Dissociation between contraction and relaxation: The possible role of phospholamban phosphorylation

Abstract

The relationship between myocardial relaxation and phosphorylation of phospholamban, an intrinsic protein of sarcoplasmic reticulum (SR), was studied in perfused rat hearts beating at constant rate and perfused at constant coronary flow. The positive inotropic effect (increase in developed tension, T, and maximal rate of rise of tension, +T) of 3 X 10(-9) and 3 X 10(-8) M isoproterenol (ISO) occurred together, with a proportionately greater increase in maximal velocity of relaxation, -T. Thus, the +T/-T ratio decreased 0.23 +/- 0.04 and 0.41 +/- 0.05 respectively. Time to half-relaxation (t1/2) and the time constant of relaxation (Tau) were also significantly decreased by ISO. Phospholamban phosphorylation (in pmol 32Pi/mg SR protein) increased from 23 +/- 3.3 (control) to 42 +/- 2.3 (3 X 10(-9) M ISO) and to 186 +/- 19.3 (3 X 10(-8) M ISO). When the negative inotropic action of nifedipine was just offset by either Ca2+ (N-Ca2+) or ISO (N-I), relaxation was faster when ISO was present. Perfusion with N-I significantly decreased +T/-T 0.18 +/- 0.05, t1/2 14 +/- 3 ms and Tau 1.4 +/- 0.2 ms. Phospholamban phosphorylation significantly increased from 23 +/- 3.3 to 40 +/- 4.9 pmol 32 Pi/mg SR protein. N-Ca2+ did not elicit any significant change in these parameters nor in phospholamban phosphorylation. Thus, phospholamban phosphorylation appears closely related to myocardial relaxation and may be one of the important mechanisms by which contractility and relaxation are dissociated in vivo.

Frequency-encoding Thr17 Phospholamban Phosphorylation Is Independent of Ser16 Phosphorylation in Cardiac Myocytes

“…In the same cells, however, no significant PKAmediated PLB phosphorylation at Ser 16 was observed at any stimulation frequency. The pattern of Ser 16 and Thr 17 phosphorylation induced by electrical stimulation alone was thus opposite to that in quiescent cells induced by ␤-adrenergic stimulation, suggesting that CaMKII-mediated Thr 17 PLB phosphorylation can occur independent of any prior Ser 16 PLB phosphorylation in intact cardiac myocytes.…”

“…Specifically, PLB in its dephosphorylated state is an inhibitor of the SR Ca 2ϩ pump (2). Phosphorylation of PLB at Ser 16 and Thr 17 by PKA and CaMKII removes this inhibition by increasing the affinity of the pump for Ca 2ϩ (1)(2)(3). In the beating mammalian heart, ␤-adrenergic stimulation increases both PKA-and CaMKIImediated phosphorylation of Ser 16 and Thr 17 (4 -6).…”

“…These previous studies in perfused hearts or in vivo have provided several lines of evidence leading to the concept that Ser 16 phosphorylation is a prerequisite for phosphorylation of Thr 17 and that Ser 16 phosphorylation is largely responsible for ␤-adrenergic modulation of cardiac relaxation (4, 5, 11, 12, 14 -18). Recent studies in transgenic mice overexpressing the Ser 16 3 Ala 16 PLB mutant have further demonstrated that prevention of Ser 16 phosphorylation abolishes Thr 17 phosphorylation and attenuates ␤-adrenergic responses (19). Thus, it is widely accepted that phosphorylation of PLB at Ser 16 is obligatory for Thr 17 phosphorylation and that Ser 16 phosphorylation is the dominant molecular event responsible for accelerated cardiac relaxation.…”

“…Recent studies in transgenic mice overexpressing the Ser 16 3 Ala 16 PLB mutant have further demonstrated that prevention of Ser 16 phosphorylation abolishes Thr 17 phosphorylation and attenuates ␤-adrenergic responses (19). Thus, it is widely accepted that phosphorylation of PLB at Ser 16 is obligatory for Thr 17 phosphorylation and that Ser 16 phosphorylation is the dominant molecular event responsible for accelerated cardiac relaxation. However, in vitro studies in the isolated SR membranes have consistently indicated that Ser 16 and Thr 17 can be readily and independently phosphorylated by PKA and CaMKII, respectively, and that when both are phosphorylated, there is an additive interaction (20,21).…”

“…Thus, it is widely accepted that phosphorylation of PLB at Ser 16 is obligatory for Thr 17 phosphorylation and that Ser 16 phosphorylation is the dominant molecular event responsible for accelerated cardiac relaxation. However, in vitro studies in the isolated SR membranes have consistently indicated that Ser 16 and Thr 17 can be readily and independently phosphorylated by PKA and CaMKII, respectively, and that when both are phosphorylated, there is an additive interaction (20,21). The apparent discrepancy between in vivo and in vitro PLB phosphorylation is yet to be reconciled.…”