Phospholamban is the regulator of the Ca'+-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to 8-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (±dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca +-ATPase for Ca`in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the (-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the heart's contractile responses to f-adrenergic agonists. (Circ Res. 1994; 75:401-409.) Key Words * phospholamban * gene targeting . sarcoplasmic reticulum * cardiac contractility * l3-agonists C ardiac 8-adrenergic stimulation is associated with increases in the force of contraction and in the rates of rise and fall of force. These changes are mediated by increases in cAMP levels, which lead to phosphorylation of key regulatory proteins that may act as effectors of the adrenergic stimulation. One of these phosphoproteins is phospholamban, the regulator of the Ca`+-ATPase in cardiac sarcoplasmic reticulum (SR). Dephosphorylated phospholamban is an inhibitor of the Ca2`-ATPase activity, and phosphorylation relieves this inhibition.' The inhibition has been suggested to involve physical direct interaction between the two proteins,23 followed by conformational changes in the SR Ca2`-ATPase.
Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FGF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.
Phospholamban (PLB) can be phosphorylated at Ser 16by cyclic AMP-dependent protein kinase and at Thr 17 by Ca 2؉ -calmodulin-dependent protein kinase during -agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr 17 phosphorylation and attenuation of the -agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Phospholamban (PLB)1 is a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLB is an inhibitor of the affinity of SERCA2 for Ca 2ϩ , and phosphorylation of PLB during -adrenergic stimulation relieves its inhibitory effects on SERCA2 (1, 2). The physiological importance of PLB has been elucidated through the generation of genetically engineered mouse models with alterations in cardiac PLB expression levels (3, 4). Ablation of PLB was associated with significantly enhanced Ca 2ϩ affinity of SERCA2 and myocardial performance (3, 5, 6). The elevated basal contractile parameters could be minimally stimulated by -agonists (3, 7), whereas there were no alterations in the -receptor signaling pathway or the phosphorylation states of other major cardiac phosphoproteins (8). On the other hand, overexpression of PLB was associated with significant depression of contractile parameters, which could be reversed upon phosphorylation of PLB during -agonist stimulation (4). These results indicate that PLB is a key regulator of cardiac function and a prominent mediator of the -adrenergic effects in the myocardium.In vitro studies have shown that PLB can be phosphorylated on Ser 10 by protein kinase C, Ser 16 by cAMP-dependent protein kinase (PKA), and Thr 17 by Ca 2ϩ -calmodulin-dependent protein kinase (CaMKII) (1, 9, 10). Each phosphorylation is associated with stimulation of the apparent affinity of SERCA2 for Ca 2ϩ . In vivo studies have shown that only Ser 16 and Thr 17 are phosphorylated in cardiac myocytes or perfused hearts (11, 12), whereas phosphorylation of PLB by protein kinase C has not been detected in vivo. Phosphorylation of PLB by PKA and CaMKII occurs during -agonist exposure, although the relative contribution of each phosphorylation to the cardiac stimulatory effects is not presently clear. Each phosphorylation appears to occur independently of the other (13-16). Some studies have reported additive effects of PKA and CaMKII phosphorylation of PLB on SR Ca 2ϩ transport (13,14,17,18), whereas others (16, 19) have proposed that maximal stimulation of the Ca 2ϩ pump occurs by phosphorylation at a single site, and additional phosphorylation of the other site does not further stimulate the pump activity.Several in vivo studies have shown that Ser 16 phosphorylation or dephosphorylation precede...
Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca 2؉ -ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser 16 and Thr 17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser 16 3 Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wildtype phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32 P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser 16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser 16 is a prerequisite for Thr 17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the -agonist stimulatory responses in the mammalian heart. Phospholamban (PLB)1 is a regulator of the affinity of the cardiac sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase for Ca 2ϩ . Dephosphorylated PLB is an inhibitor, and phosphorylation of PLB removes its inhibitory effects on the SR Ca 2ϩ -ATPase. Recently, the critical role of PLB in the regulation of cardiac contractility has been defined through gene transfer (1) and knockout (2) technology in the mouse. Cardiac-specific overexpression of PLB was associated with decreases in the affinity of the SR Ca 2ϩ -ATPase for Ca 2ϩ and depressed cardiac function, whereas PLB deficiency resulted in increased Ca 2ϩ affinity of the Ca 2ϩ -ATPase and enhanced myocardial performance. Furthermore, the stimulatory effects to -adrenergic agonists were more pronounced in the PLB-overexpressing hearts, whereas these effects were attenuated in the PLB-knockout hearts compared with wild types (1, 2). These studies suggested that PLB plays a prominent role in the heart's responses to -agonists. However, PLB is phosphorylated on both Ser 16 and Thr 17 during isoproterenol stimulation (3) and the relative contribution of each site in the altered contractile responses of the heart is not presently well known. In vitro studies have shown that Ser 16 is phosphorylated by cAMP-dependent protein kinase, whereas Thr 17 is phosphorylated by Ca 2ϩ -calmodulin-dependent protein kinase (4). Phosphorylation of each site occurs in an independent manner, although it is not presently clear whether the stimulatory effects of the two phosphorylations on SR Ca...
Phospholamban ablation is associated with significant increases in the sarcoplasmic reticulum Ca(2+)-ATPase activity and the basal cardiac contractile parameters. To determine whether the observed phenotype is due to loss of phospholamban alone or to accompanying compensatory mechanisms, hearts from phospholamban-deficient and age-matched wild-type mice were characterized in parallel. There were no morphological alterations detected at the light microscope level. Assessment of the protein levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, myosin, actin, troponin I, and troponin T revealed no significant differences between phospholamban-deficient and wild-type hearts. However, the ryanodine receptor protein levels were significantly decreased (25%) upon ablation of phospholamban, probably in an attempt to regulate the release of Ca2+ from the sarcoplasmic reticulum, which had a significantly higher diastolic Ca2+ content in phospholamban-deficient compared with wild-type hearts (16.0 +/- 2.2 versus 8.6 +/- 1.0 mmol Ca2+/kg dry wt, respectively). The increases in Ca2+ content were specific to junctional sarcoplasmic reticulum stores, as there were no alterations in the Ca2+ content of the mitochondria or A band. Assessment of ATP levels revealed no alterations, although oxygen consumption increased (1.6-fold) to meet the increased ATP utilization in the hyperdynamic phospholamban-deficient hearts. The increases in oxygen consumption were associated with increases (2.2-fold) in the active fraction of the mitochondrial pyruvate dehydrogenase, suggesting increased tricarboxylic acid cycle turnover and ATP synthesis. 31P nuclear magnetic resonance studies demonstrated decreases in phosphocreatine levels and increases in ADP and AMP levels in phospholamban-deficient compared with wild-type hearts. However, the creatine kinase activity and the creatine kinase reaction velocity were not different between phospholamban-deficient and wild-type hearts. These findings indicate that ablation of phospholamban is associated with downregulation of the ryanodine receptor to compensate for the increased Ca2+ content in the sarcoplasmic reticulum store and metabolic adaptations to establish a new energetic steady state to meet the increased ATP demand in the hyperdynamic phospholamban-deficient hearts.
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