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Mechanical and relaxation restitution represent the restoration of contractile force and relaxation, respectively, in premature beats having progressively longer extrasystolic intervals (ESI); these phenomena are related to intracellular activator Ca(2+) by poorly defined mechanisms. We tested the hypothesis that the level of phospholamban [which modulates the affinity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase for Ca(2+), and thus the SR Ca(2+) load] may be an important determinant of both mechanical and relaxation restitution. Five mice with ablation of the phospholamban (PLB) gene (PLBKO), eight isogenic wild-type controls (129SvJ), eleven mice with PLB overexpression (PLBOE), and nine isogenic wild-type (FVB/N) controls were anesthetized and instrumented with a 1.4-Fr Millar catheter in the left ventricle and a 1-Fr pacemaker in the right atrium. At a cycle length of 200 ms, extrastimuli with increasing ESI were introduced, and the peak rates of left ventricular isovolumic contraction (+/-dP/dt(max)) were normalized and fit to monoexponential equations. In a subset, the protocols were repeated after ryanodine (4 ng/g) was administered to deplete SR Ca(2+) stores. The time constant of mechanical restitution in PLBKO was significantly shorter [6.3 +/- 1.2 (SE) vs. 47.7 +/- 7.6 ms] and began earlier (50 +/- 10 vs. 70 +/- 19 ms) than in 129SvJ. In contrast, the time constant of mechnical restitution was significantly longer (80.3 +/- 7.6 vs. 54.1 +/- 9.2 ms) in PLBOE than in FVB/N. The time constant of relaxation restitution was less in PLBKO than in 129SvJ (26.2 +/- 9.9 vs. 44.6 +/- 3.3, P < 0.05) but was similar in PLBOE and FVB/N (21.1 +/- 6.3 vs. 20.5 +/- 5.7 ms). Intravenous ryanodine decreased significantly the time constants of mechanical restitution in PLBOE, 129SvJ, and FVB/N but was lethal in PLBKO. In contrast, ryanodine increased the time constant of relaxation restitution. Thus 1) the phospholamban level is a critical determinant of mechanical restitution and (to a lesser extent) relaxation restitution in these transgenic models, and 2) ryanodine differentially affects mechanical and relaxation restitution. Furthermore, our data suggest a dissociation of processes within the SR that govern contraction and relaxation.

Phospholamban levels regulate cardiac sarcoplasmic reticulum Ca2+ pump activity and myocardial contractility. To determine whether and to what extent phospholamban modulates the force-frequency relation and ventricular relaxation in vivo, we studied transgenic mice overexpressing phospholamban (PLBOE), gene-targeted mice without phospholamban (PLBKO), and isogenic wild-type controls. Contractility was assessed by the peak rate of left ventricular (LV) isovolumic contraction (+dP/dtmax), and diastolic function was assessed by both the peak rate (-dP/dtmax) and the time constant (tau) of isovolumic LV relaxation, using a high-fidelity LV catheter. Incremental atrial pacing was used to generate heart rate vs. -dP/dtmax (force-frequency) relations. Biphasic force-frequency relations were produced in all animals, and the critical heart rate (HRcrit) was taken as the heart rate at which dP/dtmax was maximal. The average LV +dP/dtmax increased in both PLBKO and PLBOE compared with their isogenic controls (both P < 0.05). The HRcrit for LV +dP/dtmax was significantly higher in PLBKO (427 +/- 20 beats/min) compared with controls (360 +/- 18 beats/min), whereas the HRcrit in PLBOE (340 +/- 30 beats/min) was significantly lower compared with that in isogenic controls (440 +/- 25 beats/min). The intrinsic heart rates were significantly lower, and the HRcrit and the +/-dP/dtmax at HRcrit were significantly greater in FVB/N than in SvJ control mice. We conclude that 1) the level of phospholamban is a critical negative determinant of the force-frequency relation and myocardial contractility in vivo, and 2) contractile parameters may differ significantly between strains of normal mice.

Calsequestrin is a high capacity Ca2+-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 Ca2+ transient amplitude was also depressed (45%), although the SR Ca2+ storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca2+ current density and prolongation of this channel's inactivation kinetics without changes in Na+-Ca2+ exchanger current density. Furthermore, there were increases in protein levels of SR Ca2+-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 Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release, leading to depressed contractility in the mammalian heart.

Phospholamban gene transcript levels are much lower in murine atria as compared to murine ventricles and this reduced phospholamban expression has been suggested to result in enhanced atrial contractile parameters. To delineate the functional role of phospholamban in murine atrium, the contractile parameters of isolated muscles from phospholamban knockout and cardiac-specific phospholamban overexpression mice along with their isogenic wild-type controls were evaluated. Assessment of the times (ms) to peak tension development and to half-relaxation of developed tension, as well as the rates (mg/s) of tension development and relaxation in paced atrial muscles, revealed that phospholamban ablation was associated with enhanced rates of relaxation with no significant effect on contraction rate, while phospholamban overexpression (three-fold) was associated with depressed rates of both contraction and relaxation. Isoproterenol stimulation resulted in significant increases in the rates of developed tension and relaxation in both phospholamban deficient and phospholamban overexpression atria, indicating that the beta-adrenergic pathway was functional in these muscles. These findings suggest that phospholamban is an important modulator of atrial contractility and its responses to beta-adrenergic agonists.