Wendy Keung - Academia.edu (original) (raw)

Papers by Wendy Keung

Research paper thumbnail of Morphometric Analysis of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes: Determining the Maturation State of a Population by Quantifying Parameters in Individual Cells

Stem cells international, 2015

Quantitative methods were established to determine the level of maturation of human embryonic ste... more Quantitative methods were established to determine the level of maturation of human embryonic stem cell-derived ventricular cardiomyocytes (hESC-vCMs) that were treated with different metabolic stimulants (i.e., isoproterenol and oleic acid) during early differentiation. Cells were double-immunolabeled with α-actinin and COX IV antibodies, to label the myofibrils and mitochondria, respectively, after which images were acquired via confocal microscopy. In order to determine the extent of differentiation, image analysis protocols were then used to quantify cell shape and area, as well as the degree of myofibrillar organization and intercalation of mitochondria between the myofibrils within the cells. We demonstrated that oleic acid or isoproterenol alone, or a combination of the two, induced a more elongated hESC-vCM phenotype than the untreated controls. In addition, cells treated with isoproterenol alone exhibited a similar level of myofibrillar organization as the controls, but tho...

Research paper thumbnail of Activating PPARα Prevents Post-Ischemic Contractile Dysfunction in Hypertrophied Neonatal Hearts

Circulation research, Jan 14, 2015

Post-ischemic contractile dysfunction is a contributor to morbidity and mortality following the s... more Post-ischemic contractile dysfunction is a contributor to morbidity and mortality following the surgical correction of congenital heart defects (CHDs) in neonatal patients. Pre-existing hypertrophy in the newborn heart can exacerbate these ischemic injuries, which may partly be due to a decreased energy supply to the heart resulting from low fatty acid β-oxidation rates. We determined whether stimulating fatty acid β-oxidation with GW7647, a peroxisome proliferator activated receptor-α (PPARα) activator, would improve cardiac energy production and post-ischemic functional recovery in neonatal rabbit hearts subjected to volume overload-induced cardiac hypertrophy. Volume-overload cardiac hypertrophy was produced in 7-day-old rabbits via an aorto-caval shunt, following which, the rabbits were treated with or without GW7647 (3 mg/kg/day) for 14 days. Biventricular working hearts were subjected to 35 min of aerobic perfusion, 25 min of global no-flow ischemia, and 30 min of aerobic repe...

Research paper thumbnail of Nongenomic responses to 17beta-estradiol in male rat mesenteric arteries abolish intrinsic gender differences in vascular responses

British journal of pharmacology, 2005

The aim of the present study was to investigate the gender differences in the acute effects of 17... more The aim of the present study was to investigate the gender differences in the acute effects of 17beta-estradiol on the rat superior mesenteric artery. Isometric tension was measured in rings of mesenteric arteries from both male and female Sprague-Dawley rats. Relaxation to acetylcholine was not significantly different between arteries (with endothelium) from male and female rats in the absence or presence of 17beta-estradiol. After blockade of endothelium-dependent hyperpolarizations with apamin (0.3 microM) plus charybdotoxin (0.1 microM), acute exposure to 17beta-estradiol (1 nM) for 30 min resulted in enhancement of relaxation to acetylcholine in arteries from male but not female rats. After acute exposure to 17beta-estradiol, mesenteric arteries from male rats were more sensitive to sodium nitroprusside than arteries from female rats. Contractions of mesenteric arteries to phenylephrine and 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619) were greater in...

Research paper thumbnail of Accumulation of ceramide in slow-twitch muscle contributes to the development of insulin resistance in the obese JCR:LA-cp rat

Experimental Physiology, 2015

The aim was to determine whether the accumulation of ceramide contributes to skeletal muscle insu... more The aim was to determine whether the accumulation of ceramide contributes to skeletal muscle insulin resistance in the JCR obese rat. r What is the main finding and its importance?

Research paper thumbnail of Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

Circulation. Cardiovascular genetics, Jan 10, 2015

-Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represe... more -Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes ( V: CMs), but hESC- V: CMs are developmentally immature. Previous attempts to profile hESC- V: CMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived V: CMs, human fetal (hF) and adult (hA) V: and atrial ( A: ) CMs. -Using 2D-Differential-In-Gel Electrophoresis (DIGE), 121 differentially expressed (>1.5-fold, p<0.05) proteins were detected. The dataset implicated a role of the peroxisome proliferator-activated receptor alpha (PPARA) signalling in cardiac maturation. Consistently, WY-14643, a PPARA agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine (T3) increased the dynamic tension developed in engineered human ventricular cardiac microtissue (hvCMT) by 3-folds, signifying their maturation. -We conclude that the PPARA and thyroid hormone pathways modulate the metabolism and maturation of hESC- V: CMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

Research paper thumbnail of Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs

Circulation. Arrhythmia and electrophysiology, 2015

Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a poten... more Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitor...

Research paper thumbnail of Abstracts-Acute Impairment of Contraction in Porcine Coronary Arteries by 17b Estradiol Involves both the Cyclic AMP and the Cyclic GMP Pathways

Research paper thumbnail of Non-genomic vascular effect of 17β-estradiol involves activation of adenylyl cyclase and protein kinase G in the rat mesenteric artery

Research paper thumbnail of INHIBITION OF MITOCHONDRIAL FATTY ACID UPTAKE IMPROVES CARDIAC INSULIN SENSITIVITY IN DIET-INDUCED OBESE MICE

Research paper thumbnail of The vasorelaxation effect of osthole, derived from Radix Angelicae Pubescentis, in porcine coronary artery

Research paper thumbnail of Identification of Myosin Light Chain Kinase as a Novel Myocardial 5'-AMP Activated Protein Kinase Kinase

Research paper thumbnail of Phytoestrogens and Cardiovascular Disorders

Progress in Experimental Cardiology, 2004

Dietary intake of phytoestrogens has been associated with a reduction in risk of cardiovascular d... more Dietary intake of phytoestrogens has been associated with a reduction in risk of cardiovascular disorders including coronary heart disease, hypertension and atherosclerosis. Phytoestrogens have long been found to be structurally similar to the female sex hormone, 17ß-estradiol, and have a wide range of estrogenic effects. Like 17ß-estradiol, phytoestrogens such as genistein have been suggested to exert its cardioprotective actions through

Research paper thumbnail of Molecular Changes in Fatty Acid Oxidation in the Failing Heart

Molecular Defects in Cardiovascular Disease, 2011

Research paper thumbnail of Transcriptome-Guided Functional Analyses Reveal Novel Biological Properties and Regulatory Hierarchy of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes Crucial for Maturation

Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); howev... more Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or nonchamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed I KATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Research paper thumbnail of Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake an... more Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptin's Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptin's anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptinmediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptin's signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptin's anorectic signaling cascade.

Research paper thumbnail of Epigenetic Regulation of the Electrophysiological Phenotype of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes: Insights for Driven Maturation and Hypertrophic Growth

Stem Cells and Development, 2013

Epigenetic regulation is implicated in embryonic development and the control of gene expression i... more Epigenetic regulation is implicated in embryonic development and the control of gene expression in a cell-specific manner. However, little is known about the role of histone methylation changes on human cardiac differentiation and maturation. Using human embryonic stem cells (hESCs) and their derived ventricular (V) cardiomyocytes (CMs) as a model, we examined trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) on promoters of genes associated with cardiac electrophysiology, contraction, and Ca(2+) handling. To avoid ambiguities due to heterogeneous chamber-specific types, hESC-derived ventricular cardiomyocytes (VCMs) were selected by dual zeocin-GFP expression under the transcriptional control of the MLC2v promoter and confirmed electrophysiologically by its signature action potential phenotype. High levels of H3K4me3 are present on pluripotency genes in hESCs with an absence of H3K27me3. Human ESC-VCMS, relative to hESCs, were characterized by a profound loss of H3K27me3 and an enrichment of H3K4me3 marks on cardiac-specific genes, including MYH6, MYH7, MYL2, cTNT, and ANF. Gene transcripts encoding key voltage-gated ion channels and Ca(2+)-handling proteins in hESC-VCMs were significantly increased, which could be attributed to a distinct pattern of differential H3K4me3 and H3K27me3 profiles. Treatment of hESC-VCMs with the histone deacetylase inhibitor valproic acid increased H3K4me3 on gene promoters, induced hypertrophic growth (as gauged by cell volume and capacitance), and augmented cardiac gene expression, but it did not affect electrophysiological properties of these cells. Hence, cardiac differentiation of hESCs involves a dynamic shift in histone methylation, which differentially affects VCM gene expression and function. We conclude that the epigenetic state of hESC-VCMs is dynamic and primed to promote growth and developmental maturation, but that proper environmental stimuli with chromatin remodeling will be required to synergistically trigger global CM maturation to a more adult-like phenotype.

Research paper thumbnail of A Simple, Cost-Effective but Highly Efficient System for Deriving Ventricular Cardiomyocytes from Human Pluripotent Stem Cells

Stem Cells and Development, 2014

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source... more Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.

Research paper thumbnail of Developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties of human pluripotent stem cell-derived cardiomyocytes

Stem Cell Research & Therapy, 2014

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are... more Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are abundant sources of cardiomyocytes (CMs) for cell replacement therapy and other applications such as disease modeling, drug discovery and cardiotoxicity screening. However, hPSC-derived CMs display immature structural, electrophysiological, calcium-handling and metabolic properties. Here, we review various biological as well as physical and topographical cues that are known to associate with the development of native CMs in vivo to gain insights into the development of strategies for facilitated maturation of hPSC-CMs. Full list of author information is available at the end of the article widely accepted that their functional and structural properties are immature in multiple aspects, with embryonic-or fetal-like electrophysiological, calcium-handling and metabolic signatures. Here, we review recent efforts that have been made to understand the different biological cues for driving maturation.

Research paper thumbnail of Inhibition of Serine Palmitoyl Transferase I Reduces Cardiac Ceramide Levels and Increases Glycolysis Rates following Diet-Induced Insulin Resistance

PLoS ONE, 2012

Objective: Diet-induced obesity (DIO) leads to an accumulation of intra-myocardial lipid metaboli... more Objective: Diet-induced obesity (DIO) leads to an accumulation of intra-myocardial lipid metabolites implicated in causing cardiac insulin resistance and contractile dysfunction. One such metabolite is ceramide, and our aim was to determine the effects of inhibiting de novo ceramide synthesis on cardiac function and insulin stimulated glucose utilization in mice subjected to DIO.

Research paper thumbnail of Differential effects of central ghrelin on fatty acid metabolism in hypothalamic ventral medial and arcuate nuclei

Physiology & Behavior, 2013

Fatty acid metabolism is an important pathway involved in the hypothalamus-mediated control of fo... more Fatty acid metabolism is an important pathway involved in the hypothalamus-mediated control of food intake. Previous studies using whole hypothalamic tissue lysates have shown that fatty acid metabolism plays a key role in ghrelin's effect on feeding. Here, we report site-specific effects of central ghrelin on fatty acid metabolism in two critical hypothalamic nuclei, the ventral medial nucleus (VMN) and the arcuate nucleus (Arc). Intracerebroventricular administration of ghrelin to rats activates AMP-activated protein kinase in both the VMN and the Arc, while ghrelin treatment has a site-specific effect on fatty acid metabolic pathways in these two nuclei. In the VMN, central ghrelin increases the phosphorylation level of ACC, indicating the decrease in activity, and decreases the level of malonyl-CoA (the product of ACC). Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase-1 (CPT-1) that is a key enzyme in mitochondrial fatty acid oxidation. Consistent with this action of malonyl-CoA on CPT-1, central ghrelin treatment increases the activity of CPT-1 in the VMN. In contrast, in the Arc, neither malonyl-CoA level nor CPT-1 activity is affected following central ghrelin. Taken together, our data suggest ghrelin exerts differential effects on fatty acid metabolic pathways in the VMN and the Arc.

Research paper thumbnail of Morphometric Analysis of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes: Determining the Maturation State of a Population by Quantifying Parameters in Individual Cells

Stem cells international, 2015

Quantitative methods were established to determine the level of maturation of human embryonic ste... more Quantitative methods were established to determine the level of maturation of human embryonic stem cell-derived ventricular cardiomyocytes (hESC-vCMs) that were treated with different metabolic stimulants (i.e., isoproterenol and oleic acid) during early differentiation. Cells were double-immunolabeled with α-actinin and COX IV antibodies, to label the myofibrils and mitochondria, respectively, after which images were acquired via confocal microscopy. In order to determine the extent of differentiation, image analysis protocols were then used to quantify cell shape and area, as well as the degree of myofibrillar organization and intercalation of mitochondria between the myofibrils within the cells. We demonstrated that oleic acid or isoproterenol alone, or a combination of the two, induced a more elongated hESC-vCM phenotype than the untreated controls. In addition, cells treated with isoproterenol alone exhibited a similar level of myofibrillar organization as the controls, but tho...

Research paper thumbnail of Activating PPARα Prevents Post-Ischemic Contractile Dysfunction in Hypertrophied Neonatal Hearts

Circulation research, Jan 14, 2015

Post-ischemic contractile dysfunction is a contributor to morbidity and mortality following the s... more Post-ischemic contractile dysfunction is a contributor to morbidity and mortality following the surgical correction of congenital heart defects (CHDs) in neonatal patients. Pre-existing hypertrophy in the newborn heart can exacerbate these ischemic injuries, which may partly be due to a decreased energy supply to the heart resulting from low fatty acid β-oxidation rates. We determined whether stimulating fatty acid β-oxidation with GW7647, a peroxisome proliferator activated receptor-α (PPARα) activator, would improve cardiac energy production and post-ischemic functional recovery in neonatal rabbit hearts subjected to volume overload-induced cardiac hypertrophy. Volume-overload cardiac hypertrophy was produced in 7-day-old rabbits via an aorto-caval shunt, following which, the rabbits were treated with or without GW7647 (3 mg/kg/day) for 14 days. Biventricular working hearts were subjected to 35 min of aerobic perfusion, 25 min of global no-flow ischemia, and 30 min of aerobic repe...

Research paper thumbnail of Nongenomic responses to 17beta-estradiol in male rat mesenteric arteries abolish intrinsic gender differences in vascular responses

British journal of pharmacology, 2005

The aim of the present study was to investigate the gender differences in the acute effects of 17... more The aim of the present study was to investigate the gender differences in the acute effects of 17beta-estradiol on the rat superior mesenteric artery. Isometric tension was measured in rings of mesenteric arteries from both male and female Sprague-Dawley rats. Relaxation to acetylcholine was not significantly different between arteries (with endothelium) from male and female rats in the absence or presence of 17beta-estradiol. After blockade of endothelium-dependent hyperpolarizations with apamin (0.3 microM) plus charybdotoxin (0.1 microM), acute exposure to 17beta-estradiol (1 nM) for 30 min resulted in enhancement of relaxation to acetylcholine in arteries from male but not female rats. After acute exposure to 17beta-estradiol, mesenteric arteries from male rats were more sensitive to sodium nitroprusside than arteries from female rats. Contractions of mesenteric arteries to phenylephrine and 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619) were greater in...

Research paper thumbnail of Accumulation of ceramide in slow-twitch muscle contributes to the development of insulin resistance in the obese JCR:LA-cp rat

Experimental Physiology, 2015

The aim was to determine whether the accumulation of ceramide contributes to skeletal muscle insu... more The aim was to determine whether the accumulation of ceramide contributes to skeletal muscle insulin resistance in the JCR obese rat. r What is the main finding and its importance?

Research paper thumbnail of Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

Circulation. Cardiovascular genetics, Jan 10, 2015

-Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represe... more -Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes ( V: CMs), but hESC- V: CMs are developmentally immature. Previous attempts to profile hESC- V: CMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived V: CMs, human fetal (hF) and adult (hA) V: and atrial ( A: ) CMs. -Using 2D-Differential-In-Gel Electrophoresis (DIGE), 121 differentially expressed (>1.5-fold, p<0.05) proteins were detected. The dataset implicated a role of the peroxisome proliferator-activated receptor alpha (PPARA) signalling in cardiac maturation. Consistently, WY-14643, a PPARA agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine (T3) increased the dynamic tension developed in engineered human ventricular cardiac microtissue (hvCMT) by 3-folds, signifying their maturation. -We conclude that the PPARA and thyroid hormone pathways modulate the metabolism and maturation of hESC- V: CMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

Research paper thumbnail of Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs

Circulation. Arrhythmia and electrophysiology, 2015

Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a poten... more Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitor...

Research paper thumbnail of Abstracts-Acute Impairment of Contraction in Porcine Coronary Arteries by 17b Estradiol Involves both the Cyclic AMP and the Cyclic GMP Pathways

Research paper thumbnail of Non-genomic vascular effect of 17β-estradiol involves activation of adenylyl cyclase and protein kinase G in the rat mesenteric artery

Research paper thumbnail of INHIBITION OF MITOCHONDRIAL FATTY ACID UPTAKE IMPROVES CARDIAC INSULIN SENSITIVITY IN DIET-INDUCED OBESE MICE

Research paper thumbnail of The vasorelaxation effect of osthole, derived from Radix Angelicae Pubescentis, in porcine coronary artery

Research paper thumbnail of Identification of Myosin Light Chain Kinase as a Novel Myocardial 5'-AMP Activated Protein Kinase Kinase

Research paper thumbnail of Phytoestrogens and Cardiovascular Disorders

Progress in Experimental Cardiology, 2004

Dietary intake of phytoestrogens has been associated with a reduction in risk of cardiovascular d... more Dietary intake of phytoestrogens has been associated with a reduction in risk of cardiovascular disorders including coronary heart disease, hypertension and atherosclerosis. Phytoestrogens have long been found to be structurally similar to the female sex hormone, 17ß-estradiol, and have a wide range of estrogenic effects. Like 17ß-estradiol, phytoestrogens such as genistein have been suggested to exert its cardioprotective actions through

Research paper thumbnail of Molecular Changes in Fatty Acid Oxidation in the Failing Heart

Molecular Defects in Cardiovascular Disease, 2011

Research paper thumbnail of Transcriptome-Guided Functional Analyses Reveal Novel Biological Properties and Regulatory Hierarchy of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes Crucial for Maturation

Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); howev... more Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or nonchamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed I KATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Research paper thumbnail of Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake an... more Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptin's Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptin's anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptinmediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptin's signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptin's anorectic signaling cascade.

Research paper thumbnail of Epigenetic Regulation of the Electrophysiological Phenotype of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes: Insights for Driven Maturation and Hypertrophic Growth

Stem Cells and Development, 2013

Epigenetic regulation is implicated in embryonic development and the control of gene expression i... more Epigenetic regulation is implicated in embryonic development and the control of gene expression in a cell-specific manner. However, little is known about the role of histone methylation changes on human cardiac differentiation and maturation. Using human embryonic stem cells (hESCs) and their derived ventricular (V) cardiomyocytes (CMs) as a model, we examined trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) on promoters of genes associated with cardiac electrophysiology, contraction, and Ca(2+) handling. To avoid ambiguities due to heterogeneous chamber-specific types, hESC-derived ventricular cardiomyocytes (VCMs) were selected by dual zeocin-GFP expression under the transcriptional control of the MLC2v promoter and confirmed electrophysiologically by its signature action potential phenotype. High levels of H3K4me3 are present on pluripotency genes in hESCs with an absence of H3K27me3. Human ESC-VCMS, relative to hESCs, were characterized by a profound loss of H3K27me3 and an enrichment of H3K4me3 marks on cardiac-specific genes, including MYH6, MYH7, MYL2, cTNT, and ANF. Gene transcripts encoding key voltage-gated ion channels and Ca(2+)-handling proteins in hESC-VCMs were significantly increased, which could be attributed to a distinct pattern of differential H3K4me3 and H3K27me3 profiles. Treatment of hESC-VCMs with the histone deacetylase inhibitor valproic acid increased H3K4me3 on gene promoters, induced hypertrophic growth (as gauged by cell volume and capacitance), and augmented cardiac gene expression, but it did not affect electrophysiological properties of these cells. Hence, cardiac differentiation of hESCs involves a dynamic shift in histone methylation, which differentially affects VCM gene expression and function. We conclude that the epigenetic state of hESC-VCMs is dynamic and primed to promote growth and developmental maturation, but that proper environmental stimuli with chromatin remodeling will be required to synergistically trigger global CM maturation to a more adult-like phenotype.

Research paper thumbnail of A Simple, Cost-Effective but Highly Efficient System for Deriving Ventricular Cardiomyocytes from Human Pluripotent Stem Cells

Stem Cells and Development, 2014

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source... more Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.

Research paper thumbnail of Developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties of human pluripotent stem cell-derived cardiomyocytes

Stem Cell Research & Therapy, 2014

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are... more Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, are abundant sources of cardiomyocytes (CMs) for cell replacement therapy and other applications such as disease modeling, drug discovery and cardiotoxicity screening. However, hPSC-derived CMs display immature structural, electrophysiological, calcium-handling and metabolic properties. Here, we review various biological as well as physical and topographical cues that are known to associate with the development of native CMs in vivo to gain insights into the development of strategies for facilitated maturation of hPSC-CMs. Full list of author information is available at the end of the article widely accepted that their functional and structural properties are immature in multiple aspects, with embryonic-or fetal-like electrophysiological, calcium-handling and metabolic signatures. Here, we review recent efforts that have been made to understand the different biological cues for driving maturation.

Research paper thumbnail of Inhibition of Serine Palmitoyl Transferase I Reduces Cardiac Ceramide Levels and Increases Glycolysis Rates following Diet-Induced Insulin Resistance

PLoS ONE, 2012

Objective: Diet-induced obesity (DIO) leads to an accumulation of intra-myocardial lipid metaboli... more Objective: Diet-induced obesity (DIO) leads to an accumulation of intra-myocardial lipid metabolites implicated in causing cardiac insulin resistance and contractile dysfunction. One such metabolite is ceramide, and our aim was to determine the effects of inhibiting de novo ceramide synthesis on cardiac function and insulin stimulated glucose utilization in mice subjected to DIO.

Research paper thumbnail of Differential effects of central ghrelin on fatty acid metabolism in hypothalamic ventral medial and arcuate nuclei

Physiology & Behavior, 2013

Fatty acid metabolism is an important pathway involved in the hypothalamus-mediated control of fo... more Fatty acid metabolism is an important pathway involved in the hypothalamus-mediated control of food intake. Previous studies using whole hypothalamic tissue lysates have shown that fatty acid metabolism plays a key role in ghrelin's effect on feeding. Here, we report site-specific effects of central ghrelin on fatty acid metabolism in two critical hypothalamic nuclei, the ventral medial nucleus (VMN) and the arcuate nucleus (Arc). Intracerebroventricular administration of ghrelin to rats activates AMP-activated protein kinase in both the VMN and the Arc, while ghrelin treatment has a site-specific effect on fatty acid metabolic pathways in these two nuclei. In the VMN, central ghrelin increases the phosphorylation level of ACC, indicating the decrease in activity, and decreases the level of malonyl-CoA (the product of ACC). Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase-1 (CPT-1) that is a key enzyme in mitochondrial fatty acid oxidation. Consistent with this action of malonyl-CoA on CPT-1, central ghrelin treatment increases the activity of CPT-1 in the VMN. In contrast, in the Arc, neither malonyl-CoA level nor CPT-1 activity is affected following central ghrelin. Taken together, our data suggest ghrelin exerts differential effects on fatty acid metabolic pathways in the VMN and the Arc.