Maximal Inhibition of SERCA2 Ca2+ Affinity by Phospholamban in Transgenic Hearts Overexpressing a Non-phosphorylatable Form of Phospholamban

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca2+ affinity of the SR Ca2+-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca2+ uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca2+ with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (≥2.6:1) exhibited increases in associated with cardiac of a non-phosphorylatable form of phospholamban in transgenic hearts in saturation of the functional phospholamban:SERCA2 at and of the SR are phospholamban in vivo. Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca2+ affinity of the SR Ca2+-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca2+ uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca2+ with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (≥2.6:1) exhibited increases in associated with cardiac of a non-phosphorylatable form of phospholamban in transgenic hearts in saturation of the functional phospholamban:SERCA2 at and of the SR are phospholamban in vivo. phospholamban sarcoplasmic reticulum SR Ca2+-ATPase Phospholamban a to with and the apparent Ca2+ affinity of the sarcoplasmic reticulum (SR) Ca2+-ATPase mechanism of and functional of in cardiac of the expression of protein in cardiac SR levels of expression in and a the the of in is at cardiac the apparent affinity of the SR Ca2+-ATPase for Ca2+ and phosphorylation of in to inhibition of SERCA2 is at protein and at protein at of is associated with of the of SR at or of phosphorylation at a cardiac protein which is to phosphorylation of protein apparent affinity of the SERCA2 for Ca2+ is the phosphorylation of is changes in the in the of to SERCA2, associated with in SR Ca2+ as of in and increases in the in the of SR and in in are associated with increases in the of SR and the is to and to the of and in transgenic in or levels of exhibited increased of SR and cardiac compared with was obtained the levels of and the apparent affinity of SERCA2 for Ca2+ as as the of and in hearts or and mice the functional of in cardiac in the of Ca2+ the SR of in of cardiac SR is of and of and SERCA2 in the of and SERCA2 a of to SERCA2, was a and the functional of SERCA2 was a in and of affinity of a of to SERCA2 the of a to the indicated a of of to of SERCA2 in of in the hearts of transgenic mice or in SR and is a of Ca2+ in the SR which is was a the levels of and the of SERCA2 for Ca2+ in and in transgenic hearts the was to was of the in the SR were the to determine the functional of to SERCA2, which is associated with maximal inhibition of the affinity of SERCA2 for transgenic mice a mutant form of (S16A,T17A) were of mutant which the of at the of to in vivo. Assessment of SR Ca2+ uptake in the transgenic hearts revealed increased inhibition of the affinity of SERCA2 for Ca2+ with increased expression of of the was obtained at expression levels cardiac was in transgenic hearts a compensatory to the of in of the of the and of the in mice of were for the was to the the the and the was the to transgenic mice was a which and the was of as the a mutant to of the and a were to the mutant the of the and the and was to the which the in the was with and of a which was the the mutant were was the and the of the in the and of expression was in the as which was of the the with and the was the and for of mice to transgenic mice to the Transgenic mice the were and of as the was of transgenic hearts lines of transgenic mice and mutant were to transgenic levels of mutant Transgenic or were obtained was and and a was to the and the of the was to the a and Transgenic levels of transgenic were chosen to the levels were to immunoblotting of cardiac and in SR was as a of to hearts was or transgenic mice and at in and cardiac were to the levels of SERCA2, calsequestrin, calreticulin, ryanodine and in and transgenic To determine the mutant form of was the SR of in SR were of the cardiac were at and the were in and as the were was to a of and at was in and at was in and at protein of and were the as a and were with of and were and or SERCA2, calsequestrin, calreticulin, and and to for for SERCA2, calsequestrin, calreticulin, ryanodine and were with SERCA2 ryanodine and and with or of was a and the of phosphorylation a at or at were were and were with and and with of was a and the Ca2+ hearts were in and at hearts were and in and of Ca2+ uptake in were obtained and as protein or protein phosphorylation was as in cardiac of and transgenic the phosphorylation was in of the in the phosphorylation of and SERCA2 was generated in the of the and were obtained was obtained was obtained and were obtained were and were obtained the for were the of the the is the of the of of the are expressed as were for of were the in maximal inhibition of the affinity of SERCA2 for Ca2+ is obtained at expression levels are 2.6-fold or in the functional of is in vivo. of transgenic with of levels of a non-phosphorylatable form of in to the of in SR of the was the which is and in of in was chosen in transgenic mice increased phosphorylation of compensatory mechanism in the increased phosphorylation the of the affinity of SERCA2 for Ca2+ and prevent of or in in expression the and Quantitative of cardiac and SR transgenic mice revealed 1.8-, 2.6-, 3.7-, and 4.7-fold increases in protein levels compared with and the SR was of increased the mice for of the of the SR Ca2+ indicated the of SERCA2 for Ca2+ was increased the maximal of Ca2+ was in and with in and hearts is a of the maximal of the SERCA2 the levels of in with or were the Ca2+ was a to Maximal increases in were in hearts 2.6-fold or a of SERCA2 the and the levels in the indicated of the SR Ca2+ are in SR functional of was to a of cardiac in and hearts the of to SERCA2 was to of the and SERCA2 levels is for cardiac the functional of in to and to the in the levels of in SR of in transgenic hearts or cardiac revealed inhibition of the affinity of SERCA2 for the is and a of the SR Ca2+ is in the SR To determine the of of SR Ca2+ in generated a of transgenic lines with increasing levels of expression in the and the of inhibition of SR Ca2+ This to determine the of to inhibition of the SR Ca2+ and the of increases in the associated with in the was in the hearts of and mice and and in SR Ca2+ and of the with the obtained in was a of Ca2+ which was in in in the and to the cardiac in hearts revealed levels of phosphorylation at and increased expression and of a protein a of is in the and to inhibition of changes in the changes in the levels of phosphorylation in the of in cardiac and with increases in the 2.6-fold in of a associated with increased expression of This compensatory mechanism in the transgenic hearts with of a non-phosphorylatable form of the of SERCA2 are and SERCA2 the to form in the SR to or SERCA2 to of as as and in SR and and revealed in the of SERCA2 to to and and phosphorylation of is associated with increases in This phosphorylation and of SERCA2 is with the increased inhibition of SERCA2 in expression the form of is the of the SR Ca2+ and in the and the of the functional protein expression with the of SERCA2 the the or the affinity of for SERCA2 compared with the of for a SERCA2 uptake and a of in of a non-phosphorylatable form of in transgenic hearts in saturation of the functional which was associated with inhibition of the affinity of SERCA2 for Ca2+ and of cardiac saturation was obtained at a of for of the SR Ca2+ are with and in of and SERCA2 in the of the SR the and of SERCA2 with Phospholamban a to with and the apparent Ca2+ affinity of the sarcoplasmic reticulum (SR) Ca2+-ATPase mechanism of and functional of in cardiac of the expression of protein in cardiac SR levels of expression in and a the the of in is at cardiac the apparent affinity of the SR Ca2+-ATPase for Ca2+ and phosphorylation of in to inhibition of SERCA2 is at protein and at protein at of is associated with of the of SR at or of phosphorylation at a cardiac protein which is to phosphorylation of protein apparent affinity of the SERCA2 for Ca2+ is the phosphorylation of is changes in the in the of to SERCA2, associated with in SR Ca2+ as of in and increases in the in the of SR and in in are associated with increases in the of SR and the is to and to the of and in transgenic in or levels of exhibited increased of SR and cardiac compared with was obtained the levels of and the apparent affinity of SERCA2 for Ca2+ as as the of and in hearts or and mice the functional of in cardiac in the of Ca2+ the SR of in of cardiac SR is of and of and SERCA2 in the of and SERCA2 a of to SERCA2, was a and the functional of SERCA2 was a in and of affinity of a of to SERCA2 the of a to the indicated a of of to of SERCA2 in of in the hearts of transgenic mice or in SR and is a of Ca2+ in the SR which is was a the levels of and the of SERCA2 for Ca2+ in and in transgenic hearts the was to was of the in the SR were the to determine the functional of to SERCA2, which is associated with maximal inhibition of the affinity of SERCA2 for transgenic mice a mutant form of (S16A,T17A) were of mutant which the of at the of to in vivo. 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in SR was as a of to hearts was or transgenic mice and at in and cardiac were to the levels of SERCA2, calsequestrin, calreticulin, ryanodine and in and transgenic To determine the mutant form of was the SR of in SR were of the cardiac were at and the were in and as the were was to a of and at was in and at was in and at protein of and were the as a and were with of and were and or SERCA2, calsequestrin, calreticulin, and and to for for SERCA2, calsequestrin, calreticulin, ryanodine and were with SERCA2 ryanodine and and with or of was a and the of phosphorylation a at or at were were and were with and and with of was a and the Ca2+ hearts were in and at hearts were and in and of Ca2+ uptake in were obtained and as protein or protein phosphorylation was as in cardiac of and transgenic the phosphorylation was in of the in the phosphorylation of and SERCA2 was generated in the of the and were obtained was obtained was obtained and were obtained were and were obtained the for were the of 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as a of to hearts was or transgenic mice and at in and cardiac were to the levels of SERCA2, calsequestrin, calreticulin, ryanodine and in and transgenic To determine the mutant form of was the SR of in SR were of the cardiac were at and the were in and as the were was to a of and at was in and at was in and at protein of and were the as a and were with of and were and or SERCA2, calsequestrin, calreticulin, and and to for for SERCA2, calsequestrin, calreticulin, ryanodine and were with SERCA2 ryanodine and and with or of was a and the of phosphorylation a at or at were were and were with and and with of was a and the Quantitative immunoblotting of cardiac and in SR was as a of to hearts was or transgenic mice and at in and cardiac were to the levels of SERCA2, calsequestrin, calreticulin, ryanodine and in and transgenic To determine the mutant form of was the SR of in SR were of the cardiac were at and the were in and as the were was to a of and at was in and at was in and at protein of and were the as a and were with of and were and or SERCA2, calsequestrin, calreticulin, and and to for for SERCA2, calsequestrin, calreticulin, ryanodine and were with SERCA2 ryanodine and and with or of was a and the of phosphorylation a at or at were were and were with and and with of was a and the SR Ca2+ hearts were in and at hearts were and in and of Ca2+ uptake in were obtained and as hearts were in and at hearts were and in and of Ca2+ uptake in were obtained and as protein or protein phosphorylation was as in cardiac of and transgenic the phosphorylation was in of the in the phosphorylation protein or protein phosphorylation was as in cardiac of and transgenic the phosphorylation was in of the in the phosphorylation of and SERCA2 was generated in the of the and were obtained was obtained was obtained and were obtained of and SERCA2 was generated in the of the and were obtained was obtained was obtained and were obtained were and were obtained the for were the of the the is the of the of of the were and were obtained the for were the of the the is the of the of of the are expressed as were for of were are expressed as were for of were the in maximal inhibition of the affinity of SERCA2 for Ca2+ is obtained at expression levels are 2.6-fold or in the functional of is in vivo. of transgenic with of levels of a non-phosphorylatable form of in to the of in SR of the was the which is and in of in was chosen in transgenic mice increased phosphorylation of compensatory mechanism in the increased phosphorylation the of the affinity of SERCA2 for Ca2+ and prevent of or in in expression the and Quantitative of cardiac and SR transgenic mice revealed 1.8-, 2.6-, 3.7-, and 4.7-fold increases in protein levels compared with and the SR was of increased the mice for of the of the SR Ca2+ indicated the of SERCA2 for Ca2+ was increased the maximal of Ca2+ was in and with in and hearts is a of the maximal of the SERCA2 the levels of in with or were the Ca2+ was a to Maximal increases in were in hearts 2.6-fold or a of SERCA2 the and the levels in the indicated of the SR Ca2+ are in SR functional of was to a of cardiac in and hearts the of to SERCA2 was to of the and SERCA2 levels is for cardiac the functional of in to and to the in the levels of in SR of in transgenic hearts or cardiac revealed inhibition of the affinity of SERCA2 for the is and a of the SR Ca2+ is in the SR To determine the of of SR Ca2+ in generated a of transgenic lines with increasing levels of expression in the and the of inhibition of SR Ca2+ This to determine the of to inhibition of the SR Ca2+ and the of increases in the associated with in the was in the hearts of and mice and and in SR Ca2+ and of the with the obtained in was a of Ca2+ which was in in in the and to the cardiac in hearts revealed levels of phosphorylation at and increased expression and of a protein a of is in the and to inhibition of changes in the changes in the levels of phosphorylation in the of in cardiac and with increases in the 2.6-fold in of a associated with increased expression of This compensatory mechanism in the transgenic hearts with of a non-phosphorylatable form of the of SERCA2 are and SERCA2 the to form in the SR to or SERCA2 to of as as and in SR and and revealed in the of SERCA2 to to and and phosphorylation of is associated with increases in This phosphorylation and of SERCA2 is with the increased inhibition of SERCA2 in expression the form of is the of the SR Ca2+ and in the and the of the functional protein expression with the of SERCA2 the the or the affinity of for SERCA2 compared with the of for a SERCA2 uptake and a of in of a non-phosphorylatable form of in transgenic hearts in saturation of the functional which was associated with inhibition of the affinity of SERCA2 for Ca2+ and of cardiac saturation was obtained at a of for of the SR Ca2+ are with and in of and SERCA2 in the of the SR the and of SERCA2 with This the in maximal inhibition of the affinity of SERCA2 for Ca2+ is obtained at expression levels are 2.6-fold or in the functional of is in vivo. of transgenic with of levels of a non-phosphorylatable form of in to the of in SR of the was the which is and in of in was chosen in transgenic mice increased phosphorylation of compensatory mechanism in the increased phosphorylation the of the affinity of SERCA2 for Ca2+ and prevent of or in in expression the and Quantitative of cardiac and SR transgenic mice revealed 1.8-, 2.6-, 3.7-, and 4.7-fold increases in protein levels compared with and the SR was of increased the mice for of the of the SR Ca2+ indicated the of SERCA2 for Ca2+ was increased the maximal of Ca2+ was in and with in and hearts is a of the maximal of the SERCA2 the levels of in with or were the Ca2+ was a to Maximal increases in were in hearts 2.6-fold or a of SERCA2 the and the levels in the indicated of the SR Ca2+ are in SR functional of was to a of cardiac in and hearts the of to SERCA2 was to of the and SERCA2 levels is for cardiac the functional of in to and to the in the levels of in SR of in transgenic hearts or cardiac revealed inhibition of the affinity of SERCA2 for the is and a of the SR Ca2+ is in the SR To determine the of of SR Ca2+ in generated a of transgenic lines with increasing levels of expression in the and the of inhibition of SR Ca2+ This to determine the of to inhibition of the SR Ca2+ and the of increases in the associated with in the was in the hearts of and mice and and in SR Ca2+ and of the with the obtained in was a of Ca2+ which was in in in the and to the cardiac in hearts revealed levels of phosphorylation at and increased expression and of a protein a of is in the and to inhibition of changes in the changes in the levels of phosphorylation in the of in cardiac and with increases in the 2.6-fold in of a associated with increased expression of This compensatory mechanism in the transgenic hearts with of a non-phosphorylatable form of the of SERCA2 are and SERCA2 the to form in the SR to or SERCA2 to of as as and in SR and and revealed in the of SERCA2 to to and and phosphorylation of is associated with increases in This phosphorylation and of SERCA2 is with the increased inhibition of SERCA2 in expression the form of is the of the SR Ca2+ and in the and the of the functional protein expression with the of SERCA2 the the or the affinity of for SERCA2 compared with the of for a SERCA2 uptake and a of in of a non-phosphorylatable form of in transgenic hearts in saturation of the functional which was associated with inhibition of the affinity of SERCA2 for Ca2+ and of cardiac saturation was obtained at a of for of the SR Ca2+ are with and in of and SERCA2 in the of the SR the and of SERCA2 with for the for of the transgenic for the and for the are to and for of