The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function.

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.Smooth muscle contraction is initiated by the phosphorylation of the regulatory myosin light chain (MLC 20 ) 1 by the calcium/calmodulin-dependent activation of the myosin light chain kinase (MLCK) (1). Relaxation is effected by the dephosphorylation of MLC 20 by the smooth muscle myosin phosphatase (SMMP). Complexity is brought to this system by accessory proteins and signaling pathways that regulate the smooth muscle contractile state (reviewed in Refs. 2 and 3). The SMMP is a target of signals that are positive and negative modulators of smooth muscle tone. SMMP is a heterotrimeric protein composed of the 37-kDa catalytic subunit (PP1c␦), the 130/133-kDa myosin targeting subunit (MYPT1, also referred to as MBS), and the 21-kDa M21 subunit (4 -6). MYPT1 targets the catalytic subunit to MLC 20 (7,8) and in this way confers substrate specificity to the phosphatase, whereas the function of the M21 subunit is unknown. Activation of the Rho kinase signaling pathway leads to phosphorylation of the MYPT1 subunit, resulting in inhibition of myosin phosphatase activity and an increase in smooth muscle tone (9 -13). This signaling pathway is thought to determine the calcium-sensitizing effect of ␣-adrenergic stimulation, for example, in which greater force is produced at a given calcium concentration th...

Background In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP activated protein kinase (PKG). Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases (PDE) including PDE-5A. We hypothesized that a PDE-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular (LV) diastolic distensibility by phosphorylating titin. Methods and Results 8 elderly dogs with experimental hypertension (OH) and 4 young normal (YN) dogs underwent measurement of the end-diastolic pressure (EDP) volume (EDV) relationship (EDPVR) during caval occlusion at baseline, after sildenafil and after BNP infusion. To assess diastolic distensibility independent of load/extrinsic forces, the EDV at a common EDP on the sequential EDPVRs was measured (LV capacitance). In a separate group of dogs (n=7 OH and 7 YN), serial full thickness LV biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/ml, P<0.001). LV diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 ml, P<0.001). Changes were similar in OH and YN dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chains −1 or −2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion Acute cGMP enhancing treatment with sildenafil and BNP improves LV diastolic distensibility in vivo, in part, by phosphorylating titin.

MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.Force regulation in smooth muscle is dependent on the activities of myosin light chain (MLC) 1 kinase and MLC phosphatase (3, 4). The activity of MLC kinase is regulated by Ca 2ϩ -calmodulin (3), whereas MLC phosphatase was originally thought to be constitutively active and unregulated (5). However, there is abundant evidence that the activity of MLC phosphatase can be both inhibited to produce Ca 2ϩ sensitization (reviewed in Refs. 5-7) or an increase in force at a constant [Ca 2ϩ ] and enhanced to produce Ca 2ϩ desensitization (8) or a decrease in force at a constant [Ca 2ϩ ]. NO is the classical agent to produce Ca 2ϩ desensitization (1, 9, 10). Recent evidence (11) suggests that NO produces vasodilatation by activating the soluble pool of guanylate cyclase, which in turn produces cGMP and leads to the activation of type I cGMP-dependent protein kinase (PKGI). PKGI mediates smooth muscle cell relaxation by several mechanisms. It has been demonstrated that PKGI acts on the maxi K ϩ channel to produce hyperpolarization of the smooth muscle (12), decreases Ca 2ϩ flux (13,14), and also activates MLC phosphatase (1, 15) to decrease the level of MLC 20 phosphorylation and to produce smooth muscle relaxation. In addition, PKGI-dependent pathways for vasodilatation may also include phosphorylation of telokin (16, 17) and HSP20 (18).MLC phosphatase is a holoenzyme consisting of a catalytic subunit (PP1c␦); a myosin-binding subunit (MBS), which is also referred to as the myosin-targeting subunit (MYPT1); and a 20-kDa subunit of unknown function (5). The activation of MLC phosphatase by PKGI is hypothesized to be due to a leucine zipper-leucine zipper (LZ-LZ) interaction of the N-terminal LZ of PKGI␣ and the C-terminal LZ of the MBS of MLC phosphatase (1, 2). The MBS has four major isoforms, which are produced by alternative RNA splicing of two different exons (5). Tissue-specific and developmentally regulated alternative splicing of a 123-bp central exon produces a 41-amino acid central insert (19). Alternative splicing of the 31-bp 3Ј-exon is responsible for the expression of LZ ϩ or LZ Ϫ MBS isoforms (5). Specifically, exclusion of the 3Ј-exon shifts the reading frame of the MBS transcript to encode a C-terminal LZ (2).We previously demonstrated that sensitivity to cGMP-mediated relaxation correlates with the relative expression of LZ ϩ / LZ Ϫ MBS isoforms (2), which is consistent with the activation of MLC phosphatase activity resulting from a LZ-LZ interaction of PKGI␣ with the MBS (1). In this study, we tested the hypothesis that cGMP-dependent activation of MLC phosphatase activity and smooth muscle vasodilatation are due to a LZ-LZ interaction of PKGI␣ and MBS by changing the expression of the MBS isoform, in isolation, and determining the effect on cGMP-mediated MLC 20 dephosphorylation in primary cultured smooth muscle cells (SMCs). MATERIALS AND METHODSCloning of the Chicken MBS of the MLC Phosphatase cDNA Fragment-...

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The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Recent studies have demonstrated that nonmuscle (NM) myosin II forms filaments and can generate and maintain force in smooth muscle tissue [Lofgren et al. (2003) J Gen Physiol 121:301-310; Morano et al. (2000) Nat Cell Biol 2:371-375]. To further investigate the mechanical contribution of NM myosin to force maintenance during smooth muscle contraction, we utilized a selective inhibitor of the NM myosin ATPase, blebbistatin [Straight et al. (2003) Science 299:1743-1747]. Force and myosin light chain (MLC(20)) phosphorylation were measured during KCl stimulation of small strips of intact mouse bladder and aorta at 22 degrees C. The bladder strips contracted with a typical phasic force response, characterized by a large, rapid, transient increase in force followed by a decline to a lower, steady-state level. The addition of blebbistatin did not alter the peak force, but decreased force maintenance. KCl depolarization of aortic strips resulted in a tonic contraction; force increased to a sustained steady state. Similar to the bladder tissue, blebbistatin substantially decreased the steady-state force in the aorta. Blebbistatin did not influence the MLC(20) phosphorylation transient in either tissue type. Additionally, blebbistatin did not change the maximum shortening velocity (V (max)) during KCl depolarization of the aorta. Our results also suggest that NMIIA and NMIIB isoforms are differentially expressed. The expression of NMIIA is more prominent in the bladder, while NMIIB expression is predominant in the aorta. These results suggest that NM myosin contributes to the mechanism of force maintenance in smooth muscle, and could suggest that the expression of NMIIB is a factor for determining the tonic contractile phenotype.

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