examined the transcript expression of MMPs and TIMPs in human cardiac fibroblasts (HCF) and myocytes (HCM) after Ang II or Ang(1-7) stimulation, and analysed the antagonistic effects of Ang(1-7) to Ang II. The results show that Ang II decreased transcript expression of MMP-1, MMP-2, TIMP-1, TIMP-2 and TIMP-3, but upregulated MMP-9 expression in the HCF cells. Transcript expression of MMP-9 and TIMP-2 was downregulated by Ang(1-7) in the same cells. In the HCM cells, Ang II induced MMP-1 and MMP-9 overexpression but MMP-2 was downregulated. All of the examined MMPs and TIMPs, except MMP-9, were markedly decreased by Ang(1-7). In the studies of antagonistic effects of Ang(1-7) to Ang II, Ang(1-7) counteracted the effects of Ang II-mediated regulation on MMP-9 and TIMP-1 in the HCF cells compared with the control group. The regulations of all examined MMPs by Ang II were reversed to basal expression by Ang(1-7) in the HCM cells. Our results suggest that Ang(1-7) and Ang II have opposite and antagonistic effects on regulation of transcription of MMPs and TIMPs in primary cultures of human cardiocytes. These effects lead to increased ratios of MMPs to TIMPs after Ang II stimulation and decreased ratios of MMPs to TIMPs after Ang(1-7) stimulation; effects which may partly depend of the type of cardiac cells. These results suggest a potential role for Ang(1-7) in attenuatating cardiac damage in Ang II-induced ECM remodelling. Less
Sustained pressure overload causes cardiac hypertrophy and the transition to heart failure. We show here that dietary supplementation with physiologically relevant levels... More
Sustained pressure overload causes cardiac hypertrophy and the transition to heart failure. We show here that dietary supplementation with physiologically relevant levels of copper (Cu) reverses preestablished hypertrophic cardiomyopathy caused by pressure overload induced by ascending aortic constriction in a mouse model. The reversal occurs in the continued presence of pressure overload. Sustained pressure overload leads to decreases in cardiac Cu and vascular endothelial growth factor (VEGF) levels along with suppression of myocardial angiogenesis. Cu supplementation replenishes cardiac Cu, increases VEGF, and promotes angiogenesis. Systemic administration of anti-VEGF antibody blunts Cu regression of hypertrophic cardiomyopathy. In cultured human cardiomyocytes, Cu chelation blocks insulin-like growth factor (IGF)-1- or Cu-stimulated VEGF expression, which is relieved by addition of excess Cu. Both IGF-1 and Cu activate hypoxia-inducible factor (HIF)-1alpha and HIF-1alpha gene silencing blocks IGF-1- or Cu-stimulated VEGF expression. HIF-1alpha coimmunoprecipitates with a Cu chaperone for superoxide dismutase-1 (CCS), and gene silencing of CCS, but not superoxide dismutase-1, prevents IGF-1- or Cu-induced HIF-1alpha activation and VEGF expression. Therefore, dietary Cu supplementation improves the condition of hypertrophic cardiomyopathy at least in part through CCS-mediated HIF-1alpha activation of VEGF expression and angiogenesis. Less
Accumulation of advanced glycation endproduct (AGE) has been implicated in the pathogenesis of diabetic complications. However, the precise role and mechanism behind AGE-... More
Accumulation of advanced glycation endproduct (AGE) has been implicated in the pathogenesis of diabetic complications. However, the precise role and mechanism behind AGE-associated diabetic heart injury are not fully clear. This study was designed to evaluate the effect of AGE on accumulation of reactive oxygen species (ROS), apoptosis, mitogen-activated protein kinase (MAPK) activation and nuclear O-GlcNAcylation in fetal human cardiac myocytes. Myocytes were maintained for 24-72 h in a defined culture medium containing high glucose, the AGE carbon precursor methylglyoxal (MG), and MG-AGE derived from MG and bovine serum albumin (BSA). Generation of ROS was detected by 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by caspase-3 activity and quantitative DNA fragmentation. Both high glucose (25.5 mM) and MG (200 microM) significantly enhanced ROS and AGE formation with greater effects elicited by MG. Both high glucose and MG-AGE significantly facilitated apoptosis with a more predominant effect from MG-AGE. In addition, phosphorylation of MAPK cascade [extracellular signal-regulated kinase-1/2 (ERK1/2) and p38] and nuclear O-GlcNAcylation were enhanced in MG-AGE-treated myocytes, similar to those elicited by high glucose. MG-AGE-induced phosphorylation of ERK1/2 and p38 was nullified by neutralizing AGE with specific anti-AGE antibody but not nonspecific antiserum. Collectively, these results indicated that AGE or its precursor MG may trigger ROS generation, apoptosis, MAPK activation and nuclear O-GlcNAcylation in human cardiac myocytes, in a manner reminiscent of high extracellular glucose. Less
Our previous studies demonstrated that light-induced vascular relaxation (photorelaxation) was mediated by a tissue source of nitric oxide that was independent of endothe... More
Our previous studies demonstrated that light-induced vascular relaxation (photorelaxation) was mediated by a tissue source of nitric oxide that was independent of endothelial nitric oxide synthase (eNOS), but sensitive to inhibitors of soluble guanylate cyclase, extracellular nitric oxide scavengers and possessed the properties of a nitrosothiol. In the present study we describe High Performance Liquid Chromatography and spectrofluorometric techniques that allowed us to measure tissue levels of the nitrosothiol, S-nitrosoglutathione and its modulation in mouse aortic tissues, smooth muscle cells and human umbilical vein endothelial cells (HUVECs) following exposure to exogenous S-nitrosoglutathione, light and chemical stimuli. Basal levels of S-nitrosoglutathione were similar in control mouse aortae and HUVECs and the store size could be enhanced by exposure of tissues/cells to nitric oxide solution. No basal S-nitrosoglutathione was detected in tissue from diabetic db/db mice; however, ultraviolet light was still able to elicit relaxation of aortic tissues. Ultraviolet light induced the release of nitric oxide from the S-nitrosoglutathione store with an associated increase in the concentration of nitrite. The release of nitric oxide from the store in HUVECs was modulated by extracellular oxidative stress induced by xanthine/xanthine oxidase and also, in an atropine-sensitive process, by acetylcholine, as well as by the calcium ionophore, ionomycin. These interventions resulted in a reduced S-nitrosoglutathione store and elevated levels of nitrite. These data suggest that endothelial and vascular smooth muscle cells possess stores of nitric oxide that, in part, exist in the form of S-nitrosoglutathione. Furthermore, these stores, albeit small, may provide an additional mechanism for the regulation of vascular tone, especially under conditions, such as diabetes, in which nitric oxide generation or bioavailability is compromised; however, additional studies are required to determine not only whether there are additional chemical storage forms of nitric oxide, but also the location of such stores. Less
The human EGF receptor (HER) 2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, and its inhibition may explain the increased incidence of cardiomyo... More
The human EGF receptor (HER) 2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, and its inhibition may explain the increased incidence of cardiomyopathy associated with the anti-HER2 monoclonal antibody trastuzumab (Genentech, South San Francisco, CA), particularly in patients with prior exposure to cardiotoxic chemotherapies e.g., anthracyclines. Here, we show that GW2974 (HER2/EGF receptor tyrosine kinase inhibitor), but not trastuzumab, activates AMP-activated protein kinase (AMPK), initiating a metabolic stress response in human cardiomyocytes that protects against TNFalpha-induced cell death. GW2974 stimulates calcium dependent fatty acid oxidation in vitro and in the myocardium of GW2974-treated rodents. Calcium chelation or siRNA-targeted AMPK knockdown blocks GW2974 induced fatty acid oxidation. In addition, inhibition of AMPK by a specific inhibitor resulted in increased killing of cardiomyocytes. Elucidating the effects of HER2-targeted therapies on AMPK may predict for risk of cardiomyopathy and provide a novel HER2-targeted strategy designed to protect myocardium from the pro-apoptotic effects of pro-inflammatory cytokines released in response to cardiac injury by chemotherapy or acute ischemia. Less
Acetaldehyde, the major metabolite of ethanol, which is far more toxic and reactive than ethanol, may be responsible for alcohol-induced cardiac damage. This study was de... More
Acetaldehyde, the major metabolite of ethanol, which is far more toxic and reactive than ethanol, may be responsible for alcohol-induced cardiac damage. This study was designed to examine the impact of facilitated acetaldehyde metabolism using transfection of human aldehyde dehydrogenase-2 (ALDH2) transgene on acetaldehyde- and ethanol-induced cell injury. Fetal human cardiac myocytes were transfected with ALDH2, the efficacy of which was verified by flow cytometry, Western blot and ALDH2 activity assays. Generation of reactive oxygen species (ROS) was detected using 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride (DAPI) fluorescence microscopy, quantitative DNA fragmentation ELISA and caspase 3 activity. Acetaldehyde and ethanol elicited overt ROS generation and apoptosis in human cardiac myocytes following 24-48 h of incubation. Immunostaining revealed activation of the MAP kinase cascades ERK1/2, SAPK/JNK and p38 MAP kinase in acetaldehyde-treated myocytes. Interestingly, ALDH2 transgene significantly attenuated acetaldehyde-induced ROS generation, apoptosis and phosphorylation of ERK1/2 and SAPK/JNK. Time-dependent response (0-12 h) revealed ROS accumulation and activation of MAP kinases prior to acetaldehyde-induced apoptosis. In addition, acetaldehyde-induced ROS generation and apoptosis were antagonized by non-enzymatic antioxidants. Our results suggested that ALDH2 transgene overexpression may effectively alleviate acetaldehyde-elicited cell injury through an ERK1/2 and SPAK/JNK-dependent mechanism. Our data are consistent with the notion of acetaldehyde as a contributor to alcoholic cardiomyopathy and implicate the therapeutic potential of ALDH2 enzyme in alcoholic complications. Less
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