Abstract
Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.
Highlights
Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins
We compared the primary sequence of known myofilament phosphoproteins from different species with the cardiac myosin light chain kinase (cMLCK) consensus sequence in regulatory light chain (RLC) and identified serines 22/23 in the cardiac specific N-terminal extension (NTE) of human cardiac troponin I as potential substrates (Fig. 1A, red)
Myosin light chain kinases have long been considered “dedicated kinases” that phosphorylate the myosin heavy chain-associated RLCs [13], recent research has suggested that MLCKs can phosphorylate other targets; e.g. the skeletal isoform of MLCK has been shown to regulate skeletal muscle myogenesis via phosphorylation of the transcription factor MEF2c [20], and smooth muscle MLCK has been suggested recently to phosphorylate a multitude of proteins that, interestingly, overlap with PKA signaling targets in nonmuscle cells [21]
Summary
We compared the primary sequence of known myofilament phosphoproteins from different species with the cMLCK consensus sequence in RLC and identified serines 22/23 in the cardiac specific N-terminal extension (NTE) of human cardiac troponin I (hcTnI) as potential substrates (Fig. 1A, red). To achieve a homogeneously low cTnI phosphorylation level, we first exchanged trabeculae overnight with unphosphorylated human or rat cardiac troponin, followed by PP treatment directly before the mechanical experiments and subsequent incubation with Ca2ϩ/CaM/cMLCK. Incubation of rat cTn-exchanged and PP-dephosphorylated trabeculae with Ca2ϩ/CaM-activated cMLCK increased the calcium sensitivity of force by 0.07 Ϯ 0.01 pCa (mean Ϯ S.E., n ϭ 5) (Fig. 5A, Tables S1 and S2) but had no effect on the steepness of the force calcium relation (nH of 4.76 Ϯ 0.46 versus 4.44 Ϯ 0.42, mean Ϯ S.E.), in good agreement with results obtained previously from native rat ventricular trabeculae [7]. SDS-PAGE (see Fig. 1B), we could not directly determine the level of troponin I phosphorylation, but it was estimated to be ϳ0.3 mol Pi/mol, assuming a troponin exchange efficiency of 70%
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