Adenylyl Cyclase Type VI Gene Transfer Reduces Phospholamban Expression in Cardiac Myocytes via Activating Transcription Factor 3

Abstract

Cardiac-directed expression of adenylyl cyclase type VI (ACVI) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with ACVI expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by ACVI in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overexpression of ACVI down-regulated mRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. We determined that the cAMP-responsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by ACVI. Overexpression of ACVI did not alter the expression of CREB, CREM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of ACVI increased expression of ATF3 protein, a suppressor of transcription. Following ACVI gene transfer, when cardiac myocytes were stimulated with isoproterenol or NKH477, a water-soluble forskolin analog that directly stimulates AC, expression of ATF3 protein was increased even more, which correlated with reduced expression of PLB. We then that ATF3 protein the cAMP-responsive-like element on the promoter and that overexpression of ATF3 in cardiac myocytes promoter that ACVI effects on gene transcription that are not directly on cAMP Cardiac-directed expression of adenylyl cyclase type VI (ACVI) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with ACVI expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by ACVI in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overexpression of ACVI down-regulated mRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. We determined that the cAMP-responsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by ACVI. Overexpression of ACVI did not alter the expression of CREB, CREM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of ACVI increased expression of ATF3 protein, a suppressor of transcription. Following ACVI gene transfer, when cardiac myocytes were stimulated with isoproterenol or NKH477, a water-soluble forskolin analog that directly stimulates AC, expression of ATF3 protein was increased even more, which correlated with reduced expression of PLB. We then that ATF3 protein the cAMP-responsive-like element on the promoter and that overexpression of ATF3 in cardiac myocytes promoter that ACVI effects on gene transcription that are not directly on cAMP cyclase AC, adenylyl adenylyl cyclase type of CREB, transcription CREM, element AC, adenylyl adenylyl cyclase type of CREB, transcription CREM, element is the in the in cardiac myocytes and that the of a on the of cardiac myocytes cAMP and expression of type VI (ACVI) effects on function in and these effects of ACVI are not is that the increased intracellular levels of is in heart which that agents that increase cAMP are for the pharmacological agents that the or the of cAMP increase cardiac function not In contrast, a in cardiac myocytes, improves cardiac function, and increases survival in when expression is is the by these of which are associated with increased intracellular of the and expression in heart that the salutary effects of expression on not directly with cAMP be whether ACVI gene of cAMP transcription of genes in in cardiac myocytes, focused on how ACVI and that is the by agents that directly cAMP We found that in the of or down-regulated the expression of genes the element phospholamban an inhibitor of the sarcoplasmic reticulum was down-regulated by ACVI gene transfer, a of increased expression of transcription a suppressor in the protein In contrast, of with isoproterenol or of with increased expression and did not alter expression of ATF3 of was the genes on the of or and that rat cardiac myocytes the were in these stimulated with the forskolin analog with ACVI and protein of the cardiac myocytes was in the of were the genes on the of and promoter was rat by in the and and the was a was and the were by promoter was the by and and in of the gene the the in the was the for the element were and and myocytes neonatal were were the with protein or ACVI with an the of the ACVI for protein with isoproterenol or was for in or gene and of cardiac myocytes was and were in and a of was and were and and the was cultured cardiac was of the with was In the experiments are in and the and the were that the of transcription of the promoter a promoter or a promoter was a and with or cardiac ATF3 a on promoter of the element in the promoter was associated with of promoter and the of ATF3 was myocytes were with for were with were with for and with for of ACVI and for were with and then for with a that was with or were by and of cardiac was the by were or protein was a a of was of was on a mRNA was by that rat were myocytes were in and the were in and in of were on in a by with were and in a for were by and were by and inhibitor were on for and in a for was and in was in the and were by in the of the with of in the were with the and of in and for were then and in was in a and an with was by with for the with were by with the or by with the and then for are were by for were conducted and was conducted when by for with for was when of by of whether overexpression of pharmacological of gene expression in cardiac myocytes, gene of of genes that ACVI gene was associated with reduced expression of and In contrast, expression of these genes was increased by with gene on mRNA neonatal rat cardiac myocytes was in the of were with the on that expression was reduced by ACVI gene increased by pharmacological of and expression be increased by by ACVI gene neonatal rat cardiac myocytes with with with a water-soluble forskolin analog that directly stimulates adenylyl the the gene for We expression by and found that mRNA was reduced by ACVI gene cardiac myocytes and that with was associated with in mRNA of an that the that the effects on were expression by ACVI gene was increased was associated with a in mRNA In reduced protein was reduced by overexpression of ACVI In contrast, increased mRNA expression expression. mRNA was reduced by overexpression of ACVI. rat cardiac myocytes were with of ACVI or of neonatal rat cardiac myocytes was in and with mRNA was down-regulated by overexpression of ACVI increased by with were was gene a of that the that the effects on were AC, protein expression was reduced by ACVI. of protein of neonatal rat cardiac myocytes were and by protein was an isoproterenol and and and and and that ACVI gene is associated with reduced protein isoproterenol was associated with a of protein and down-regulation of protein and or rat promoter in a is element in that is a element in the a the of ACVI on the of the In a the promoter when with the the of the with reduced the of the the was and the promoter was the gene and in of element was associated with of and the of ACVI on the promoter was that the element in the promoter an in down-regulation of by the of on of the with a expression a of cardiac myocytes with the did not promoter even in the of that for of the was increase in with were in and or In contrast, the ACVI expression in and stimulated with the the ACVI expression in and stimulated with of cardiac myocytes with the promoter which have that the in that the of ACVI is the element in the of transcription by is not the element in the of ATF3 the by which ACVI the expression of transcription in the in ACVI gene We found that did not alter the expression of CREB, CREM, ATF1, ATF2, and ATF4 proteins. the expression of ATF3 was increased and Following ACVI gene transfer, ATF3 protein expression was increased by isoproterenol or and which correlated with a in expression ATF3 expression was In contrast, of cardiac myocytes with isoproterenol or on ATF3 expression and and of was increased by ACVI gene increased a with isoproterenol or and of proteins. were in with or and by of protein was of the neonatal rat cardiac isoproterenol ATF3 protein expression was increased by ACVI gene expression of ATF3 cardiac myocytes were with or and for the cardiac myocytes were stimulated with isoproterenol or for ATF3 protein, by increased expression with that increased ATF3 protein was in the ATF3 in cardiac myocytes by We of ATF3 gene in protein in the of myocytes ACVI gene with the for the expression of ACVI and of ACVI and of ATF3 in myocytes and ACVI or ATF3 in myocytes of on that ACVI gene gene expression increased expression of whether ATF3 a the of the with cardiac myocytes, were of these was found when an was the was when the was with the the the did not an these and a or suppressor by with or suppressor of was in cardiac myocytes these did not the with cardiac myocytes and element in the the was with of cardiac myocytes with or were in ACVI in in the of and and and and were with and were with and then the was the a was in the a was in the correlated with in the of and a was with of and the by that ATF3 protein the element in the the of of the with the were in the were with an of type not with the of and protein was found the and these that ATF3 the found in the whether ATF3 directly promoter In a the was cardiac myocytes with an ATF3 expression ATF3 promoter by when the element was ATF3 promoter that ATF3 not the element the promoter the of and the have determined the effects of ACVI gene on gene transcription. We found that ACVI expression and increases the expression of a suppressor in the with isoproterenol or NKH477, expression is and ATF3 expression is We that ATF3 the element in the promoter of and the of the In contrast, the or of effects on and on ATF3 and of promoter of the gene have and rat promoter of rat is in a of rat a phospholamban promoter element and the element was not in the promoter were were not We have and the element in the rat and that is critical for the of the of element of promoter We that ACVI gene the expression of increased expression of ATF3 with the element of of by an in the of sarcoplasmic reticulum by sarcoplasmic reticulum of or increased sarcoplasmic reticulum and cardiac survival and cardiac function In contrast, overexpression of an increase in promoter was associated with and expression of in and expression ACVI expression be have a on cardiac function and be an by which AC, in that increase intracellular a in the and of of ATF3 is by and and and In was found that expression of ATF3 was by forskolin forskolin did not the expression of ATF3 in cardiac of ATF3 be by protein the was the in the is expression of or were the ACVI and were not with that the is the of ATF3 is an with experiments in which ATF3 is or overexpression of ATF3 in and and of ATF3 with increased In cardiac myocytes, ATF3 overexpression was associated with reduced an in in ATF3 in and ATF3 is in cardiac myocytes and expression of ATF3 in was associated with and and of ATF3 increased ATF3 be when in in the is associated with and reduced function. in ACVI of for of not or effects the ATF3 overexpression ACVI the of increases the expression of ATF3 and increases of when stimulated with isoproterenol or gene transcription or ATF3 in or be of how a and an in ACVI. the of ACVI. and are for of these in with or genes of overexpression of ACVI in neonatal rat cardiac myocytes gene transcription in a increased expression of ATF3 protein, a of ACVI on transcription is the effects of or adenylyl cyclase pharmacological that ACVI effects on gene transcription that are not directly on cAMP cyclase AC, adenylyl adenylyl cyclase type of CREB, transcription CREM, element AC, adenylyl adenylyl cyclase type of CREB, transcription CREM, element is the in the in cardiac myocytes and that the of a on the of cardiac myocytes cAMP and expression of type VI (ACVI) effects on function in and these effects of ACVI are not is that the increased intracellular levels of is in heart which that agents that increase cAMP are for the pharmacological agents that the or the of cAMP increase cardiac function not In contrast, a in cardiac myocytes, improves cardiac function, and increases survival in when expression is is the by these of which are associated with increased intracellular of the and expression in heart that the salutary effects of expression on not directly with cAMP be whether ACVI gene of cAMP transcription of genes in function. in cardiac myocytes, focused on how ACVI and that is the by agents that directly cAMP We found that in the of or down-regulated the expression of genes the element phospholamban an inhibitor of the sarcoplasmic reticulum was down-regulated by ACVI gene transfer, a of increased expression of transcription a suppressor in the protein In contrast, of with isoproterenol or of with increased expression and did not alter expression of ATF3 of was the genes on the of or and that rat cardiac myocytes the were in these stimulated with the forskolin analog with ACVI and protein of the cardiac myocytes was in the of were the genes on the of and promoter was rat by in the and and the was a was and the were by promoter was the by and and in of the gene the the in the was the for the element were and and myocytes neonatal were were the with protein or ACVI with an the of the ACVI for protein with isoproterenol or was for in or gene and of cardiac myocytes was and were in and a of was and were and and the was cultured cardiac was of the with was In the experiments are in and the and the were that the of myocytes were with for were with were with for and with for of ACVI and for were with and then for with a that was with or were by and of cardiac was the by were or protein was a a of was of was on a mRNA was by that rat were myocytes were in and the were in and in of were on in a by with were and in a for were by and were by and inhibitor were on for and in a for was and in was in the and were by in the of the with of in the were with the and of in and for were then and in was in a and an with was by with for the with were by with the or by with the and then for are were by for were conducted and was conducted when by for with for was when of was the genes on the of or and that rat cardiac myocytes the were in these stimulated with the forskolin analog with ACVI and protein of the cardiac myocytes was in the of were the genes on the of and promoter was rat by in the and and the was a was and the were by promoter was the by and and in of the gene the the in the was the for the element were and and myocytes neonatal were were the with protein or ACVI with an the of the ACVI for protein with isoproterenol or was for in or gene and of cardiac myocytes was and were in and a of was and were and and the was cultured cardiac was of the with was In the experiments are in and the and the were that the of myocytes were with for were with were with for and with for of ACVI and for were with and then for with a that was with or were by and of cardiac was the by were or protein was a a of was of was on a mRNA was by that rat were myocytes were in and the were in and in of were on in a by with were and in a for were by and were by and inhibitor were on for and in a for was and in was in the and were by in the of the with of in the were with the and of in and for were then and in was in a and an with was by with for the with were by with the or by with the and then for are were by for were conducted and was conducted when by for with for was when of by of whether overexpression of pharmacological of gene expression in cardiac myocytes, gene of of genes that ACVI gene was associated with reduced expression of and In contrast, expression of these genes was increased by with the the gene for We expression by and found that mRNA was reduced by ACVI gene cardiac myocytes and that with was associated with in mRNA of an that the that the effects on were expression by ACVI gene was increased was associated with a in mRNA In reduced protein was reduced by overexpression of ACVI In contrast, increased mRNA expression expression. mRNA was reduced by overexpression of ACVI. rat cardiac myocytes were with of ACVI or of neonatal rat cardiac myocytes was in and with mRNA was down-regulated by overexpression of ACVI increased by with were was gene a of that the that the effects on were AC, protein expression was reduced by ACVI. of protein of neonatal rat cardiac myocytes were and by protein was an isoproterenol and and and and and that ACVI gene is associated with reduced protein isoproterenol was associated with a of protein and down-regulation of protein and or rat promoter in a is element in that is a element in the a the of ACVI on the of the In a the promoter when with the the of the with reduced the of the the was and the promoter was the gene and in of element was associated with of and the of ACVI on the promoter was that the element in the promoter an in down-regulation of by the of on of the with a expression a of cardiac myocytes with the did not promoter even in the of that for of the was increase in with were in and or In contrast, the ACVI expression in and stimulated with the the ACVI expression in and stimulated with of cardiac myocytes with the promoter which have that the in that the of ACVI is the element in the of transcription by is not the element in the of ATF3 the by which ACVI the expression of transcription in the in ACVI gene We found that did not alter the expression of CREB, CREM, ATF1, ATF2, and ATF4 proteins. the expression of ATF3 was increased and Following ACVI gene transfer, ATF3 protein expression was increased by isoproterenol or and which correlated with a in expression ATF3 expression was In contrast, of cardiac myocytes with isoproterenol or on ATF3 expression and and of was increased by ACVI gene increased a with isoproterenol or and of proteins. were in with or and by of protein was of the neonatal rat cardiac isoproterenol ATF3 protein expression was increased by ACVI gene expression of ATF3 cardiac myocytes were with or and for the cardiac myocytes were stimulated with isoproterenol or for ATF3 protein, by increased expression with that increased ATF3 protein was in the ATF3 in cardiac myocytes by We of ATF3 gene in protein in the of myocytes ACVI gene with the for the expression of ACVI and of ACVI and of ATF3 in myocytes and ACVI or ATF3 in myocytes of on that ACVI gene gene expression increased expression of whether ATF3 a the of the with cardiac myocytes, were of these was found when an was the was when the was with the the the did not an these and a or suppressor by with or suppressor of was in cardiac myocytes these did not the with cardiac myocytes and element in the the was with of cardiac myocytes with or were in ACVI in in the of and and and and were with and were with and then the was the a was in the a was in the correlated with in the of and a was with of and the by that ATF3 protein the element in the the of of the with the were in the were with an of type not with the of and protein was found the and these that ATF3 the found in the whether ATF3 directly promoter In a the was cardiac myocytes with an ATF3 expression ATF3 promoter by when the element was ATF3 promoter that ATF3 not the element the promoter of by of whether overexpression of pharmacological of gene expression in cardiac myocytes, gene of of genes that ACVI gene was associated with reduced expression of and In contrast, expression of these genes was increased by with the the gene for We expression by and found that mRNA was reduced by ACVI gene cardiac myocytes and that with was associated with in mRNA of an that the that the effects on were expression by ACVI gene was increased was associated with a in mRNA In reduced protein was reduced by overexpression of ACVI In contrast, increased mRNA expression or rat promoter in a is element in that is a element in the a the of ACVI on the of the In a the promoter when with the the of the with reduced the of the the was and the promoter was the gene and in of element was associated with of and the of ACVI on the promoter was that the element in the promoter an in down-regulation of by ACVI. In the of on of the with a expression a of cardiac myocytes with the did not promoter even in the of that for of the was increase in with were in and or In contrast, the ACVI expression in and stimulated with the the ACVI expression in and stimulated with of cardiac myocytes with the promoter which have that the in that the of ACVI is the element in the of transcription by is not the element in the Overexpression of ATF3 the by which ACVI the expression of transcription in the in ACVI gene We found that did not alter the expression of CREB, CREM, ATF1, ATF2, and ATF4 proteins. the expression of ATF3 was increased and Following ACVI gene transfer, ATF3 protein expression was increased by isoproterenol or and which correlated with a in expression ATF3 expression was In contrast, of cardiac myocytes with isoproterenol or on ATF3 expression and and of was increased by ACVI gene increased a with isoproterenol or and that increased ATF3 protein was in the ATF3 in cardiac myocytes by We of ATF3 gene in of on that ACVI gene gene expression increased expression of whether ATF3 a the of the with cardiac myocytes, were of these was found when an was the was when the was with the the the did not an these and a or suppressor by with or suppressor of was in cardiac myocytes these did not the with cardiac myocytes and the of of the with the were in the were with an of type not with the of and protein was found the and these that ATF3 the found in the whether ATF3 directly promoter In a the was cardiac myocytes with an ATF3 expression ATF3 promoter by when the element was ATF3 promoter that ATF3 not the element the promoter the of and the have determined the effects of ACVI gene on gene transcription. We found that ACVI expression and increases the expression of a suppressor in the with isoproterenol or NKH477, expression is and ATF3 expression is We that ATF3 the element in the promoter of and the of the In contrast, the or of effects on and on ATF3 and of promoter of the gene have and rat promoter of rat is in a of rat a phospholamban promoter element and the element was not in the promoter were were not We have and the element in the rat and that is critical for the of the of element of promoter We that ACVI gene the expression of increased expression of ATF3 with the element of of by an in the of sarcoplasmic reticulum by sarcoplasmic reticulum of or increased sarcoplasmic reticulum and cardiac survival and cardiac function In contrast, overexpression of an increase in promoter was associated with and expression of in and expression ACVI expression be have a on cardiac function and be an by which AC, in that increase intracellular a in the and of of ATF3 is by and and and In was found that expression of ATF3 was by forskolin forskolin did not the expression of ATF3 in cardiac of ATF3 be by protein the was the in the is expression of or were the ACVI and were not with that the is the of ATF3 is an with experiments in which ATF3 is or overexpression of ATF3 in and and of ATF3 with increased In cardiac myocytes, ATF3 overexpression was associated with reduced an in in ATF3 in and ATF3 is in cardiac myocytes and expression of ATF3 in was associated with and and of ATF3 increased ATF3 be when in in the is associated with and reduced function. in ACVI of for of not or effects the ATF3 overexpression ACVI the of increases the expression of ATF3 and increases of when stimulated with isoproterenol or gene transcription or ATF3 in or be of how a and an in ACVI. the of ACVI. and are for of these in with or genes of overexpression of ACVI in neonatal rat cardiac myocytes gene transcription in a increased expression of ATF3 protein, a of ACVI on transcription is the effects of or adenylyl cyclase pharmacological that ACVI effects on gene transcription that are not directly on cAMP the of and the have determined the effects of ACVI gene on gene transcription. We found that ACVI expression and increases the expression of a suppressor in the with isoproterenol or NKH477, expression is and ATF3 expression is We that ATF3 the element in the promoter of and the of the In contrast, the or of effects on and on ATF3 expression. and of promoter of the gene have and rat promoter of rat is in a of rat a phospholamban promoter element and the element was not in the promoter were were not We have and the element in the rat and that is critical for the of the of element of promoter We that ACVI gene the expression of increased expression of ATF3 with the element of PLB. of by an in the of sarcoplasmic reticulum by sarcoplasmic reticulum of or increased sarcoplasmic reticulum and cardiac survival and cardiac function In contrast, overexpression of an increase in promoter was associated with and expression of in and expression ACVI expression be have a on cardiac function and be an by which AC, in that increase intracellular a in the and of of ATF3 is by and and and In was found that expression of ATF3 was by forskolin forskolin did not the expression of ATF3 in cardiac of ATF3 be by protein the was the in the is expression of or were the ACVI and were not with that the is the of ATF3 is an with experiments in which ATF3 is or overexpression of ATF3 in and and of ATF3 with increased In cardiac myocytes, ATF3 overexpression was associated with reduced an in in ATF3 in and ATF3 is in cardiac myocytes and expression of ATF3 in was associated with and and of ATF3 increased ATF3 be when in in the is associated with and reduced function. in ACVI of for of not or effects the ATF3 overexpression ACVI the of increases the expression of ATF3 and increases of when stimulated with isoproterenol or gene transcription or ATF3 in or be of how a and an in ACVI. the of ACVI. and are for of these in with or genes of ACVI. In overexpression of ACVI in neonatal rat cardiac myocytes gene transcription in a increased expression of ATF3 protein, a of ACVI on transcription is the effects of or adenylyl cyclase pharmacological that ACVI effects on gene transcription that are not directly on cAMP We and for the and for the and