The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary cent... more The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2′ or C3′ is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2′ was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.
Proceedings of the National Academy of Sciences, 2020
The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its struct... more The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.
Background: Bicuspid aortic valve (BAV), the most common congenital heart defect affecting 1% to ... more Background: Bicuspid aortic valve (BAV), the most common congenital heart defect affecting 1% to 2% of the population, is a major risk factor for premature aortic valve disease and accounts for the majority of valve replacement. The genetic basis and mechanisms of BAV etiology and pathogenesis remain largely undefined. Methods: Cardiac structure and function was assessed in mice lacking a Gata6 allele. Human GATA6 gene variants were analyzed in 452 BAV cases from the BAV consortium and 1849 controls from the Framingham GWAS (Genome Wide Association Study). GATA6 expression was determined in mice and human tissues using quantitative real-time polymerase chain reaction and immunohistochemistry. Mechanistic studies were carried out in cultured cells. Results: Gata6 heterozygous mice have highly penetrant right-left (RL)-type BAV, the most frequent type in humans. GATA6 transcript levels are lower in human BAV compared with normal tricuspid valves. Mechanistically, Gata6 haploinsufficie...
Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of g... more Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of growth factors. The identity of these factors, however, remains largely unknown, and we hypothesized here that the secreted C1q-TNF-related protein-9 (CTRP9) might act as endothelial derived protein to modulate heart remodeling in response to pressure overload. To examine the source of cardiac CTRP9 and its function during pressure overload. CTRP9 was mainly derived from myocardial capillary endothelial cells. CTRP9 mRNA expression was enhanced in hypertrophic human hearts and in mouse hearts after transverse aortic constriction (TAC). CTRP9 protein was more abundant in the serum of patients with severe aortic stenosis and in murine hearts after TAC. Interestingly, heterozygous and especially homozygous (KO) C1qtnf9 gene-deleted (CTRP9 knock-out) mice were protected from the development of cardiac hypertrophy, left ventricular dilatation and dysfunction during TAC. CTRP9 overexpression, i...
Transgenic mice that overexpress human type 1 angiotensin II receptor (AT1R) in the heart develop... more Transgenic mice that overexpress human type 1 angiotensin II receptor (AT1R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT1R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT1R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT1R overexpression on ventricular L-type Ca2+ currents ( ICaL), cell shortening, and Ca2+ handling in 50-day and 6-mo-old male AT1R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT1R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, ICaL, and Cav1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT1R mice compared with controls. In addition, Ca2+ transients and caffeine-induced Ca2+ transients were reduced whereas the time to 90% Ca2+ ...
In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the ... more In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony–stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit–dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL– or dn-SCL– expressing cells. c-kit mRNA, c-kit promoter activity, and the response to ...
In vertebrates, heart development is a complex process requiring proper differentiation and inter... more In vertebrates, heart development is a complex process requiring proper differentiation and interaction between myocardial and endocardial cells. Significant progress has been made in elucidating the molecular events underlying myocardial cell differentiation. In contrast, little is known about the development of the endocardial lineage that gives rise to cardiac valves and septa. We have used a novel in vitro model to identify the molecular hierarchy of endocardial differentiation and the role of transcription factor GATA5 in endocardial development. The results indicate that GATA5 is induced at an early stage of endothelial-endocardial differentiation prior to expression of such early endocardial markers as Tie2 and ErbB3. Inhibition of either GATA5 expression or NF-ATc activation, blocks terminal differentiation at a pre-endocardial stage and GATA5 and NF-ATc synergistically activate endocardial transcription. The data reveal that transcription factor GATA5 is required for differ...
In vertebrates, heart development is a complex process requiring proper differentiation and inter... more In vertebrates, heart development is a complex process requiring proper differentiation and interaction between myocardial and endocardial cells. Significant progress has been made in elucidating the molecular events underlying myocardial cell differentiation. In contrast, little is known about the development of the endocardial lineage that gives rise to cardiac valves and septa. We have used a novel in vitro model to identify the molecular hierarchy of endocardial differentiation and the role of transcription factor GATA5 in endocardial development. The results indicate that GATA5 is induced at an early stage of endothelial-endocardial differentiation prior to expression of such early endocardial markers as Tie2 and ErbB3. Inhibition of either GATA5 expression or NF-ATc activation, blocks terminal differentiation at a pre-endocardial stage and GATA5 and NF-ATc synergistically activate endocardial transcription. The data reveal that transcription factor GATA5 is required for differ...
The G1 cyclins play a pivotal role in regulation of cell differentiation and proliferation. The m... more The G1 cyclins play a pivotal role in regulation of cell differentiation and proliferation. The mechanisms underlying their cellspecific roles are incompletely understood. Here, we show that a G1 cyclin, cyclin D2 (CycD2), enhances the activity of transcription factor GATA4, a key regulator of cardiomyocyte growth and differentiation. GATA4 recruits CycD2 to its target promoters, and their interaction results in synergistic activation of GATA-dependent transcription. This effect is specific to CycD2 because CycD1 is unable to potentiate activity of GATA4 and is CDK-independent. GATA4 physically interacts with CycD2 through a discreet N-terminal activation domain that is essential for the cardiogenic activity of GATA4. Human mutations in this domain that are linked to congenital heart disease interfere with CycD2-GATA4 synergy. Cardiogenesis assays in Xenopus embryos indicate that CycD2 enhances the cardiogenic function of GATA4. Together, our data uncover a role for CycD2 as a cardi...
Cardiac development is governed by a complex network of transcription factors (TFs) that regulate... more Cardiac development is governed by a complex network of transcription factors (TFs) that regulate cell fates in a spatiotemporal manner. Among these, the GATA family of zinc finger TFs plays prominent roles in regulating the development of the myocardium, endocardium, and outflow tract. This family comprises six members three of which, GATA4, 5, and 6, are predominantly expressed in cardiac cells where they activate specific downstream gene targets via interactions with one another and with other TFs and signaling molecules. Their critical function in heart formation is evidenced by the phenotypes of animal models lacking these factors and by the broad spectrum of human congenital heart diseases associated with mutations in their genes. Similarly, in the postnatal heart, these proteins play significant and nonredundant roles in cardiac function, regulating adaptive stress responses including cardiomyocyte hypertrophy and survival, as well as endothelial homeostasis and angiogenesis. As such, decreased expression of either GATA4, 5, or 6 results in impaired cardiovascular homeostasis and increased risk of premature and serious cardiovascular events such as hypertension, arrhythmia, aortopathy, and heart failure. Although a great deal of progress has been made in understanding GATA‐dependent regulatory processes in the heart, the molecular mechanisms underlying the specificity of GATA factors and their upstream regulation remain incompletely understood. The knowledge and tools developed since their discovery 25 years ago should accelerate progress toward further elucidation of their mechanisms of action in health and disease. This in turn will greatly improve diagnosis and care for the millions of individuals affected by congenital and acquired cardiac disease worldwide.
The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary cent... more The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2′ or C3′ is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2′ was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.
Proceedings of the National Academy of Sciences, 2020
The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its struct... more The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.
Background: Bicuspid aortic valve (BAV), the most common congenital heart defect affecting 1% to ... more Background: Bicuspid aortic valve (BAV), the most common congenital heart defect affecting 1% to 2% of the population, is a major risk factor for premature aortic valve disease and accounts for the majority of valve replacement. The genetic basis and mechanisms of BAV etiology and pathogenesis remain largely undefined. Methods: Cardiac structure and function was assessed in mice lacking a Gata6 allele. Human GATA6 gene variants were analyzed in 452 BAV cases from the BAV consortium and 1849 controls from the Framingham GWAS (Genome Wide Association Study). GATA6 expression was determined in mice and human tissues using quantitative real-time polymerase chain reaction and immunohistochemistry. Mechanistic studies were carried out in cultured cells. Results: Gata6 heterozygous mice have highly penetrant right-left (RL)-type BAV, the most frequent type in humans. GATA6 transcript levels are lower in human BAV compared with normal tricuspid valves. Mechanistically, Gata6 haploinsufficie...
Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of g... more Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of growth factors. The identity of these factors, however, remains largely unknown, and we hypothesized here that the secreted C1q-TNF-related protein-9 (CTRP9) might act as endothelial derived protein to modulate heart remodeling in response to pressure overload. To examine the source of cardiac CTRP9 and its function during pressure overload. CTRP9 was mainly derived from myocardial capillary endothelial cells. CTRP9 mRNA expression was enhanced in hypertrophic human hearts and in mouse hearts after transverse aortic constriction (TAC). CTRP9 protein was more abundant in the serum of patients with severe aortic stenosis and in murine hearts after TAC. Interestingly, heterozygous and especially homozygous (KO) C1qtnf9 gene-deleted (CTRP9 knock-out) mice were protected from the development of cardiac hypertrophy, left ventricular dilatation and dysfunction during TAC. CTRP9 overexpression, i...
Transgenic mice that overexpress human type 1 angiotensin II receptor (AT1R) in the heart develop... more Transgenic mice that overexpress human type 1 angiotensin II receptor (AT1R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT1R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT1R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT1R overexpression on ventricular L-type Ca2+ currents ( ICaL), cell shortening, and Ca2+ handling in 50-day and 6-mo-old male AT1R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT1R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, ICaL, and Cav1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT1R mice compared with controls. In addition, Ca2+ transients and caffeine-induced Ca2+ transients were reduced whereas the time to 90% Ca2+ ...
In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the ... more In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony–stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit–dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL– or dn-SCL– expressing cells. c-kit mRNA, c-kit promoter activity, and the response to ...
In vertebrates, heart development is a complex process requiring proper differentiation and inter... more In vertebrates, heart development is a complex process requiring proper differentiation and interaction between myocardial and endocardial cells. Significant progress has been made in elucidating the molecular events underlying myocardial cell differentiation. In contrast, little is known about the development of the endocardial lineage that gives rise to cardiac valves and septa. We have used a novel in vitro model to identify the molecular hierarchy of endocardial differentiation and the role of transcription factor GATA5 in endocardial development. The results indicate that GATA5 is induced at an early stage of endothelial-endocardial differentiation prior to expression of such early endocardial markers as Tie2 and ErbB3. Inhibition of either GATA5 expression or NF-ATc activation, blocks terminal differentiation at a pre-endocardial stage and GATA5 and NF-ATc synergistically activate endocardial transcription. The data reveal that transcription factor GATA5 is required for differ...
In vertebrates, heart development is a complex process requiring proper differentiation and inter... more In vertebrates, heart development is a complex process requiring proper differentiation and interaction between myocardial and endocardial cells. Significant progress has been made in elucidating the molecular events underlying myocardial cell differentiation. In contrast, little is known about the development of the endocardial lineage that gives rise to cardiac valves and septa. We have used a novel in vitro model to identify the molecular hierarchy of endocardial differentiation and the role of transcription factor GATA5 in endocardial development. The results indicate that GATA5 is induced at an early stage of endothelial-endocardial differentiation prior to expression of such early endocardial markers as Tie2 and ErbB3. Inhibition of either GATA5 expression or NF-ATc activation, blocks terminal differentiation at a pre-endocardial stage and GATA5 and NF-ATc synergistically activate endocardial transcription. The data reveal that transcription factor GATA5 is required for differ...
The G1 cyclins play a pivotal role in regulation of cell differentiation and proliferation. The m... more The G1 cyclins play a pivotal role in regulation of cell differentiation and proliferation. The mechanisms underlying their cellspecific roles are incompletely understood. Here, we show that a G1 cyclin, cyclin D2 (CycD2), enhances the activity of transcription factor GATA4, a key regulator of cardiomyocyte growth and differentiation. GATA4 recruits CycD2 to its target promoters, and their interaction results in synergistic activation of GATA-dependent transcription. This effect is specific to CycD2 because CycD1 is unable to potentiate activity of GATA4 and is CDK-independent. GATA4 physically interacts with CycD2 through a discreet N-terminal activation domain that is essential for the cardiogenic activity of GATA4. Human mutations in this domain that are linked to congenital heart disease interfere with CycD2-GATA4 synergy. Cardiogenesis assays in Xenopus embryos indicate that CycD2 enhances the cardiogenic function of GATA4. Together, our data uncover a role for CycD2 as a cardi...
Cardiac development is governed by a complex network of transcription factors (TFs) that regulate... more Cardiac development is governed by a complex network of transcription factors (TFs) that regulate cell fates in a spatiotemporal manner. Among these, the GATA family of zinc finger TFs plays prominent roles in regulating the development of the myocardium, endocardium, and outflow tract. This family comprises six members three of which, GATA4, 5, and 6, are predominantly expressed in cardiac cells where they activate specific downstream gene targets via interactions with one another and with other TFs and signaling molecules. Their critical function in heart formation is evidenced by the phenotypes of animal models lacking these factors and by the broad spectrum of human congenital heart diseases associated with mutations in their genes. Similarly, in the postnatal heart, these proteins play significant and nonredundant roles in cardiac function, regulating adaptive stress responses including cardiomyocyte hypertrophy and survival, as well as endothelial homeostasis and angiogenesis. As such, decreased expression of either GATA4, 5, or 6 results in impaired cardiovascular homeostasis and increased risk of premature and serious cardiovascular events such as hypertension, arrhythmia, aortopathy, and heart failure. Although a great deal of progress has been made in understanding GATA‐dependent regulatory processes in the heart, the molecular mechanisms underlying the specificity of GATA factors and their upstream regulation remain incompletely understood. The knowledge and tools developed since their discovery 25 years ago should accelerate progress toward further elucidation of their mechanisms of action in health and disease. This in turn will greatly improve diagnosis and care for the millions of individuals affected by congenital and acquired cardiac disease worldwide.
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