Abstract
Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.
Highlights
Human myometrial contractions last approximately 60 seconds and are followed by extended periods of relaxation
As an essential regulator of smooth muscle contractility [1,2], high molecular weight caldesmon (h-CaD) functions to inhibit myosin ATPase activity [7,8] as well as interact with tropomyosin [9,10,11] to block the binding site of myosin on actin. Together these two properties prevent actinmyosin cross bridge cycling, which is critical to the generation of contractile force [3,4]. h-CaD possesses binding domains for calcium (Ca2+)-calmodulin [12], and recently significantly increased myometrial h-CaD expression at $37 weeks gestation was correlated with decreased Ca2+ sensitivity, suggesting that h-CaD is a potent inhibitor of Ca2+-induced smooth muscle contraction [6]
Total ERK2 expression increased by 38% during the onset of labor, densitometric analysis revealed no change in the ratio of phospho-ERK2/total ERK2 between NIL and L laboring human myometrium (P = 0.475; Figure 2D)
Summary
Human myometrial contractions last approximately 60 seconds and are followed by extended periods of relaxation. As an essential regulator of smooth muscle contractility [1,2], h-CaD functions to inhibit myosin ATPase activity [7,8] as well as interact with tropomyosin [9,10,11] to block the binding site of myosin on actin Together these two properties prevent actinmyosin cross bridge cycling, which is critical to the generation of contractile force [3,4]. Phosphorylation of h-CaD on serine residue 789, which is a known ERK phosphorylation site [5], induces conformational changes that relieve inhibition of myosin ATPase as well as exposing the myosin binding site on actin Together these changes permit actin-myosin cross bridge cycling and the subsequent generation of contractile force. Examined h-CaD phosphorylation in vivo, and the authors reported significantly increased h-CaD phosphorylation in term laboring myometrium, statistical significance was achieved relative to non-pregnant myometrium, with no significant difference evident between term non-laboring and term laboring human myometrium
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