Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.

Shatavari Supplementation in Postmenopausal Women Improves Handgrip Strength and Increases Vastus lateralis Myosin Regulatory Light Chain Phosphorylation but Does Not Alter Markers of Bone Turnover

Shatavari has long been used as an Ayurvedic herb for women’s health, but empirical evidence for its effectiveness has been lacking. Shatavari contains phytoestrogenic compounds that bind to the estradiol receptor. Postmenopausal estradiol deficiency contributes to sarcopenia and osteoporosis. In a randomised double-blind trial, 20 postmenopausal women (68.5 ± 6 years) ingested either placebo (N = 10) or shatavari (N = 10; 1000 mg/d, equivalent to 26,500 mg/d fresh weight shatavari) for 6 weeks. Handgrip and knee extensor strength were measured at baseline and at 6 weeks. Vastus lateralis (VL) biopsy samples were obtained. Data are presented as difference scores (Week 6—baseline, median ± interquartile range). Handgrip (but not knee extensor) strength was improved by shatavari supplementation (shatavari +0.7 ± 1.1 kg, placebo −0.4 ± 1.3 kg; p = 0.04). Myosin regulatory light chain phosphorylation, a known marker of improved myosin contractile function, was increased in VL following shatavari supplementation (immunoblotting; placebo −0.08 ± 0.5 a.u., shatavari +0.3 ± 1 arbitrary units (a.u.); p = 0.03). Shatavari increased the phosphorylation of Aktser473 (Aktser473 (placebo −0.6 ± 0.6 a.u., shatavari +0.2 ± 1.3 a.u; p = 0.03) in VL. Shatavari supplementation did not alter plasma markers of bone turnover (P1NP, β-CTX) and stimulation of human osteoblasts with pooled sera (N = 8 per condition) from placebo and shatavari supplementation conditions did not alter cytokine or metabolic markers of osteoblast activity. Shatavari may improve muscle function and contractility via myosin conformational change and further investigation of its utility in conserving and enhancing musculoskeletal function, in larger and more diverse cohorts, is warranted.

Shatavari Supplementation in Postmenopausal Women Improves Handgrip Strength and Increases Vastus Lateralis Myosin Regulatory Light Chain Phosphorylation But Does Not Alter Markers of Bone Turnover: A Randomised Controlled Trial

BackgroundShatavari has long been used as an Ayurvedic herb for women’s health, but empirical evidence for its effectiveness has been lacking. Shatavari contains phytoestrogenic compounds that bind to the estradiol receptor, and may therefore benefit postmenopausal women since postmenopausal estradiol deficiency contributes to sarcopenia and osteoporosis.MethodsIn a randomised double-blind trial, 20 postmenopausal women (68.5 ± 6 y) ingested either placebo (N=10) or shatavari (N=10; 1000 mg/d, equivalent to 26,500 mg/d fresh weight shatavari) for 6 weeks. Handgrip and knee extensor strength were measured at baseline and at 6 weeks. Vastus lateralis (VL) biopsy samples were obtained. Data are presented and analysed (t test/Mann Whitney U) as difference scores (Week 6 – baseline, median ± interquartile range).ResultsHandgrip, (but not knee extensor) strength was improved by shatavari supplementation (shatavari +0.7 ± 1.1 kg, placebo -0.4 ± 1.3 kg; p=0.04). Myosin regulatory light chain phosphorylation, a known marker of improved myosin contractile function, was increased in VL following shatavari supplementation (immunoblotting; placebo -0.08 ± 0.5 a.u. shatavari +0.3 ± 1 arbitrary units (a.u.); p=0.03). Shatavari increased phosphorylation of Aktser473 (Aktser473 (placebo -0.6 ± 0.6 a.u. shatavari +0.2 ± 1.3 a.u; p=0.03) in VL. Shatavari supplementation did not alter plasma markers of bone turnover (P1NP, β-CTX) and stimulation of human osteoblasts with pooled sera (N=8 per condition) from placebo and shatavari supplementation conditions did not alter cytokine or metabolic markers of osteoblast activity.ConclusionsShatavari may improve muscle function and contractility via myosin conformational change and warrants further investigation of its utility in conserving musculoskeletal function in postmenopausal women.Trial RegistrationRetrospectively registered at clinicaltrials.gov as NCT05025917 on 30/08/21.

A new role of glutathione peroxidase 4 during human erythroblast enucleation

The selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane–oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA–mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion.

Does postactivation potentiation (PAP) increase voluntary performance?

The transient increase in torque of an electrically evoked twitch following a voluntary contraction is called postactivation potentiation (PAP). Phosphorylation of myosin regulatory light chains is the most accepted mechanism explaining the enhanced electrically evoked twitch torque. While many authors attribute voluntary postactivation performance enhancement (PAPE) to the positive effects of PAP, few actually confirmed that contraction was indeed potentiated using electrical stimulation (twitch response) at the time that PAPE was measured. Thus, this review aims to investigate if increases in voluntary performance after a conditioning contraction (CC) are related to the PAP phenomenon. For this, studies that confirmed the presence of PAP through an evoked response after a voluntary CC and concurrently evaluated PAPE were reviewed. Some studies reported increases in PAPE when PAP reaches extremely high values. However, PAPE has also been reported when PAP was not present, and unchanged/diminished performance has been identified when PAP was present. This range of observations demonstrates that mechanisms of PAPE are different from mechanisms of PAP. These mechanisms of PAPE still need to be understood and those studying PAPE should not assume that regulatory light chain phosphorylation is the mechanism for such enhanced voluntary performance. Novelty The occurrence of PAP does not necessarily mean that the voluntary performance will be improved. Improvement in voluntary performance is sometimes observed when the PAP level reaches extremely high values. Other mechanisms may be more relevant than that for PAP in the manifestation of acute increases in performance following a conditioning contraction.

Lack of influence of estrogen on myosin phosphorylation and post-tetanic potentiation in muscles from young adult C57BL mice

Estrogen influences myosin phosphorylation and post-tetanic potentiation in murine fast muscle. We tested the hypothesis that this influence is mediated by estrogen effects on skeletal myosin light chain kinase (skMLCK) activity. To this end, extensor digitorum longus muscles from female wildtype and skMLCK-absent (skMLCK−/−) mice were grouped as follows: ovariectomized with estrogen (E+), ovariectomized without estrogen (E–), sham surgery, and intact baseline. At 8 weeks of age, the ovariectomized groups were ovariectomized followed by implantation of either a 0.1 mg 17β-estradiol (E+) or placebo pellet (E–). Two weeks later, muscles were isolated and suspended in vitro (25° C) for determination of regulatory light chain phosphorylation and post-tetanic potentiation. Regulatory light chain phosphorylation was not different across conditions within either genotype although wildtype values were significantly greater than skMLCK−/− values. Consistent with this, the potentiation of concentric twitch force was similar between E+ and E– groups within each genotype but wildtype values were greater than skMLCK−/− values. However, unaltered estradiol levels following ovariectomy, likely due to previously underappreciated confounds of mouse age, development, and growth during estrogen supplementation, prevented direct testing of the hypothesis. Future studies should note the importance of estrous cycles and continuing physiological developments of young adult mice when working with ovarian hormones.

Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head–head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head–head/head–tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.