RESUMEN
The sarcomere is the smallest functional unit of myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction.
Asunto(s)
Técnicas Analíticas Microfluídicas , Modelos Biológicos , Contracción Muscular/fisiología , Sarcómeros/metabolismo , Animales , Ratones , Perfusión/métodos , Sarcómeros/químicaRESUMEN
We tested the hypotheses that 1) a decrease in activation of skeletal muscles at short sarcomere lengths (SLs) is caused by an inhibition of Ca(2+) release from the sarcoplasmic reticulum (SR), and 2) the decrease in Ca(2+) would be caused by an inhibition of action potential conduction from the periphery to the core of the fibers. Intact, single fibers dissected from the flexor digitorum brevis from mice were activated at different SLs, and intracellular Ca(2+) was imaged with confocal microscopy. Force decreased at SLs shorter than 2.1 µm, while Ca(2+) concentration decreased at SLs below 1.9 µm. The concentration of Ca(2+) at short SL was lower at the core than at the peripheries of the fiber. When the external concentration of Na(+) was decreased in the experimental media, impairing action potential conduction, Ca(2+) gradients were observed in all SLs. When caffeine was used in the experimental media, the gradients of Ca(2+) were abolished. We concluded that there is an inhibition of Ca(2+) release from the sarcoplasmic reticulum (SR) at short SLs, which results from a decreased conduction of action potential from the periphery to the core of the fibers.
Asunto(s)
Calcio/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Potenciales de Acción/fisiología , Animales , Señalización del Calcio/fisiología , Ratones , Sarcómeros/metabolismo , Sarcómeros/fisiología , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiologíaRESUMEN
Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.
Asunto(s)
Aminoaciltransferasas/metabolismo , Conectina/química , Conectina/fisiología , Miofibrillas/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Aminoaciltransferasas/genética , Animales , Módulo de Elasticidad/fisiología , Ratones , Ratones Noqueados , Proteínas Musculares/química , Proteínas Musculares/fisiología , Miofibrillas/química , Miofibrillas/ultraestructura , Estrés Mecánico , Relación Estructura-ActividadRESUMEN
Normal adult aging is associated with impaired muscle contractile function; however, to what extent cross-bridge kinetics are altered in aging muscle is not clear. We used a slacken restretch maneuver on single muscle fiber segments biopsied from the vastus lateralis of young adults (â¼23 yr), older nonathlete (NA) adults (â¼80 yr), and age-matched world class masters athletes (MA; â¼80 yr) to assess the rate of force redevelopment (ktr) and cross-bridge kinetics. A post hoc analysis was performed, and only the mechanical properties of "slow type" fibers based on unloaded shortening velocity (Vo) measurements are reported. The MA and NA were â¼54 and 43% weaker, respectively, for specific force compared with young. Similarly, when force was normalized to cross-sectional area determined via the fiber shape angularity data, both old groups did not differ, and the MA and NA were â¼43 and 48% weaker, respectively, compared with young (P < 0.05). Vo for both MA and NA old groups was 62 and 46% slower, respectively, compared with young. Both MA and NA adults had approximately two times slower values for ktr compared with young. The slower Vo in both old groups relative to young, coupled with a similarly reduced ktr, suggests impaired cross-bridge kinetics are responsible for impaired single fiber contractile properties with aging. These results challenge the widely accepted resilience of slow type fibers to cellular aging.
Asunto(s)
Envejecimiento , Atletas , Contracción Muscular , Fibras Musculares Esqueléticas , Fuerza Muscular , Músculo Cuádriceps/fisiopatología , Sarcopenia/fisiopatología , Factores de Edad , Anciano , Anciano de 80 o más Años , Biopsia , Técnica del Anticuerpo Fluorescente , Humanos , Cinética , Masculino , Fibras Musculares Esqueléticas/química , Cadenas Pesadas de Miosina/análisis , Músculo Cuádriceps/química , Sarcopenia/diagnóstico , Sarcopenia/metabolismo , Adulto JovenRESUMEN
Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical care myopathy, masticatory muscles do not lose mass. Thus, their properties may be harnessed to preserve mass. We compared masticatory and appendicular muscles responses to microgravity, using mice aboard the space shuttle Space Transportation System-135. Age- and sex-matched controls remained on the ground. After 13 days of space flight, 1 masseter (MA) and tibialis anterior (TA) were frozen rapidly for biochemical and functional measurements, and the contralateral MA was processed for morphologic measurements. Flight TA muscles exhibited 20 ± 3% decreased muscle mass, 2-fold decreased phosphorylated (P)-Akt, and 4- to 12-fold increased atrogene expression. In contrast, MAs had no significant change in mass but a 3-fold increase in P-focal adhesion kinase, 1.5-fold increase in P-Akt, and 50-90% lower atrogene expression compared with limb muscles, which were unaltered in microgravity. Myofibril force measurements revealed that microgravity caused a 3-fold decrease in specific force and maximal shortening velocity in TA muscles. It is surprising that myofibril-specific force from both control and flight MAs were similar to flight TA muscles, yet power was compromised by 40% following flight. Continued loading in microgravity prevents atrophy, but masticatory muscles have a different set point that mimics disuse atrophy in the appendicular muscle.
Asunto(s)
Músculos Masticadores/patología , Vuelo Espacial , Ingravidez/efectos adversos , Animales , Fenómenos Biomecánicos , Femenino , Expresión Génica , Masticación/fisiología , Músculos Masticadores/fisiopatología , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/fisiología , Proteínas Musculares/genética , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular/etiología , Atrofia Muscular/patología , Atrofia Muscular/fisiopatología , Miofibrillas/patología , Miofibrillas/fisiología , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Soporte de Peso/fisiologíaRESUMEN
BACKGROUND: When an activated muscle is rapidly stretched, force rises and peaks while muscle lengthens. The peak force is normally called critical-force (Pc). The mechanism behind this increase in force is not well understood, but it has been associated with crossbridges operating in different states. METHODS: Myofibrils were attached between a cantilever and a micro-needle, and activated with Ca(2+) or MgADP. During activation, the myofibrils were stretched by 3% SLo at 10 SLo·s(-1). A crossbridge model was developed to better understand the effects of MgADP in myofibrils activation. RESULTS: Despite a similar stretch magnitude, MgADP activation produced a higher Pc (1.37 ± 0.07 P/Po) than Ca(2+) activation (Pc = 1.23 ± 0.03 P/Po). These results suggest that myofibrils activated with MgADP become stiffer than myofibrils activated with Ca(2+). CONCLUSIONS: MgADP induces a fraction of crossbridges to form a "rigor-like" state that precedes ADP release, and that may not contribute to isometric forces. Such interpretation was strengthened by the results obtained with the developed crossbridge model, which showed that MgADP bias crossbridges into the rigor-like state. This state would be crucial to initiate a cooperative activation of crossbridges and actin, and to resist to unbinding from actin when the myofibrils are stretched. SIGNIFICANCE: Our results suggest a new mechanism contributing for force output during stretch, which underlies basic mechanisms of muscle contraction.
Asunto(s)
Adenosina Difosfato/metabolismo , Músculo Esquelético/fisiología , Miofibrillas/fisiología , Animales , Calcio/metabolismo , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , ConejosRESUMEN
OBJECTIVE: Skeletal muscle weakness is a prominent clinical feature in patients with rheumatoid arthritis (RA), but the underlying mechanism(s) is unknown. Here we investigate the mechanisms behind arthritis-induced skeletal muscle weakness with special focus on the role of nitrosative stress on intracellular Ca(2+) handling and specific force production. METHODS: Nitric oxide synthase (NOS) expression, degree of nitrosative stress and composition of the major intracellular Ca(2+) release channel (ryanodine receptor 1, RyR1) complex were measured in muscle. Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) and force production were assessed in single-muscle fibres and isolated myofibrils using atomic force cantilevers. RESULTS: The total neuronal NOS (nNOS) levels were increased in muscles both from collagen-induced arthritis (CIA) mice and patients with RA. The nNOS associated with RyR1 was increased and accompanied by increased [Ca(2+)]i during contractions of muscles from CIA mice. A marker of peroxynitrite-derived nitrosative stress (3-nitrotyrosine, 3-NT) was increased on the RyR1 complex and on actin of muscles from CIA mice. Despite increased [Ca(2+)]i, individual CIA muscle fibres were weaker than in healthy controls, that is, force per cross-sectional area was decreased. Furthermore, force and kinetics were impaired in CIA myofibrils, hence actin and myosin showed decreased ability to interact, which could be a result of increased 3-NT content on actin. CONCLUSIONS: Arthritis-induced muscle weakness is linked to nitrosative modifications of the RyR1 protein complex and actin, which are driven by increased nNOS associated with RyR1 and progressively increasing Ca(2+) activation.
Asunto(s)
Actinas/metabolismo , Artritis Experimental/complicaciones , Artritis Reumatoide/complicaciones , Calcio/metabolismo , Debilidad Muscular/etiología , Anciano , Animales , Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Femenino , Humanos , Ratones Endogámicos DBA , Persona de Mediana Edad , Debilidad Muscular/metabolismo , Debilidad Muscular/fisiopatología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Nitrosación , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Estrés Fisiológico/fisiologíaRESUMEN
When activated muscle fibers are stretched at low speeds [≤ 2 optimal length (L(o))/s], force increases in two phases, marked by a change in slope [critical force (P(c))] that happens at a critical sarcomere length extension (L(c)). Some studies attribute P(c) to the number of attached cross bridges before stretch, while others attribute it to cross bridges in a pre-power-stroke state. In this study, we reinvestigated the mechanisms of forces produced during stretch by altering either the number of cross bridges attached to actin or the cross-bridge state before stretch. Two sets of experiments were performed: 1) activated fibers were stretched by 3% L(o) at speeds of 1.0, 2.0, and 3.0 L(o)/s in different pCa(2+) (4.5, 5.0, 5.5, 6.0), or 2) activated fibers were stretched by 3% L(o) at 2 L(o)/s in pCa(2+) 4.5 containing either 5 µM blebbistatin(+/-) or its inactive isomer (+/+). All stretches started at a sarcomere length (SL) of 2.5 µm. When fibers were activated at a pCa(2+) of 4.5, P(c) was 2.47 ± 0.11 maximal force developed before stretch (P(o)) and decreased with lower concentrations of Ca(2+). L(c) was not Ca(2+) dependent; the pooled experiments provided a L(c) of 14.34 ± 0.34 nm/half-sarcomere (HS). P(c) and L(c) did not change with velocities of stretch. Fibers activated in blebbistatin(+/-) showed a higher P(c) (2.94 ± 0.17 P(o)) and L(c) (16.30 ± 0.38 nm/HS) than control fibers (P(c) 2.31 ± 0.08 P(o); L(c) 14.05 ± 0.63 nm/HS). The results suggest that forces produced during stretch are caused by both the number of cross bridges attached to actin and the cross bridges in a pre-power-stroke state. Such cross bridges are stretched by large amplitudes before detaching from actin and contribute significantly to the force developed during stretch.
Asunto(s)
Calcio/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Contracción Muscular , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Animales , Fenómenos Biomecánicos , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/citología , Conejos , Estrés MecánicoRESUMEN
Denervation and mitochondrial impairment are implicated in age-related skeletal muscle atrophy and may play a role in physical frailty. We recently showed that denervation modulates muscle mitochondrial function in octogenarian men, but this has not been examined in elderly women. On this basis, we tested the hypothesis that denervation plays a modulating role in mitochondrial impairment in skeletal muscle from prefrail or frail elderly (FE) women. Mitochondrial respiratory capacity and reactive oxygen species emission were examined in permeabilized myofibers obtained from vastus lateralis muscle biopsies from FE and young inactive women. Muscle respiratory capacity was reduced in proportion to a reduction in a mitochondrial marker protein in FE, and mitochondrial reactive oxygen species emission was elevated in FE versus young inactive group. Consistent with a significant accumulation of neural cell adhesion molecule-positive muscle fibers in FE (indicative of denervation), a 50% reduction in reactive oxygen species production after pharmacologically inhibiting the denervation-mediated reactive oxygen species response in FE women suggests a significant modulation of mitochondrial function by denervation. In conclusion, our data support the hypothesis that denervation plays a modulating role in skeletal muscle mitochondrial function in FE women, suggesting therapeutic strategies in advanced age should focus on the causes and treatment of denervation.
Asunto(s)
Desnervación , Anciano Frágil , Mitocondrias Musculares/metabolismo , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Anciano , Biopsia , Estudios de Casos y Controles , Femenino , Humanos , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Consumo de Oxígeno , Quebec , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Length changes of muscle fibers have previously been shown to result in a temporary reduction in fiber stiffness that is referred to as thixotropy. Understanding the mechanism of this thixotropy is important to our understanding of muscle function since there are many instances in which muscle is subjected to repeated patterns of lengthening and shortening. By applying sinusoidal length changes to one end of single permeabilized muscle fibers and measuring the force response at the opposite end, we studied the history-dependent stiffness of both relaxed and activated muscle fibers. For length change oscillations greater than 1 Hz, we observed thixotropic behavior of activated fibers. Treatment of these fibers with EDTA and blebbistatin, which inhibits myosin-actin interactions, quashed this effect, suggesting that the mechanism of muscle fiber thixotropy is cross-bridge dependent. We modeled a half-sarcomere experiencing sinusoidal length changes, and our simulations suggest that thixotropy could arise from force-dependent cross-bridge kinetics. Surprisingly, we also observed that, for length change oscillations less than 1 Hz, the muscle fiber exhibited rheopexy. In other words, the stiffness of the fiber increased in response to the length changes. Blebbistatin and EDTA did not disrupt the rheopectic behavior, suggesting that a non-cross-bridge mechanism contributes to this phenomenon.
Asunto(s)
Músculo Esquelético/fisiología , Animales , ConejosRESUMEN
The mechanisms behind the shortening-induced force depression commonly observed in skeletal muscles remain unclear, but have been associated with sarcomere length non-uniformity and/or crossbridge inhibition. The purpose of this study was twofold: (i) to evaluate if force depression is present in isolated single sarcomeres, a preparation that eliminates sarcomere length non-uniformities and (ii) to evaluate if force depression is inhibited when single sarcomeres are activated with MgADP, which biases crossbridges into a strongly-bound state. Single sarcomeres (n = 16) were isolated from rabbit psoas myofibrils using two micro-needles (one compliant, one rigid), piercing the sarcomere externally adjacent to the Z-lines. The sarcomeres were contracted isometrically and subsequently shortened, in both Ca(2+)- and MgADP-activating solutions. Shortening in Ca(2+)-activated samples resulted in a 27.44 ± 9.04% force depression when compared to isometric contractions produced at similar final sarcomere lengths (P < 0.001). There was no force depression in MgADP-activated sarcomeres (force depression = -1.79 ± 9.69%, P = 0.435). These results suggest that force depression is a sarcomeric property, and that is associated with an inhibition of myosin-actin interactions.
Asunto(s)
Adenosina Difosfato/metabolismo , Músculo Esquelético/fisiología , Sarcómeros/fisiología , Adenosina Difosfato/farmacología , Animales , Calcio/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/efectos de los fármacos , Conejos , Sarcómeros/efectos de los fármacosRESUMEN
The goal of this study was to compare the effects of Ca(2+) and MgADP activation on force development in skeletal muscles during and after imposed length changes. Single fibres dissected from the rabbit psoas were (i) activated in pCa(2+)4.5 and pCa(2+)6.0, or (ii) activated in pCa(2+)4.5 before and after administration of 10 mM MgADP. Fibres were activated in sarcomere lengths (SL) of 2.65 µm and 2.95 µm, and subsequently stretched or shortened (5%SL at 1.0 SL.s(-1)) to reach a final SL of 2.80 µm. The kinetics of force during stretch were not altered by pCa(2+) or MgADP, but the fast change in the slope of force development (P1) observed during shortening and the corresponding SL extension required to reach the change (L1) were higher in pCa(2+)6.0 (P1 = 0.22 ± 0.02 Po; L1 = 5.26 ± 0.24 nm.HS(.1)) than in pCa(2+)4.5 (P1 = 0.15 ± 0.01 Po; L1 = 4.48 ± 0.25 nm.HS(.1)). L1 was also increased by MgADP activation during shortening. Force enhancement after stretch was lower in pCa(2+)4.5 (14.9 ± 5.4%) than in pCa(2+)6.0 (38.8 ± 7.5%), while force depression after shortening was similar in both Ca(2+) concentrations. The stiffness accompanied the force behavior after length changes in all situations. MgADP did not affect the force behavior after length changes, and stiffness did not accompany the changes in force development after stretch. Altogether, these results suggest that the mechanisms of force generation during and after stretch are different from those obtained during and after shortening.
Asunto(s)
Adenosina Difosfato/farmacología , Calcio/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Animales , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Conejos , Sarcómeros/efectos de los fármacos , Sarcómeros/fisiologíaRESUMEN
The goal of this study was to evaluate if isolated sarcomeres and half-sarcomeres produce a long-lasting increase in force after a stretch is imposed during activation. Single and half-sarcomeres were isolated from myofibrils using micro-needles, which were also used for force measurements. After full force development, both preparations were stretched by different magnitudes. The sarcomere length (SL) or half-sarcomere length variations (HSL) were extracted by measuring the initial and final distances from the Z-line to the adjacent Z-line or to a region externally adjacent to the M-line of the sarcomere, respectively. Half-sarcomeres generated approximately the same amount of isometric force (29.0 ± SD 15.5â nN·µm(-2)) as single sarcomeres (32.1 ± SD 15.3â nN·µm(-2)) when activated. In both cases, the steady-state forces after stretch were higher than the forces during isometric contractions at similar conditions. The results suggest that stretch-induced force enhancement is partly caused by proteins within the half-sarcomere.
Asunto(s)
Contracción Isométrica/fisiología , Estimulación Física/métodos , Sarcómeros/fisiología , Sarcómeros/ultraestructura , Animales , Células Cultivadas , Módulo de Elasticidad/fisiología , Conejos , Estrés MecánicoRESUMEN
BACKGROUND: Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. OBJECTIVE: The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. METHODS: We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. RESULTS: In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89±9 nN/µm(2)) when compared to WT (132±11 nN/µm(2)), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K(act)), redevelopment (K(tr)), and relaxation (K(rel)). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209±31 nN/µm(2)) when compared to WT (123±20 nN/µm(2)). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K(act), but α-MHCAte1 myofibrils presented a faster K(rel) (6.11±0.41s(-1) vs 4.63±0.41 s(-1)). CONCLUSION: Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.
Asunto(s)
Aminoaciltransferasas/genética , Diafragma/fisiología , Eliminación de Gen , Contracción Muscular/genética , Miocardio , Miofibrillas/genética , Aminoaciltransferasas/deficiencia , Animales , Fenómenos Biomecánicos/genética , Modelos Animales de Enfermedad , Corazón/fisiología , Ratones , Ratones Noqueados , Contracción Miocárdica/genética , Miocardio/enzimologíaRESUMEN
When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P1 and P2) that happen at specific lengths (L1 and L2). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-power-stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca²âº concentrations (pCa²âº 4.5, 5.0, 5.5, 6.0) (nâ=â13), (ii) activated in the presence of blebbistatin (nâ=â16), and (iii) activated in the presence of blebbistatin at varying velocities (nâ=â5). In all experiments, a ramp shortening was imposed (amplitude 10%L0, velocity 1 L0â¢sarcomere length (SL)â¢s⻹), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 L0â¢s⻹). The values of P1, P2, L1, and L2 did not change with Ca²âº concentrations. Blebbistatin decreased P1, and it did not alter P2, L1, and L2. We developed a mathematical cross-bridge model comprising a load-dependent power-stroke transition and a pre-power-stroke cross-bridge state. The P1 and P2 critical points as well as the critical lengths L1 and L2 were explained qualitatively by the model, and the effects of blebbistatin inhibition on P1 were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-power-stroke state.