James T. Stull

Acta2 R149C/+ mice did not develop thoracic aortic disease despite decreased aortic contraction, likely due to increased retention of mutant SM α-actin in the TCP1 folding complex.

The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1cβ, is essential for smooth muscle contraction

Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo . In contrast, smooth muscle–specific conditional PP1cβ knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cβ to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cβ content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cβ forms in smooth muscle.

Myh11 mutations reduce force development in aortic smooth muscle vs wild-type tissues.

Nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation differ from pharmacological responses with bath-added agonist in airway smooth muscle.

MilewiczArteriosclerosis Thrombosis and Vascular Biology

Altered Smooth Muscle Cell Force Generation as a Driver of Thoracic Aortic Aneurysms and Dissections

The importance of maintaining contractile function in aortic smooth muscle cells (SMCs) is evident by the fact that heterozygous mutations in the major structural proteins or kinases controlling contraction lead to the formation of aneurysms of the ascending thoracic aorta that predispose to life-threatening aortic dissections. Force generation by SMC requires ATP-dependent cyclic interactions between filaments composed of SMC-specific isoforms of α-actin (encoded by ACTA2) and myosin heavy chain (MYH11). ACTA2 and MYH11 mutations are predicted or have been shown to disrupt this cyclic interaction predispose to thoracic aortic disease. Movement of the myosin motor domain is controlled by phosphorylation of the regulatory light chain on the myosin filament, and loss-of-function mutations in the dedicated kinase for this phosphorylation, myosin light chain kinase (MYLK) also predispose to thoracic aortic disease. Finally, a mutation in the cGMP-activated protein kinase (PRKG1) results in constitutive activation of the kinase in the absence of cGMP, thus driving SMC relaxation in part through increased dephosphorylation of the regulatory light chain and predisposes to thoracic aortic disease. Furthermore, SMCs cannot generate force without connections to the extracellular matrix through focal adhesions, and mutations in the major protein in the extracellular matrix, fibrillin-1, linking SMCs to the matrix also cause thoracic aortic disease in individuals with Marfan syndrome. Thus, disruption of the ability of the aortic SMC to generate force through the elastin-contractile units in response to pulsatile blood flow may be a primary driver for thoracic aortic aneurysms and dissections.

Cardiac myosin light chain kinase (cMLCK) and the newly characterized MLCK4 exhibit distinct biochemical regulatory properties for regulatory light chain phosphorylation in cardiac muscle.

Physiological RLC phosphorylation and force development in ileal smooth muscle depend on MLCK and MLCP activities without changes in constitutive MYPT1 phosphorylation.

Posttetanic potentiation interferes with the catchlike property during concentric force development at moderate speeds of shortening in wild-type mouse skeletal muscle.

In failing RV, α1A-subtype stimulation increased contractile force by ≈200%, whereas α1B-subtype stimulation decreased force by ≈50%.

A conditioning stimulus increased mean concentric force in wildtype and skMLCK -/- mouse muscles to 1.31-1.35 and 1.19-1.21 of pre-stimulus levels, respectively (p<0.05).

Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, while RLC phosphorylation was not changed by dobutamine or beta-blockers.

Smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation, whereas T852A mice displayed no significant changes.

A conditioning stimulus increased mean concentric force in wild-type and skMLCK(-/-) mouse muscles to 1.31-1.35 and 1.19-1.21 of prestimulus levels, respectively (p < 0.05).

MYPT1 deletion in adult mice enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries, demonstrating its role in blood pressure maintenance.

MYPT1 depletion reduced PP1cδ protein but resulted in only modest changes in bladder smooth muscle contractile and relaxation responses to KCl or carbachol, as substantial constitutive phosphorylation of MYPT1 in wild-type tissues already attenuates MLCP activity.

The Effects of Neuregulin on Cardiac Myosin Light Chain Kinase Gene-Ablated Hearts

A 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses, contrasting with bladder smooth muscle where a 50% decrease had no effect.

Signalling to contractile proteins by muscarinic and purinergic pathways in neurally stimulated bladder smooth muscle

Key points Parasympathetic nerves release the neurotransmitters ATP and acetylcholine that activate purinergic and muscarinic receptors, respectively, to initiate contraction of urinary bladder smooth muscle. Although both receptors mediate Ca 2+ influx for myosin regulatory light chain (RLC) phosphorylation necessary for contraction, the muscarinic receptor may also recruit cellular mechanisms affecting the Ca 2+ sensitivity of RLC phosphorylation. Using transgenic mice expressing Ca 2+ /calmodulin sensor myosin light chain kinase (MLCK) in smooth muscles, the effects of selective purinergic or muscarinic receptor inhibition were examined on neurally stimulated tissues in relation to signalling pathways converging on RLC phosphorylation. Purinergic‐mediated Ca 2+ signals provide the initial Ca 2+ /calmodulin activation of MLCK with muscarinic receptors supporting sustained responses. Activation of muscarinic receptors leads phosphorylation of myosin light chain phosphatase inhibitor CPR‐17 to enhance Ca 2+ sensitivity while also initiating phosphorylation‐dependent Ca 2+ desensitization of MLCK. The interplay between Ca 2+ sensitization and desensitization mechanisms fine tunes the contractile signalling module for force development. Abstract Urinary bladder smooth muscle contraction is triggered by parasympathetic nerves, which release ATP and acetylcholine (ACh) that bind to purinergic and muscarinic receptors, respectively. Neuronal signalling may thus elicit myosin regulatory light chain (RLC) phosphorylation and contraction through the combined, but distinct contributions of these receptors. Both receptors mediate Ca 2+ influx whereas muscarinic receptors may also recruit Ca 2+ ‐sensitization mechanisms. Using transgenic mice expressing calmodulin sensor myosin light chain kinase (MLCK) in smooth muscles, the effects of suramin/α,β‐methyleneATP (α,β‐meATP) (purinergic inhibition) or atropine (muscarinic inhibition) on neurally stimulated elevation of [Ca 2+ ] i , MLCK activation, force and phosphorylation of RLC, myosin light chain phosphatase (MLCP) targeting subunit MYPT1 and MLCP inhibitor protein CPI‐17 were examined. Electric field stimulation (EFS) increased [Ca 2+ ] i , MLCK activation and concomitant force in a frequency‐dependent manner. The dependence of force on [Ca 2+ ] i and MLCK activation decreased with time suggesting increased Ca 2+ sensitization in the late contractile phase. RLC and CPI‐17 phosphorylation increased upon stimulation with maximal responses at 20 Hz; both responses were attenuated by atropine, but only RLC phosphorylation was inhibited by suramin/α,β‐meATP. Antagonism of purinergic receptors suppressed maximal MLCK activation to a greater extent in the early contractile phase than in the late contractile phase; atropine had the opposite effect. A frequency‐ and time‐dependent increase in MLCK phosphorylation explained the desensitization of MLCK to Ca 2+ , since MLCK activation declined more rapidly than [Ca 2+ ] i . EFS elicited little or no effect on MYPT1 Thr696 or 850 phosphorylation. Thus, purinergic Ca 2+ signals provide the initial activation of MLCK with muscarinic receptors supporting sustained responses. Activation of muscarinic receptors recruits CPI‐17, but not MYPT1‐mediated Ca 2+ sensitization. Furthermore, nerve‐released ACh also initiates signalling cascades leading to phosphorylation‐dependent desensitization of MLCK.

Myh11(R247C/R247C) mice showed decreased aortic contraction and significantly increased neointimal formation due to increased SMC proliferation compared with wild-type mice.

Smooth muscle MLCK knockout in mice lowers basal blood pressure and blunts salt-induced hypertension.