Induction of ferroptosis promotes vascular smooth muscle cell phenotypic switching and aggravates neointimal hyperplasia in mice.

BACKGROUND: Stent implantation-induced neointima formation is a dominant culprit in coronary artery disease treatment failure after percutaneous coronary intervention. Ferroptosis, an iron-dependent regulated cell death, has been associated with various cardiovascular diseases. However, the effect of ferroptosis on neointima formation remains unclear. METHODS: The mouse common right carotid arteries were ligated for 16 or 30 days, and ligated tissues were collected for further analyses. Primary rat vascular smooth muscle cells (VSMCs) were isolated from the media of aortas of Sprague-Dawley (SD) rats and used for in vitro cell culture experiments. RESULTS: Ferroptosis was positively associated with neointima formation. In vivo, RAS-selective lethal 3 (RSL3), a ferroptosis activator, aggravated carotid artery ligation-induced neointima formation and promoted VSMC phenotypic conversion. In contrast, a ferroptosis inhibitor, ferrostatin-1 (Fer-1), showed the opposite effects in mice. In vitro, RSL3 promoted rat VSMC phenotypic switching from a contractile to a synthetic phenotype, evidenced by increased contractile markers (smooth muscle myosin heavy chain and calponin 1), and decreased synthetic marker osteopontin. The induction of ferroptosis by RSL3 was confirmed by the increased expression level of ferroptosis-associated gene prostaglandin-endoperoxide synthase 2 (Ptgs2). The effect of RSL3 on rat VSMC phenotypic switching was abolished by Fer-1. Moreover, N-acetyl-L-cysteine (NAC), the reactive oxygen species inhibitor, counteracted the effect of RSL3 on the phenotypic conversion of rat VSMCs. CONCLUSIONS: Ferroptosis induces VSMC phenotypic switching and accelerates ligation-induced neointimal hyperplasia in mice. Our findings suggest inhibition of ferroptosis as an attractive strategy for limiting vascular restenosis.

Intrauterine neuromuscular and stromal dysplasia of the bladder in retinoic acid-induced myelomeningocele fetal rats.

OBJECTIVES: To observe the changes in the bladder of fetal rats with myelomeningocele (MMC) induced by all-trans retinoic acid (atRA) during the embryonic development stages. METHODS: The fetal rat model of MMC was induced by intragastric administration of atRA to pregnant rats on embryonic day 10 (E10). Fetal rats were harvested at E16, E18, E20, and E21 for observation and further testing. Those with MMC were classified as the MMC group, while those without MMC as the RA group. The areas of MMC skin defect, the crown-rump length (CRL), and body weight in different groups were compared. The histopathological changes in the bladder were compared. The expression levels of alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMMHC), connexin 43 (Cx43), desmin, ß3 tubulin, and vesicular acetylcholine transporter (VAChT) in the bladder were investigated by immunohistochemical staining and Western blotting. Pregnant rats given intragastric administration with olive oil (OIL group) at E10 were set as the blank control group. RESULTS: A total of 415 fetal rats of different gestational ages were harvested with an MMC incidence of 56.05 % (139/248). The incidence rate increased with embryonic days (p < 0.001). Compared with the other two control groups, the CRL and bodyweight of MMC fetal rats were significantly delayed at E21 (p < 0.001). The expression levels of αSMA, SMMHC, Cx43, desmin, ß3 tubulin and VAChT in the bladder of MMC fetal rats were significantly decreased at E21 (p < 0.05). CONCLUSIONS: In atRA-induced MMC fetal rats, there is neural, muscular, and stromal dysplasia in the bladder at an early gestational age. Further investigations on neurogenic bladder secondary to MMC are applicable using this animal model.

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP.

The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75 µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5 µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.

Phenotype transitions induced by mechanical stimuli in airway smooth muscle are regulated by differential interactions of parvin isoforms with paxillin and Akt.

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.

Doxorubicin induces detrusor smooth muscle impairments through myosin dysregulation, leading to a risk of lower urinary tract dysfunction.

Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg-1·wk-1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+ channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.

RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na+/H+ exchanger.

Smooth muscle contraction is triggered when Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC20). However, blood vessels from Mlck-deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC20 to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from Rsk2-deficient mice were dilated and showed reduced myogenic tone and RLC20 phosphorylation. RSK2 phosphorylated Ser19 in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na+/H+ exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca2+ that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in Rsk2-deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca2+ transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, Rsk2-deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

PROBLEM: Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. METHODS OF STUDY: Mid-secretory phase endometrial tissues from women with RPL (n = 15) and fertile controls (n = 7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. RESULTS: CD56+ uNK number was significantly higher in women with RPL compared to controls (P < 0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2  = 0.43, P = 0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P < 0.05 and P < 0.0001, respectively), and correlated with the number of uNK cell (r2  = 0.18, P < 0.04, and r2  = 0.65, P < 0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P = 0.0027). CONCLUSION: Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

The expression and functional activities of smooth muscle myosin and non-muscle myosin isoforms in rat prostate.

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non-muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM-A/B) and myosin light chain 17 isoforms (LC17a/b ), and isoform ratios were determined via competitive RT-PCR. SM MHC and NM MHC isoforms (NMMHC-A, NMMHC-B and NMMHC-C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC17a and SM1 but with relatively equal expression of SM-A/SM-B at the mRNA level. Meanwhile, isoforms of NMMHC-A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC-A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre-contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.

Helical model of smooth muscle myosin filament and the ribbons made of caldesmon: history revisited.

In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio.

Lymphocytes influence intracranial aneurysm formation and rupture: role of extracellular matrix remodeling and phenotypic modulation of vascular smooth muscle cells.

BACKGROUND: Intracranial aneurysms (IA) are increasingly recognized as a disease driven by chronic inflammation. Recent research has identified key mediators and processes underlying IA pathogenesis, but mechanistic understanding remains incomplete. Lymphocytic infiltrates have been demonstrated in patient IA tissue specimens and have also been shown to play an important role in abdominal aortic aneurysms (AAA) and related diseases such as atherosclerosis. However, no study has systematically examined the contribution of lymphocytes in a model of IA. METHODS: Lymphocyte-deficient (Rag1) and wild-type (WT; C57BL/6 strain) mice were subjected to a robust IA induction protocol. Rates of IA formation and rupture were measured, and cerebral artery tissue was collected and utilized for histology and gene expression analysis. RESULTS: At 2 weeks, the Rag1 group had significantly fewer IA formations and ruptures than the WT group. Histological analysis of unruptured IA tissue showed robust B and T lymphocyte infiltration in the WT group, while there were no differences in macrophage infiltration, IA diameter, and wall thickness. Significant differences in interleukin-6 (IL-6), matrix metalloproteinases 2 (MMP2) and 9 (MMP9), and smooth muscle myosin heavy chain (MHC) were observed between the groups. CONCLUSIONS: Lymphocytes are key contributors to IA pathogenesis and provide a novel target for the prevention of IA progression and rupture in patients.

Capsaicin alleviates abnormal intestinal motility through regulation of enteric motor neurons and MLCK activity: Relevance to intestinal motility disorders.

SCOPE: Capsaicin is an active component of chili peppers, having diverse effects. However, the effects of capsaicin on intestinal motility are still controversial. The present study aimed to investigate the effects of capsaicin on intestinal motility disorder and uncover related mechanisms. MATERIALS AND RESULTS: A rat model with intestinal motility disorder was established in vitro through adding different stimuli into tissue bath; in vivo using constipation and diarrhea model, respectively. Capsaicin exerted dual effects on intestinal motility, i.e. the relaxation and contraction of jejunum induced by corresponding stimulus were, respectively, regulated to be normal contraction by capsaicin. The mechanisms underlined capsaicin-induced dual effects were investigated using Western blotting, qRT-PCR, and whole-cell patch clamp, respectively. Results showed that cholinergic excitatory nerves, adrenergic nerves, and neurons containing nitric oxide synthase, which are the main muscle motor neurons in enteric nervous system (ENS), are involved in capsaicin-induced dual effects. The competition for regulation of Ca(2+) influx by capsaicin induced the interaction with components of the ENS. Capsaicin significantly increased myosin light chain kinase (MLCK) expression and myosin phosphorylation extent in jejunal segments of constipation-prominent rats and significantly decreased MLCK expression and myosin phosphorylation extent in jejunal segments of diarrhea-prominent rats. CONCLUSION: In summary, capsaicin alleviates abnormal intestinal motility through regulating enteric motor neurons and MLCK activity, which is beneficial for the treatment of gastrointestinal motility disorders.

Phosphate and ADP differently inhibit coordinated smooth muscle myosin groups.

Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 µm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo--suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.

Phenotypic modulation of smooth muscle cells in lymphoedema.

BACKGROUND: Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood. Smooth muscle cells (SMCs) play pivotal roles in the pathogenesis of various vascular diseases, including atherosclerosis. OBJECTIVES: We analysed SMCs in lymphatic vessels from the lymphoedematous legs of 29 patients. METHODS: Expression of smooth muscle α-actin (SMαA) and smooth muscle myosin heavy chain (SM-MHC) isoforms SM1 and SM2 was investigated using immunohistochemistry. RESULTS: Compared with normal lymphatic vessels, all affected lymphatic vessels in chronic lymphoedema showed marked wall thickening. In addition to increases in the numbers of rows of SMαA(+) SM1(+) SMCs in the tunica media, SMCs were also observed in the subendothelial region (tunica intima). While most intimal and medial cells were positive for SMαA and SM1, staining for SM1 and particularly SM2, a marker of mature SMCs, progressively declined in lymphatic vessels in increasingly severe lymphoedema lesions. Consequently, the SM1(+) and SM2(+) cell fractions were significantly reduced in the tunica media and intima of lymphatic vessels. CONCLUSIONS: These observations indicate that the lymphatic tunica media and tunica intima consist mainly of phenotypically modulated SMCs, and that SMCs play a key role in the development of lymphoedema.

Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

Function of the head-tail junction in the activity of myosin II.

All class II myosins have the conserved amino acid sequence Pro-Leu-Leu at their head-tail junctions. We systematically altered this sequence in smooth muscle heavy meromyosin (HMM) by site-directed mutagenesis and examined the effects of these mutations on actin-myosin interactions. Deletion of the proline and second leucine did not cause any noticeable change in either actin-activated ATPase activity or actin-sliding velocity. In contrast, deletion of the two leucine residues and substitution of the first leucine with alanine resulted in a 14-fold and 5-fold decrease, respectively, in actin-activated ATPase activity. However, both these mutations did not appreciably affect actin-sliding velocity, which was consistent with a result that there was no considerable change in the ADP release rate from acto-HMM in the deletion mutant. In contrast to double-headed HMM, a single-headed subfragment-1 (S1) with a Leu-Leu deletion mutation exhibited actin activated ATPase activity similar to that by wild type S1. Our results suggest that the first leucine of the conserved Leu-Leu sequence at the head-tail junction profoundly affects the cooperativity between the two heads involved in the actin activated ATPase activity of myosin II.

Transplantation of myoblast sheets that secrete the novel peptide SVVYGLR improves cardiac function in failing hearts.

AIMS: Transplantation of myoblast sheets is a promising therapy for enhancing cardiac function after heart failure. We have previously demonstrated that a 7-amino-acid sequence (Ser-Val-Val-Tyr-Gly-Leu-Arg) derived from osteopontin (SV peptide) induces angiogenesis. In this study, we evaluated the long-term therapeutic effects of myoblast sheets secreting SV in a rat infarction model. METHODS AND RESULTS: Two weeks after ligation of the left anterior descending coronary artery, the animals were divided into the following three groups: a group transplanted with wild-type rat skeletal myoblast sheets (WT-rSkMs); a group transplanted with SV-secreting myoblast sheets (SV-rSkMs); and a control group (ligation only). We evaluated cardiac function, histological changes, and smooth muscle actin (SMA) expression through transforming growth factor-ß (TGF-ß) signalling. The ejection fraction and fractional shortening were significantly better, and the enlargement of end-systolic volume was also significantly attenuated in the SV-rSkM group. Left ventricular remodelling, including fibrosis and hypertrophy, was significantly attenuated in the SV-rSkM group, and SV secreted by the myoblast sheets promoted angiogenesis in the infarcted border area. Furthermore, many clusters of SMA-positive cells were observed in the infarcted areas in the SV-rSkM group. In vitro SMA expression was increased when SV was added to the isolated myocardial fibroblasts. Moreover, SV bound to the TGF-ß receptor, and SV treatment activated TGF-ß receptor-Smad signalling. CONCLUSION: The SV-secreting myoblast sheets facilitate a long-term improvement in cardiac function. The SV can induce differentiation of fibroblasts to myofibroblasts via TGF-ß-Smad signalling. This peptide could possibly be used as a bridge to heart transplantation or as an ideal peptide drug for cardiac regeneration therapy.

Cross-talk between Rho-associated kinase and cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin light chain phosphatase.

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Temperature dependent measurements reveal similarities between muscle and non-muscle myosin motility.

We examined the temperature dependence of muscle and non-muscle myosin (heavy meromyosin, HMM) with in vitro motility and actin-activated ATPase assays. Our results indicate that myosin V (MV) has a temperature dependence that is similar in both ATPase and motility assays. We demonstrate that skeletal muscle myosin (SK), smooth muscle myosin (SM), and non-muscle myosin IIA (NM) have different temperature dependence in ATPase compared to in vitro motility assays. In the class II myosins we examined (SK, SM, and NM) the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while for in vitro motility assays it is controversial. In MV the rate-limiting step for both in vitro motility and ATPase assays is known to be ADP release. Consequently, in MV the temperature dependence of the ADP release rate constant is similar to the temperature dependence of in vitro motility. Interestingly, the temperature dependence of the ADP release rate constant of SM and NM was shifted toward the in vitro motility temperature dependence. Our results suggest that the rate-limiting step in SK, SM, and NM may shift from attachment-limited in solution to detachment limited in the in vitro motility assay. Internal strain within the myosin molecule or by neighboring myosin motors may slow ADP release which becomes rate-limiting in the in vitro motility assay. Within this small subset of myosins examined, the in vitro sliding velocity correlates reasonably well with actin-activated ATPase activity, which was suggested by the original study by Barany (J Gen Physiol 50:197-218, 1967).

Loop 1 dynamics in smooth muscle myosin: isoform specific differences modulate ADP release.

Isoforms of the smooth muscle (SM) myosin motor domain differ in the presence or absence of a seven amino acid insert in a flexible surface loop spanning the nucleotide-binding pocket known as Loop 1. The presence of this insert leads to a two-fold increase in actin sliding velocity and ADP release rate between these isoforms, although the effect of Loop 1 on the kinetics of ADP release remains unclear. To further investigate the role of the Loop 1 insert in modulating ADP release in SM myosin we have inserted a single tryptophan residue into Loop 1 of both isoforms as a probe of local structural dynamics. By monitoring the dynamics of Loop 1 in relation to the release of ADP we have observed a unique movement of Loop 1 in the inserted isoform, preceding nucleotide release, which is absent in the non-inserted isoform. This movement is sequence dependent as alanine replacement of the insert residues abolishes the transition and slows ADP release. Thus movement of Loop 1 is a critical factor in increasing the ADP release rate in the inserted faster isoform of SM myosin.

Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue.

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1-2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators.