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The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n = 6) were 35.0 [+ or -] 2.9 nN/ [micro][m.sup.2] when stretched to an average sarcomere length of 3.4 [micro]m in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only ~25% of the passive force enhancement observed in intact myofibrils. Therefore, ~75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation. myofibrils; residual force enhancement; titin; stiffness; calcium

Cross-sectional area (CSA) is a fundamental variable in characterizing muscle mechanical properties. Typically, the CSA of a single muscle fibre is assessed by measuring either one or two diameters, and assuming the cross-section is either circular or elliptical in shape. However, fibre cross-sections have irregular shapes. The accuracy and precision of CSAs determined using circular and elliptical shape assumptions are unclear for mammalian skinned muscle fibres. Second harmonic generation imaging of skinned rabbit soleus fibres revealed that the circular assumption overstated real CSA by 5.3±25.9% whereas the elliptical assumption overstated real CSA by 2.8±6.9%. A preferred rotational alignment can bias the circular assumption, as real CSA was overstated by 22.1±24.8% when using the larger fibre diameter and understated by 11.4±13% when using the smaller fibre diameter. With 73% lower variable error and reduced bias, the elliptical assumption is superior to the circular assumption when assessing the CSA of skinned mammalian fibres., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Multiscale models aiming to connect muscle's molecular and cellular function have been difficult to develop, in part due to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single, skinned rabbit psoas muscle fibers to ramp shortenings and step stretches performed on the plateau region of the force-length relationship. We isolated myosin from the same muscles and, under similar conditions, performed single-molecule and ensemble measurements of myosin's ATP-dependent interaction with actin using laser trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of an inhibited state. The model also includes a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force after stretch (residual force enhancement). By optimizing the model fit to a subset of our fiber measurements, we specified seven unknown parameters. The model then successfully predicted the remainder of our fiber measurements and also our molecular measurements from the laser trap and in vitro motility. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale models. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combines with thick filament activation to decrease the number of cycling cross-bridges., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Aim: To determine the dose-response relationship of collagenase Clostridium histolyticum (CCH) on collagen content and the change in muscle fiber bundle stiffness after ex vivo treatment of adductor longus biopsies with CCH in children with cerebral palsy (CP)., Method: Biopsy samples of adductor longus from children with CP (classified in Gross Motor Function Classification System levels IV and V) were treated with 0 U/mL, 200 U/mL, 350 U/mL, or 500 U/mL CCH; percentage collagen reduction was measured to determine the dose-response. Peak and steady-state stresses were determined at 1%, 2.5%, 5%, and 7.5% strain increments; Young's modulus was calculated., Results: Eleven patients were enrolled (nine males, two females, mean age at surgery 6 years 5 months; range: 2-16 years). A linear CCH dose-response relationship was determined. Peak and steady-state stress generation increased linearly at 5.9/2.3mN/mm 2 , 12.4/5.3mN/mm 2 , 22.2/9.7mN/mm 2 , and 33.3/15.5mN/mm 2 at each percentage strain increment respectively. After CCH treatment, peak and steady-state stress generation decreased to 3.2/1.2mN/mm 2 , 6.5/2.9mN/mm 2 , 12.2/5.7mN/mm 2 , and 15.4/7.7mN/mm 2 respectively (p < 0.004). Young's modulus decreased from 205 kPa to 100 kPa after CCH (p = 0.003)., Interpretation: This preclinical ex vivo study provides proof of concept for the use of collagenase to decrease muscle stiffness in individuals with CP., (© 2023 Mac Keith Press.)

The cross-bridge theory predicts that muscle force is determined by muscle length and the velocity of active muscle length changes. However, before the formulation of the cross-bridge theory, it had been observed that the isometric force at a given muscle length is enhanced or depressed depending on active muscle length changes before that given length is reached. These enhanced and depressed force states are termed residual force enhancement (rFE) and residual force depression (rFD), respectively, and together they are known as the history-dependent features of muscle force production. In this review, we introduce early attempts in explaining rFE and rFD before we discuss more recent research from the past 25 years which has contributed to a better understanding of the mechanisms underpinning rFE and rFD. Specifically, we discuss the increasing number of findings on rFE and rFD which challenge the cross-bridge theory and propose that the elastic element titin plays a role in explaining muscle history-dependence. Accordingly, new three-filament models of force production including titin seem to provide better insight into the mechanism of muscle contraction. Complementary to the mechanisms behind muscle history-dependence, we also show various implications for muscle history-dependence on in-vivo human muscle function such as during stretch-shortening cycles. We conclude that titin function needs to be better understood if a new three-filament muscle model which includes titin, is to be established. From an applied perspective, it remains to be elucidated how muscle history-dependence affects locomotion and motor control, and whether history-dependent features can be changed by training., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Residual force enhancement (RFE), an increase in isometric force after active stretching of a muscle compared with the purely isometric force at the corresponding length, has been consistently observed throughout the structural hierarchy of skeletal muscle. Similar to RFE, passive force enhancement (PFE) is also observable in skeletal muscle and is defined as an increase in passive force when a muscle is deactivated after it has been actively stretched compared with the passive force following deactivation of a purely isometric contraction. These history-dependent properties have been investigated abundantly in skeletal muscle, but their presence in cardiac muscle remains unresolved and controversial. The purpose of this study was to investigate whether RFE and PFE exist in cardiac myofibrils and whether the magnitudes of RFE and PFE increase with increasing stretch magnitudes. Cardiac myofibrils were prepared from the left ventricles of New Zealand White rabbits, and the history-dependent properties were tested at three different final average sarcomere lengths (n = 8 for each), 1.8, 2, and 2.2 μm, while the stretch magnitude was kept at 0.2 μm/sarcomere. The same experiment was repeated with a final average sarcomere length of 2.2 μm and a stretching magnitude of 0.4 μm/sarcomere (n = 8). All 32 cardiac myofibrils exhibited increased forces after active stretching compared with the corresponding purely isometric reference conditions (p < 0.05). Furthermore, the magnitude of RFE was greater when myofibrils were stretched by 0.4 compared with 0.2 μm/sarcomere (p < 0.05). We conclude that, like in skeletal muscle, RFE and PFE are properties of cardiac myofibrils and are dependent on stretch magnitude., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The steady-state isometric force produced by skeletal muscle after active shortening and stretching is depressed and enhanced, respectively, compared with purely isometric force produced at corresponding final lengths and at the same level of activation. One hypothesis proposed to account for these force depression (FD) and force enhancement (FE) properties is a change in cross-bridge cycling kinetics. The rate of cross-bridge attachment (f) and/or cross-bridge detachment (g) may be altered following active shortening and active stretching, leading to FD and FE, respectively. Experiments elucidating cross-bridge kinetics in actively shortened and stretched muscle preparations and their corresponding purely isometric contractions have yet to be performed. The aim of this study was to investigate cross-bridge cycling kinetics of muscle fibres at steady-state following active shortening and stretching. This was done by determining muscle fibre stiffness and rate of active force redevelopment following a quick release-re-stretch protocol (kTR). Applying these measures to equations previously used in the literature for a two-state cross-bridge cycling model (attached/detached cross-bridges) allowed us to determine apparent f and g, the proportion of attached cross-bridges, and the force produced per cross-bridge. kTR, apparent f and g, the proportion of attached cross-bridges and the force produced per cross-bridge were significantly decreased following active shortening compared with corresponding purely isometric contractions, indicating a change in cross-bridge cycling kinetics. Additionally, we showed no change in cross-bridge cycling kinetics following active stretch compared with corresponding purely isometric contractions. These findings suggest that FD is associated with changes in cross-bridge kinetics, whereas FE is not., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Study Design: Basic science, experimental animal study., Objective: To determine the effects of Botulinum toxin type A (BTX-A) injections on the mechanical properties of skinned muscle fibers (cells) of rabbit paraspinal muscles., Summary of Background Data: BTX-A has been widely used in the treatment of disorders of muscle hyperactivity, such as spasticity, dystonia, and back pain. However, BTX-A injection has been shown to cause muscle atrophy, fat infiltration, and decreased force output in target muscles, but its potential effects on the contractile machinery and force production on the cellular level remain unknown., Methods: Nineteen-month-old, male New Zealand White Rabbits received either saline or BTX-A injections into the paraspinal muscles, equally distributed along the left and right sides of the spine at T12, L1, and L2 at 0, 8, 12, 16, 20, and 24 weeks. Magnetic resonance imaging was used to quantify muscle crosssectional area and structural changes before and at 28 weeks following the initial injection. Skinned fibers isolated from the paraspinal muscles were tested for their active and passive force-length relationships, unloaded shortening velocity, and myosin heavy chain isoforms., Results: BTX-A injections led to significant fat infiltration within the injected muscles and a greater proportion of IIa to IIx fibers. Isolated fast fibers from BTX-A injected animals had lower active force and unloaded shortening velocity compared with fibers from saline-injected control animals. Force and velocity properties were not different between groups for the slow fibers., Conclusion: Injection of BTX-A into the paraspinal rabbit muscles leads to significant alterations in the contractile properties of fast, but not slow, fibers.Level of Evidence: N/A., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The steady-state isometric force of a muscle after active stretching is greater than the steady-state force for a purely isometric contraction at the same length and activation level. The mechanisms underlying this property, termed residual force enhancement (rFE), remain unknown. When myofibrils are actively stretched while cross-bridge cycling is inhibited, rFE is substantially reduced, suggesting that cross-bridge cycling is essential to produce rFE. Our purpose was to further investigate the role of cross-bridge cycling in rFE by investigating whether fast stretching that causes cross-bridge slipping is associated with a loss of rFE. Skinned fibre bundles from rabbit psoas muscles were stretched slowly (0.08 µm s-1) or rapidly (800 µm s-1) while activated, from an average sarcomere length of 2.4 to 3.2 µm. Force was enhanced by 38±4% (mean±s.e.m) after the slow stretches but was not enhanced after the fast stretches, suggesting that proper cross-bridge cycling is required to produce rFE., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Purpose: Aerobic exercise training results in distinct structural and mechanical myocardial adaptations. In skeletal muscle, whey protein supplementation is effective in enhancing muscle adaptation following resistance exercise. However, it is unclear whether cardiac adaptation to aerobic exercise can be enhanced by systematic protein supplementation., Methods: Twelve-week-old rats were assigned to 12 weeks of either sedentary or aerobic exercise with either a standard (Sed+Standard, Ex+Standard) or high-protein (Sed+Pro, Ex+Pro) diet. Echocardiography was used to measure cardiac structural remodeling and performance. Skinned cardiac fiber bundles were used to determine the active and passive stress properties, maximum shortening velocity, and calcium sensitivity., Results: Aerobic training was characterized structurally by increases in ventricle volume (Ex+Standard, 19%; Ex+Pro, 29%) and myocardial thickness (Ex+Standard, 26%; Ex+- Pro, 12%) compared to that of baseline. Skinned trabecula r fiber bundles also had a greater unloaded shortening velocity (Sed+Standard, 1.04±0.05; Sed+Pro, 1.07±0.03; Ex- +Standard, 1.16±0.04; Ex+Pro, 1.18±0.05 FL/s) and calcium sensitivity (pCa50: Sed+Standard, 6.04±0.17; Sed+Pro, 6.08±0.19; Ex+Standard, 6.30±0.09; Ex+Pro, 6.36±0.12) in trained hearts compared to that of hearts from sedentary animals. However, the addition of a high-protein diet did not provide additional benefits to either the structural or mechanical adaptations of the myocardium., Conclusion: Therefore, it seems that a high-whey-protein diet does not significantly enhance adaptations of the heart to aerobic exercise in comparison to that of a standard diet.

The purpose of this study was to investigate the alterations with obesity, and the effects of moderate aerobic exercise or prebiotic dietary-fibre supplementation on the mechanical and biochemical properties of the tail tendon in a rat model of high-fat/high-sucrose (HFS) diet-induced obesity. Thirty-two male Sprague-Dawley rats were randomized to chow (n = 8) or HFS (n = 24) diets. After 12-weeks, the HFS fed rats were further randomized into sedentary (HFS sedentary, n = 8), exercise (HFS + E, n = 8) or prebiotic fibre supplementation (HFS + F, n = 8) groups. After another 12-weeks, rats were sacrificed, and one tail tendon was isolated and tested. Stress-relaxation and stretch-to-failure tests were performed to determine mechanical properties (peak, steady-state, yield and failure stresses, Young's modulus, and yield and failure strains) of the tendons. The hydroxyproline content was also analyzed. The HFS sedentary and HFS + F groups had higher final body masses and fat percentages compared to the chow and HFS + E groups. Yield strain was reduced in the HFS sedentary rats compared to the chow rats. Peak and steady-state stresses, failure strain, Young's modulus, and hydroxyproline content were not different across groups. Although the HFS + E group showed higher failure stress, yield stress, and yield strain compared to the HFS sedentary group, HFS + F animals did not produce differences in the properties of the tail tendon compared to the HFS sedentary group. These results indicate that exposure to a HFS diet led to a reduction in the yield strain of the tail tendon and aerobic exercise, but not fibre supplementation, attenuated these diet-related alterations to tendon integrity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Our purpose was to use small-angle X-ray diffraction to investigate the structural changes within sarcomeres at steady-state isometric contraction following active lengthening and shortening, compared to purely isometric contractions performed at the same final lengths. We examined force, stiffness, and the 1,0 and 1,1 equatorial and M3 and M6 meridional reflections in skinned rabbit psoas bundles, at steady-state isometric contraction following active lengthening to a sarcomere length of 3.0 µm (15.4% initial bundle length at 7.7% bundle length/s), and active shortening to a sarcomere length of 2.6 µm (15.4% bundle length at 7.7% bundle length/s), and during purely isometric reference contractions at the corresponding sarcomere lengths. Compared to the reference contraction, the isometric contraction after active lengthening was associated with an increase in force (i.e., residual force enhancement) and M3 spacing, no change in stiffness and the intensity ratio I 1,1 /I 1,0 , and decreased lattice spacing and M3 intensity. Compared to the reference contraction, the isometric contraction after active shortening resulted in decreased force, stiffness, I 1,1 /I 1,0 , M3 and M6 spacings, and M3 intensity. This suggests that residual force enhancement is achieved without an increase in the proportion of attached cross-bridges, and that force depression is accompanied by a decrease in the proportion of attached cross-bridges. Furthermore, the steady-state isometric contraction following active lengthening and shortening is accompanied by an increase in cross-bridge dispersion and/or a change in the cross-bridge conformation compared to the reference contractions.

Introduction: Aerobic and resistance exercise training results in distinct structural changes of the heart. The mechanics of how cardiac cells adapt to resistance training and the benefits to cells when combining aerobic and resistance exercise remains largely unknown. The purpose of this study was to compare mechanical adaptations of skinned cardiac fiber bundles after chronic resistance, aerobic and combined exercise training in rats. We hypothesized that differences in mechanical function on the fiber bundle level coincide with differences previously reported in the structure of the heart., Method: Twelve-week-old rats were assigned to (i) an aerobic running group (n = 6), (ii) a ladder climbing resistance group (n = 6), (iii) a combination group subjected to aerobic and resistance training (n = 6), or (iv) a sedentary (control) group (n = 5). Echocardiography was used to measure cardiac structural remodeling. Skinned cardiac fiber bundles were used to determine active and passive force properties, maximal shortening velocity, and calcium sensitivity., Results: Aerobically trained animals had 43%-49% greater ventricular volume and myocardial thickness, and a 4%-17% greater shortening velocity and calcium sensitivity compared with control group rats. Resistance-trained rats had 37%-71% thicker ventricular walls, a 56% greater isometric force production, a 9% greater shortening velocity, and a 4% greater calcium sensitivity compared with control group rats. The combination exercise-trained rats had 25%-43% greater ventricular volume and myocardial wall thickness, a 55% greater active force production, a 7% greater shortening velocity, and a 60% greater cross-bridge cooperativity compared with control group rats., Conclusions: The heart adapts differently to each exercise modality, and a combination of aerobic and resistance training may have the greatest benefit for cardiac health and performance., (Copyright © 2021 by the American College of Sports Medicine.)

Purpose: The purpose of this study was to investigate the effects of a high-fat high-sucrose (HFHS) diet on previously reported adaptations of cardiac morphological and contractile properties to resistance training., Methods: Twelve-week-old rats participated in 12-weeks of resistance exercise training and consumed an HFHS diet. Echocardiography and skinned cardiac muscle fiber bundle testing were performed to determine the structural and mechanical adaptations., Results: Compared to chow-fed sedentary animals, both HFHS- and chow-fed resistance-trained animals had thicker left ventricular walls. Isolated trabecular fiber bundles from chow-fed resistance-trained animals had greater force output, shortening velocities, and calcium sensitivities than those of chow-fed sedentary controls. However, trabeculae from the HFHS resistance-trained animals had greater force output but no change in unloaded shortening velocity or calcium sensitivity than those of the chow-fed sedentary group animals., Conclusion: Resistance exercise training led to positive structural and mechanical adaptations of the heart, which were partly offset by the HFHS diet.

Muscle force is enhanced during shortening when shortening is preceded by an active stretch. This phenomenon is known as the stretch-shortening cycle (SSC) effect. For some stretch-shortening conditions this increase in force during shortening is maintained following SSCs when compared to the force following a pure shortening contraction. It has been suggested that the residual force enhancement property of muscles, which comes into play during the stretch phase of SSCs may contribute to the force increase after SSCs. Knowing that residual force enhancement is associated with a substantial reduction in metabolic energy per unit of force, it seems reasonable to assume that the metabolic energy cost per unit of force is also reduced following a SSC. The purpose of this study was to determine the energy cost per unit of force at steady-state following SSCs and compare it to the corresponding energy cost following pure shortening contractions of identical speed and magnitude. We hypothesized that the energy cost per unit of muscle force is reduced following SSCs compared to the pure shortening contractions. For the SSC tests, rabbit psoas fibers ( n = 12) were set at an average sarcomere length (SL) of 2.4 μm, activated, actively stretched to a SL of 3.2 μm, and shortened to a SL of 2.6 or 3.0 μm. For the pure shortening contractions, the same fibers were activated at a SL of 3.2 μm and actively shortened to a SL of 2.6 or 3.0 μm. The amount of ATP consumed was measured over a 40 s steady-state total isometric force following either the SSCs or the pure active shortening contractions. Fiber stiffness was determined in an additional set of 12 fibers, at steady-state for both experimental conditions. Total force, ATP consumption, and stiffness were greater following SSCs compared to the pure shortening contractions, but ATP consumption per unit of force was the same between conditions. These results suggest that the increase in total force observed following SSCs was achieved with an increase in the proportion of attached cross-bridges and titin stiffness. We conclude that muscle efficiency is not enhanced at steady-state following SSCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Joumaa, Fukutani and Herzog.)

Decrements in contractile function resulting from obesity are thought to be major reasons for the link between obesity and cardiovascular disease, while exercise has been shown to improve cardiac muscle contractile function. The purpose of this study was to evaluate cardiac contractile properties following obesity induction and the potential protective effect of exercise. Twelve-week-old rats ( n = 30) were organized into either a chow diet or a high-fat, high-sucrose (HFHS) diet group. Following 12 weeks of obesity induction the HFHS group animals were stratified and grouped into sedentary (HFHS+Sed) and exercise (HFHS+Ex) groups for an additional 12 weeks. Following 24 weeks of diet intervention, with 12 weeks of aerobic exercise (25 m/min, 30 min/day, 5 days/week) for the HFHS+Ex group, skinned cardiac fibre bundle testing was used to evaluate cardiac contractile properties. Body fat and mass were significantly greater in the HFHS-fed animals compared with the chow controls ( p  < 0.043). Hearts from rats in the HFHS+Sed group had significantly greater mass ( p  < 0.03), significantly slower maximum shortening velocity ( p  = 0.001), and tended to have lower calcium sensitivity ( p  = 0.077) and a lower proportion of α-myosin heavy chain composition ( p  = 0.074) than the sedentary chow animals. However, 12 weeks of moderate aerobic exercise partially prevented these decrements in contractile properties. Novelty Cardiac muscle from animals exposed to an obesogenic diet for 24 weeks had impaired contractile properties compared with controls. Obesity-induced impairment of contractile properties of the heart were partially prevented by a 12-week aerobic exercise regime.

Advanced age has been shown to result in decreased compliance, shortening velocity, and calcium sensitivity of the heart muscle. Even though cardiac health has been studied extensively in elderly populations, relatively little is known about cardiac health and age for the first part of adulthood. The purpose of this study was to compare cardiac contractile properties across the first year of life in rats (between 17-53 weeks), corresponding to early to mid-adulthood. Hearts were harvested from rats aged 17-, 24-, 36-, and 53-weeks. Skinned cardiac trabecular fibre bundle testing was used to evaluate active and passive force properties, maximum shortening velocity, calcium sensitivity, and myosin heavy chain isoforms. Maximum active stress production was not different between age groups. Calcium sensitivity increased progressively, while shortening velocity remained unchanged after an increase from 17-and 24-weeks. Passive stiffness decreased between 17- and 24-weeks, but then increased progressively through to 53-weeks. Thus, many of the observed detrimental changes in systolic function (reduced shortening velocity and calcium sensitivity) associated with aging, do not seem to occur in early to mid-adulthood, while early signs of increased diastolic stiffness manifest within 53 weeks of age and may represent a first sign of decreasing heart function and health., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In skeletal muscle, steady-state force is consistently greater following active stretch than during a purely isometric contraction at the same length (residual force enhancement; RFE). Similarly, when deactivated, the force remains higher following active stretch than following an isometric condition (passive force enhancement; PFE). RFE and PFE have been associated with the sarcomere protein titin, but skeletal and cardiac titin have different structures, and results regarding RFE in cardiac muscle have been inconsistent and contradictory. Therefore, the purpose of this study was to determine if cardiac muscle exhibits RFE and PFE. Skinned fibre bundles (n = 10) were activated isometrically at a sarcomere length of 2.2 μm and actively stretched by 15% of their length. The resultant active and passive forces were compared to the corresponding forces obtained for purely isometric contractions at the long length. RFE was observed in all fibre bundles, averaging 5.5 ± 2.5% (ranging from 2.3 to 9.4%). PFE was observed in nine of the ten bundles, averaging 11.1 ± 6.5% (ranging from -2.1 to 18.7%). Stiffness was not different between the active isometric and the force enhanced conditions, but was higher following deactivation from the force-enhanced compared to the isometric reference state. We conclude that there is RFE and PFE in cardiac muscle. We speculate that cardiac muscle has the same RFE capability as skeletal muscle, and that the most likely mechanism for the RFE and PFE is the engagement of a passive structural element during active stretching., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Childhood obesity is a major risk factor for heart disease during adulthood, independent of adulthood behaviours. Therefore, it seems that childhood obesity leads to partly irreversible decrements in cardiac function. Little is known about how obesity during maturation affects the mechanical properties of the heart. The purpose of this study was to evaluate contractile properties in developing hearts from animals with dietary-induced obesity (high-fat high-sucrose diet). We hypothesized that obesity induced during adolescence results in decrements in cardiac contractile function. Three-week-old rats ( n = 16) were randomized into control (chow) or dietary-induced obesity (high-fat high-sucrose diet) groups. Following 14 weeks on the diet, skinned cardiac trabeculae fibre bundle testing was performed to evaluate active and passive force, maximum shortening velocity, and calcium sensitivity. Rats in the high-fat high-sucrose diet group had significantly larger body mass and total body fat percentage. There were no differences in maximal active or passive properties of hearts between groups. Hearts from the high-fat high-sucrose diet rats had significantly slower maximum shortening velocities and lower calcium sensitivity than controls. Decreased shortening velocity and calcium sensitivity in hearts of obese animals may constitute increased risk of cardiac disease in adulthood. Novelty Cardiac muscle from animals exposed to an obesogenic diet during development had lower shortening velocity and calcium sensitivity than those from animals fed a chow diet. These alterations in mechanical function may be a mechanism for the increased risk of cardiac disease observed in adulthood.

Background: Cerebral palsy (CP) is the most common cause of childhood disability, typified by a static encephalopathy with peripheral musculoskeletal manifestations-most commonly related to spasticity-that are progressive with age. Hip displacement is one of the most common manifestations, observed to lead to painful degenerative arthritis over time. Despite the key role that spasticity-related adductor muscle contractures are thought to play in the development of hip displacement in CP, basic science research in this area to date has been limited. This study was initiated to correlate hip adductor muscle changes intrinsic to the sarcomere-specifically, titin isoforms and sarcomere length-to the severity of hip displacement in children with spastic cerebral palsy., Methods: Single gracilis muscle biopsies were obtained from children with CP (Gross Motor Function Classification System (GMFCS) III-V; n = 10) who underwent adductor muscle release surgery for the treatment of hip displacement. Gel electrophoresis was used to estimate titin molecular weight. Sarcomere lengths were measured from muscle fascicles using laser diffraction. The severity of hip displacement was determined by measuring by Reimers migration percentage (MP) from anteroposterior pelvic x-rays. Correlation analyses between titin, sarcomere lengths, and MP were performed., Results: The mean molecular weight of titin was 3588 kDa. The mean sarcomere length was 3.51 μm. Increased MP was found to be associated with heavier isoforms of titin (R 2 = 0.65, p < 0.05) and with increased sarcomere lengths (R 2 = 0.65, p < 0.05). Heavier isoforms of titin were also associated with increased sarcomere lengths (R 2 = 0.80, p < 0.05)., Conclusions: Our results suggest that both larger titin isoforms and sarcomere lengths are positively correlated with increased severity of hip displacement and may represent adaptations in response to concomitant increases in spasticity and muscle shortening., Trial Registration: As this study does not report the results of a health care intervention on human participants, it has not been registered.

During isometric contractions, the optimal length of skeletal muscles increases with decreasing activation. The underlying mechanism for this phenomenon is thought to be linked to length dependence of Ca 2+ sensitivity. Muscular dystrophy with myositis ( mdm ), a recessive titin mutation in mice, was used as a tool to study the role of titin in activation dependence of optimal length and length dependence of Ca 2+ sensitivity. We measured the shift in optimal length between tetanic and twitch stimulation in mdm and wild-type muscles, and the length dependence of Ca 2+ sensitivity at short and long sarcomere lengths in mdm and wild-type fiber bundles. The results indicate that the mdm mutation leads to a loss of activation dependence of optimal length without the expected change in length dependence of Ca 2+ sensitivity, demonstrating that these properties are not linked, as previously suggested. Furthermore, mdm muscles produced maximum tetanic stress during sub-optimal filament overlap at lengths similar to twitch contractions in both genotypes, but the difference explains less than half of the observed reduction in active force of mdm muscles. Mdm muscles also exhibited increased electromechanical delay, contraction and relaxation times, and decreased rate of force development in twitch contractions. We conclude that the small deletion in titin associated with mdm in skeletal muscles alters force production, suggesting an important regulatory role for titin in active force production. The molecular mechanisms for titin's role in regulating muscle force production remain to be elucidated., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The worldwide trajectory of increasing obesity rates is a major health problem precipitating a rise in the prevalence of a variety of co-morbidities and chronic diseases. Tendinopathy, in weight and non-weight bearing tendons, in individuals with overweight or obesity has been linked to metabolic dysfunction resulting from obesity. Exercise and dietary fibre supplementation (DFS) are common countermeasures to combat obesity and therefore it seems reasonable to assume that they might protect tendons from structural and mechanical damage in a diet-induced obesity (DIO) model. The purpose of this study was to determine the effects of a DIO, DIO combined with moderate exercise, DIO combined with DFS (prebiotic oligofructose), and DIO combined with moderate exercise and DFS on the mechanical and biochemical properties of the rat tail tendon. Twenty-four male Sprague-Dawley rats, fed a high-fat/high-sucrose diet were randomized into a sedentary, a moderate exercise, a DFS, or a moderate exercise combined with DFS group for 12 weeks. Additionally, six lean age-matched animals were included as a sedentary control group. DIO in combination with exercise alone and with exercise and DFS reduced the Young's Modulus but not the collagen content of the rat tail tendons compared to lean control animals. However, no differences in the mechanical and biochemical properties of the rat tail tendon were detected between the DIO and the lean control group, suggesting that DIO by itself did not impact the tail tendon. It seems that longer DIO exposure periods may be needed to develop overt differences in our DIO model., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cerebral palsy (CP) is the result of a static brain lesion which causes spasticity and muscle contracture. The source of the increased passive stiffness in patients is not understood and while whole muscle down to single muscle fibres have been investigated, the smallest functional unit of muscle (the sarcomere) has not been. Muscle biopsies (adductor longus and gracilis) from pediatric patients were obtained (CP n = 9 and control n = 2) and analyzed for mechanical stiffness, in-vivo sarcomere length and titin isoforms. Adductor longus muscle was the focus of this study and the results for sarcomere length showed a significant increase in length for CP (3.6 µm) compared to controls (2.6 µm). Passive stress at the same sarcomere length for CP compared to control was significantly lower in CP and the elastic modulus for the physiological range of muscle was lower in CP compared to control (98.2 kPa and 166.1 kPa, respectively). Our results show that CP muscle at its most reduced level (the myofibril) is more compliant compared to normal, which is completely opposite to what is observed at higher structural levels (single fibres, muscle fibre bundles and whole muscle). It is noteworthy that at the in vivo sarcomere length in CP, the passive forces are greater than normal, purely as a functional of these more compliant sarcomeres operating at long lengths. Titin isoforms were not different between CP and non-CP adductor longus but titin:nebulin was reduced in CP muscle, which may be due to titin loss or an over-expression of nebulin in CP muscles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the role of titin in preventing the development of sarcomere length nonuniformities following activation and after active and passive stretch by determining the effect of partial titin degradation on sarcomere length nonuniformities and force in passive and active myofibrils. Selective partial titin degradation was performed using a low dose of trypsin. Myofibrils were set at a sarcomere length of 2.4 µm and then passively stretched to sarcomere lengths of 3.4 and 4.4 µm. In the active condition, myofibrils were set at a sarcomere length of 2.8 µm, activated, and actively stretched by 1 µm/sarcomere. The extent of sarcomere length nonuniformities was calculated for each sarcomere as the absolute difference between sarcomere length and the mean sarcomere length of the myofibril. Our main finding is that partial titin degradation does not increase sarcomere length nonuniformities after passive stretch and activation compared with when titin is intact but increases the extent of sarcomere length nonuniformities after active stretch. Furthermore, when titin was partially degraded, active and passive stresses were substantially reduced. These results suggest that titin plays a crucial role in actively stretched myofibrils and is likely involved in active and passive force production.

The positive effects of chronic endurance exercise training on health and performance have been well documented. These positive effects have been evaluated primarily at the structural level, and work has begun to evaluate mechanical adaptations of the myocardium. However, it remains poorly understood how the volume of exercise training affects cardiac adaptation. To gain some understanding, we subjected 3-mo-old Sprague-Dawley rats ( n = 23) to treadmill running for 11 wk at one of three exercise volumes (moderate, high, and extra high). Following training, hearts were excised and mechanical testing was completed on skinned trabecular fiber bundles. Performance on a maximal fitness test was dose dependent on training volume, where greater levels of training led to greater performance. No differences were observed between animals from any group for maximal active stress and passive stress at a sarcomere length of 2.2 µm. Heart mass and passive stress at sarcomere lengths beyond 2.4 µm increased in a dose-dependent manner for animals in the control and moderate- and high-duration groups. However, hearts from animals in the extra high-duration group presented with inhibited responses for heart mass and passive stress, despite performing greatest on a graded treadmill fitness test. These results suggest that heart mass and passive stress adapt in a dose-dependent manner, until exercise becomes excessive and adaptation is inhibited. Our findings are in agreement with the beneficial role exercise has in cardiac adaptation. However, excessive exercise comes with risks of maladaptation, which must be weighed against the desire to increase performance. NEW & NOTEWORTHY For the first time, we present findings on cardiac trabecular muscle passive stiffness and show the effect of excessive exercise on the heart. We demonstrated that heart mass increases with exercise until a maximum, after which greater exercise volume results in inhibited adaptation. At paraphysiological lengths, passive stiffness increases with exercise but to a lesser degree with excessive training. Despite greater performance on graded exercise tests, animals in the highest trained group exhibited possible maladaptation.

Increased muscle force during stretch-shortening cycles (SSCs) has been widely examined. However, the mechanisms causing increased muscle force in SSCs remain unknown. The purpose of this study was to determine the influence of residual force enhancement and elongation of attached cross-bridges on the work enhancement in SSCs. For the Control condition, skinned rabbit soleus fibers were elongated passively from an average sarcomere length of 2.4 to 3.0  μ m, activated and then actively shortened to 2.4  μ m. For the Transition condition, fibers were elongated actively from an average sarcomere length of 2.4 to 3.0  μ m. Two seconds after the end of active lengthening, fibers were actively shortened to 2.4  μ m. In the SSC condition, fibers were lengthened actively from an average sarcomere length of 2.4 to 3.0  μ m, and then immediately shortened actively to 2.4  μ m. Increased muscle force in the SSCs was quantified by the increase in mechanical work during active shortening compared to the mechanical work measured during the purely active shortening contractions. Work enhancement was significantly greater in the SSC compared to the Transition conditions. This difference was associated with the pause given between the active lengthening and shortening phase in the Transition test, which likely resulted in a reduction of the average elongation of the attached cross-bridges caused by active stretching. Since some work enhancement was still observed in the Transition condition, another factor, for example the stretch-induced residual force enhancement, must also have contributed to the work enhancement in SSCs., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

In actively stretched skeletal muscle sarcomeres, titin-based force is enhanced, increasing the stiffness of active sarcomeres. Titin force enhancement in sarcomeres is vastly reduced in mdm , a genetic mutation with a deletion in titin. Whether loss of titin force enhancement is associated with compensatory mechanisms at higher structural levels of organization, such as single fibres or entire muscles, is unclear. The aim of this study was to determine whether mechanical deficiencies in titin force enhancement are also observed at the fibre level, and whether mechanisms compensate for the loss of titin force enhancement. Single skinned fibres from control and mutant mice were stretched actively and passively beyond filament overlap to observe titin-based force. Mutant fibres generated lower contractile stress (force divided by cross-sectional area) than control fibres. Titin force enhancement was observed in control fibres stretched beyond filament overlap, but was overshadowed in mutant fibres by an abundance of collagen and high variability in mechanics. However, titin force enhancement could be measured in all control fibres and most mutant fibres following short stretches, accounting for ∼25% of the total stress following active stretch. Our results show that the partial loss of titin force enhancement in myofibrils is not preserved in all mutant fibres and this mutation likely affects fibres differentially within a muscle. An increase in collagen helps to reestablish total force at long sarcomere lengths with the loss in titin force enhancement in some mutant fibres, increasing the overall strength of mutant fibres., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In the cross-bridge theory, contractile force is produced by cross-bridges that form between actin and myosin filaments. However, when a contracting muscle is stretched, its active force vastly exceeds the force that can be attributed to cross-bridges. This unexplained, enhanced force has been thought to originate in the giant protein titin, which becomes stiffer in actively compared with passively stretched sarcomeres by an unknown mechanism. We investigated this mechanism using a genetic mutation (mdm) with a small but crucial deletion in the titin protein. Myofibrils from normal and mdm mice were stretched from sarcomere lengths of 2.5 to 6.0 μm. Actively stretched myofibrils from normal mice were stiffer and generated more force than passively stretched myofibrils at all sarcomere lengths. No increase in stiffness and just a small increase in force were observed in actively compared with passively stretched mdm myofibrils. These results are in agreement with the idea that titin force enhancement stiffens and stabilizes the sarcomere during contraction and that this mechanism is lost with the mdm mutation., (© 2016. Published by The Company of Biologists Ltd.)

In humans, enhanced joint range of motion is observed after static stretch training and results either from an increased stretch tolerance or from a change in the biomechanical properties of the muscle-tendon unit. We investigated the effects of an intermittent stretch training on muscle biomechanical and structural variables. The left plantarflexors muscles of seven anesthetized New Zealand (NZ) White rabbits were passively and statically stretched three times a week for 4 wk, while the corresponding right muscles were used as nonstretched contralateral controls. Before and after the stretching protocol, passive torque produced by the left plantarflexor muscles as a function of the ankle angle was measured. The left and right plantarflexor muscles were harvested from dead rabbits and used to quantify possible changes in muscle structure. Significant mass and serial sarcomere number increases were observed in the stretched soleus but not in the plantaris or medial gastrocnemius. This difference in adaptation between the plantarflexors is thought to be the result of their different fiber type composition and pennation angles. Neither titin isoform nor collagen amount was modified in the stretched compared with the control soleus muscle. Passive torque developed during ankle dorsiflexion was not modified after the stretch training on average, but was decreased in five of the seven experimental rabbits. Thus, an intermittent stretching program similar to those used in humans can produce a change in the muscle structure of NZ White rabbits, which was associated in some rabbits with a change in the biomechanical properties of the muscle-tendon unit., (Copyright © 2015 the American Physiological Society.)

Isometric force after active stretch of muscles is higher than the purely isometric force at the corresponding length. This property is termed residual force enhancement. Active force in skeletal muscle depends on calcium attachment characteristics to the regulatory proteins. Passive force has been shown to influence calcium attachment characteristics, specifically the sarcomere length dependence of calcium sensitivity. Since one of the mechanisms proposed to explain residual force enhancement is the increase in passive force that results from engagement of titin upon activation and stretch, our aim was to test if calcium sensitivity of residual force enhancement was different from that of its corresponding purely isometric contraction and if such a difference was related to the molecular spring titin. Force-pCa curves were established in rabbit psoas skinned fibers for reference and residual force-enhanced states at a sarcomere length of 3.0 μm 1) in a titin-intact condition, 2) after treatment with trypsin to partially eliminate titin, and 3) after treatment with trypsin and osmotic compression with dextran T-500 to decrease the lattice spacing in the absence of titin. The force-pCa curves of residual force enhancement were shifted to the left compared with their corresponding controls in titin-intact fibers, indicating increased calcium sensitivity. No difference in calcium sensitivity was observed between reference and residual force-enhanced contractions in trypsin-treated and osmotically compressed trypsin-treated fibers. Furthermore, calcium sensitivity after osmotic compression was lower than that observed for residual force enhancement in titin-intact skinned fibers. These results suggest that titin-based passive force regulates the increase in calcium sensitivity of residual force enhancement by a mechanism other than reduction of the myofilament lattice spacing., (Copyright © 2014 the American Physiological Society.)

Ever since the 1950s, muscle force regulation has been associated with the cross-bridge interactions between the two contractile filaments, actin and myosin. This gave rise to what is referred to as the "two-filament sarcomere model". This model does not predict eccentric muscle contractions well, produces instability of myosin alignment and force production on the descending limb of the force-length relationship, and cannot account for the vastly decreased ATP requirements of actively stretched muscles. Over the past decade, we and others, identified that a third myofilament, titin, plays an important role in stabilizing the sarcomere and the myosin filament. Here, we demonstrate additionally how titin is an active participant in muscle force regulation by changing its stiffness in an activation/force dependent manner and by binding to actin, thereby adjusting its free spring length. Therefore, we propose that skeletal muscle force regulation is based on a three filament model that includes titin, rather than a two filament model consisting only of actin and myosin filaments.