Ca²⁺ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

Abstract

Key points• Blood flow to our organs is maintained within a defined range to provide an adequate supply of nutrients and remove waste products by contraction and relaxation of smooth muscle cells of resistance arteries and arterioles.• The ability of these cells to contract in response to an increase in intravascular pressure, and to relax following a reduction in pressure (the 'myogenic response'), is critical for appropriate control of blood flow, but our understanding of its mechanistic basis is incomplete.• Small arteries of skeletal muscles were used to test the hypothesis that myogenic constriction involves two enzymes, Rho-associated kinase and protein kinase C, which evoke vasoconstriction by activating the contractile protein, myosin, and by reorganizing the cytoskeleton.• Knowledge of the mechanisms involved in the myogenic response contributes to understanding of how blood flow is regulated and will help to identify the molecular basis of dysfunctional control of arterial diameter in disease.Abstract The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca 2+ entry via voltage-gated Ca 2+ channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC 20 ) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca 2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca 2+ sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC 20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC 20 was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC 20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca 2+ sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC 20 pho...

Cytoskeletal Reorganization Evoked by Rho-associated kinase- and Protein Kinase C-catalyzed Phosphorylation of Cofilin and Heat Shock Protein 27, Respectively, Contributes to Myogenic Constriction of Rat Cerebral Arteries

“…20 , Calponin, or Caldesmon Phosphorylation-RMCA constriction under conditions in which a detectable change in the level of LC 20 phosphorylation was not apparent was specifically examined to assess the contribution of thin filament regulatory phosphoproteins and cytoskeletal reorganization to force development. Phospho-LC 20 content of RMCAs pressurized to 100 mm Hg was previously shown to be ϳ50% (18), consistent with the maximal level of 50 -55% detected for rat skeletal muscle arterioles pressurized to between 80 and 140 mm Hg (19). Based on these data, the level of phospho-LC 20 was quantified using RMCA segments flash frozen at 10, 80, and 120 mm Hg once vessel diameter had stabilized at the relevant intraluminal pressure (this required 30 s to 3 min in different vessels).…”

“…Previous studies postulated that PKC might phosphorylate CPI-17 and/or MYPT1, leading to an inhibition of MLCP activity, Ca 2ϩ sensitization of the contractile filaments, increased LC 20 phosphorylation, and greater force at constant [Ca 2ϩ ] i (6,8,94). However, this is unlikely because no evidence of a pressure-dependent increase in the level of phospho-CPI-17 or PKC-dependent MYPT1 phosphorylation was detected in the myogenic response of rat cerebral or gracilis arteries (18,19,52). That the pressure-dependent increase in HSP27 phosphorylation and decline in G-actin content were blocked by GF109203X treatment in this study indicates that PKC inhibitors may suppress myogenic constriction through inhibition of PKC-mediated HSP27 phosphorylation and actin polymerization.…”

“…Myogenic constriction is accompanied by: (i) E m depolarization leading to Ca 2ϩ influx through voltage-gated Ca 2ϩ channels (14), increased Ca 2ϩ wave activity (i.e. increased frequency and number of cells exhibiting waves (15), but see (16)), and increased LC 20 phosphorylation (17)(18)(19) that is suppressed by the MLCK inhibitor, ML-7 (17); as well as (ii) increased inhibitory MYPT1-T855 phosphorylation that is suppressed by blockers of ROK activity, including Y27632 and H1152, with an associated loss of myogenic constriction (18,19). Although inhibition of PKC suppresses myogenic constriction (20,21), no change in CPI-17 phosphorylation was detected, excluding a role for PKC-mediated suppression of MLCP activity (18,19).…”

Regulation of Smooth Muscle Dystrophin and Synaptopodin 2 Expression by Actin Polymerization and Vascular Injury

“…Actin polymerization as such directly regulates smooth muscle contractility and remodeling in resistance arteries. 13,14,27 Furthermore, the effect of actin polymerization for gene transcription via MRTF has been shown to be involved in multiple and SMCs. 10,[28][29][30] Herein, we demonstrate that the ratio of filamentous to globular actin is dramatically decreased in phenotypically modulated SMCs.…”

“…12 Apart from its role in transcriptional regulation, actin dynamics is known to be crucial for smooth muscle contraction and an abnormal increase in actin polymerization may thus result in hypercontractility of arteries leading to inward remodeling and hypertension. 13,14 Furthermore, we have previously demonstrated that actin dynamics is an important factor for stretch sensing in vascular smooth muscle. [15][16][17] In the present study, we hypothesized that genes whose transcription is dependent on actin polymerization are also involved in the regulation of smooth muscle function and vascular disease.…”