Threonine-17 phosphorylation of phospholamban: a key determinant of frequency-dependent increase of cardiac contractility

Multiple studies have shown that phospholamban (PLN) plays a key role in regulation of frequency-dependent increase of cardiac contraction, a hallmark of the contractile reserve in myocardium. However, the mechanisms underlying this relationship remain elusive. Phosphorylation of PLN occurs on residues: serine-16 (Ser16) and threonine-17 (Thr17) in vivo. In isolated wild-type cardiomyocytes, we found that increases of stimulation frequency from 0.5 to 5 Hz were associated with increased Thr17 phosphorylation of PLN and cardiac contractility. To further delineate the role of PLN phosphorylation in the frequency-dependent increases of cardiac function, three transgenic mouse models, expressing wild-type, Ser16Ala (S16A), or Thr17Ala (T17A) mutant PLN in the null background were generated. Transgenic lines expressing similar levels of wild-type or mutant PLN were selected and isolated cardiomyocytes were paced from 0.5 to 5 Hz. Upon increases in pacing frequency, the fractional shortening (FS) and rates of contraction (+dL/dt) and relaxation (–dL/dt) increased in wild-type and S16A mutant PLN cardiomyocytes. In contrast, in myocytes expressing the T17A mutant PLN, there were no increases in FS and ±dL/dt upon increasing the frequency of stimulation. The time to 50% peak shortening (TTP50) and to 50% relaxation (TTR50) were also abbreviated to a much higher extent (two-fold) in wild-type and S16A mutant compared to T17A mutant PLN cardiomyocytes. These results indicate that Thr17 phosphorylation of PLN is the major contributor to frequency-dependent increases of contractile and relaxation parameters in mouse cardiomyocytes, although some increases in these parameters occur even in the absence of PLN phosphorylation. Thus, the positive force–frequency relationship in cardiomyocytes is mechanistically and mainly related to PLN phosphorylation.