Phospholamban is the regulator of the cardiac sarcoplasmic reticulum (SR) Ca 2 ϩ -ATPase activity and an important modulator of basal contractility in the heart. To determine whether all the SR Ca 2 ϩ -ATPase enzymes are subject to regulation by phospholamban in vivo, transgenic mice were generated which overexpressed phospholamban in the heart, driven by the cardiac-specific ␣ -myosin heavy chain promoter. Quantitative immunoblotting revealed a twofold increase in the phospholamban protein levels in transgenic hearts compared to wild type littermate hearts. The transgenic mice showed no phenotypic alterations and no changes in heart/body weight, heart/lung weight, and cardiomyocyte size. Isolated unloaded cardiac myocytes from transgenic mice exhibited diminished shortening fraction (63%) and decreased rates of shortening (64%) and relengthening (55%) compared to wild type (

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Calsequestrin is a high capacity Ca 2؉-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca 2؉ transient amplitude was also depressed (45%), although the SR Ca 2؉ storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca 2؉ current density and prolongation of this channel's inactivation kinetics without changes in Na ؉ -Ca 2؉ exchanger current density. Furthermore, there were increases in protein levels of SR Ca 2؉ -ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca 2؉ capacity, but decreases in Ca 2؉ -induced SR Ca 2؉ release, leading to depressed contractility in the mammalian heart.

These data demonstrate that blocking the α(4) β(7) integrin exclusively yields gut-selective antiinflammatory activity in primates.

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...