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Genome-Wide Association Study on Muscle Stiffness Identified Novel Locus for Predisposition to Muscle Strain Injury.

PURPOSE: We aimed to screen the entire genome for genetic variants associated with passive muscle stiffness, which has been suggested as a risk factor for muscle strain injury. METHODS: This genome-wide association study (GWAS) on passive muscle stiffness included 350 physically active young Japanese individuals. Three hamstring constituents were measured using ultrasound shear wave elastography. Skeletal muscle transcriptomes were compared across the genotypes of GWAS-identified variants in 48 healthy Japanese individuals. Association between GWAS-identified variants and history of muscle strain injury was examined in 1428 Japanese athletes. RESULTS: Two loci on chromosome 11 demonstrated a genome-wide significant association with passive muscle stiffness of the biceps femoris long head (rs12807854 T/C: P = 5.19 × 10 -10 , rs78405694 T/C: P = 2.09 × 10 -8 ; linear regression analysis adjusted for sex, age, and stretching exercise habits). Skeletal muscle RNA sequencing revealed significantly elevated expression of extracellular matrix-related genes in muscles carrying stiffness-increasing alleles of these variants. Among athletes, rs12807854 T/C was significantly associated with a history of muscle strain injury ( P = 0.0254; logistic regression analysis adjusted for age, sex, competitive level, and main sport). Carriers of the C allele, associated with increased muscle stiffness, exhibited a heightened risk of muscle strain injury (odds ratio = 1.62; 95% confidence interval = 1.06-2.47 per C allele increase). By contrast, rs78405694 did not show a significant association with muscle strain injury in this population. CONCLUSIONS: A novel locus associated with passive muscle stiffness and muscle strain injury was identified. Elucidating the detailed mechanisms linking the identified locus to passive muscle stiffness may lead to the development of new strategies to prevent muscle strain injuries.

Humans

Measurements of muscle stiffness, the electromyogram and activity in single muscle spindles of human flexor muscles following conditioning by passive stretch or contraction.

In experiments on adult human subjects we examined the effect on passive mechanical properties of a muscle by conditioning it with either an isometric contraction or passive muscle extension. The test measurement was the amount of muscle displacement (stiffness) and the accompanying EMG in response to a brief torque pulse. Two muscles were tested, flexor digitorum profundus (FDP) and brachialis. In FDP the discharge of single muscle spindles was recorded as well. After muscle extension and return to the initial length, passive stiffness was less than after an isometric contraction. The changes in stiffness were accompanied by changes in pattern of EMG and in the responses of muscle spindles. It is suggested that in resting muscle there are stable cross bridges between actin and myosin filaments of muscle fibres which largely determine the passive stiffness. Muscle extension leads to detachment of these cross bridges which then re-form at the longer length. Return of the muscle to its starting length leads to development of slack in muscle fibres because, stiffened by the presence of the stable cross bridges, they are unable to shorten. Slack in muscle fibres lowers their measured stiffness. Muscle contraction, on the other hand, will result in any preexisting slack being taken up by the actively shortening muscle fibres, thereby raising muscle stiffness. Stiffness in intrafusal fibres is likely to follow a similar pattern to that in extrafusal fibres, leading to changes in stretch responsiveness of muscle spindles and consequently in the reflex EMG. It is concluded that the changes in stiffness and accompanying reflexes observed in this study are likely to be seen, at least under some conditions, in normal movements.

Adult

Effect of knee and hip joint positions on passive stiffness of the rectus femoris and vastus lateralis in healthy individuals.

Passive muscle stiffness is a key determinant of musculoskeletal function and is influenced by structural components such as titin, connective tissue, and fascia. However, the effects of joint position, muscle depth, and sex on quadriceps passive stiffness remain unclear. To investigate the passive stiffness of the rectus femoris (RF) and vastus lateralis (VL) under different joint configurations, muscle depths, and between sexes using shear wave elastography (SWE). Thirty-six healthy young adults (18 men and 18 women) participated in this randomized crossover study. Passive stiffness was assessed in four positions of knee flexion: supine with 60&#xb0; (SUP60), supine with 20&#xb0; (SUP20), sitting with 60&#xb0; (SIT60), and sitting with 20&#xb0; (SIT20). SWE measurements (m/s) were obtained from 30 regions of interest (ROIs) per muscle, categorized into superficial, intermediate, and deep levels. Data were analyzed using Generalized Estimating Equations (GEE). A significant effect of position was observed, with higher stiffness values in the SUP60 condition for both RF and VL (p&#x2009;<&#x2009;0.001). Superficial regions consistently exhibited greater stiffness compared to intermediate and deep regions across all positions (p&#x2009;<&#x2009;0.001). Additionally, men demonstrated significantly higher stiffness values than women (p&#x2009;<&#x2009;0.001). Significant interactions were found between position and muscle, as well as position and depth. Quadriceps passive stiffness is influenced by joint position, muscle depth, and sex. The SUP60 position elicits the highest stiffness, while superficial muscle regions are consistently stiffer. These findings highlight the non-uniform mechanical behavior of the quadriceps and may have implications for clinical assessment, rehabilitation, and exercise prescription. Clinical trial registration: This study was registered at Clinicaltrials.gov in June 06th, 2023. Register number NCT05905406. Link to access https//clinicaltrials.gov/study/NCT05905406.

Humans

Calcium sensitivity of isometric tension in intact papillary muscles and chemically skinned trabeculae in different models of hypertensive hypertrophy.

STUDY OBJECTIVE - The aim was to examine the contractile state, the inotropic response to [Ca2+]e and the Ca2+ sensitivity of the contractile proteins in different models of hypertensive hypertrophy in an early stage of evolution (3-4 weeks). DESIGN - Renal hypertension was induced by placing a silver clip around the left renal artery. The contralateral kidney was either removed (1K-1C) or left untouched (2K-1C). Hypertension through sodium overload was produced by administration of deoxycorticosterone and 1% NaCl drinking water. (DOCA rats). Active and passive length-tension curves were performed to evaluate basal contractility at Lmax and passive stiffness of cardiac muscle. The inotropic responsiveness to [Ca2+]e and the Ca2+ sensitivity of the contractile proteins were also evaluated. EXPERIMENTAL MATERIAL - Papillary muscles and skinned trabeculae from the left ventricle of male Wistar hypertensive and age matched normotensive rats were used. MEASUREMENTS AND RESULTS - Cardiac hypertrophy was similar in all hypertensive groups. In 2K-1C and 1K-1C rats, basal contractility was not significantly different from controls. In DOCA rats, developed tension and time to peak tension (TTP) were significantly greater than controls. The inotropic response to [Ca2+]e was depressed in 2K-1C and increased in DOCA rats. In DOCA rats, increasing [Ca2+]e produced an increase in TTP greater than in controls. No differences were detected in muscle passive stiffness or in Ca2+ sensitivity of the contractility proteins among the different groups. CONCLUSIONS - In the earlier stages of hypertensive hypertrophy, differences in basal contractile state and/or inotropic responsiveness appear to be more related to the initiating cause of hypertensive hypertrophy than to the degree of hypertrophy itself. These differences cannot be attributed to changes in Ca2+ sensitivity of the contractile system.

Animals

Effect of age on passive elastic stiffness of rat heart muscle.

A thick-wall spherical model for the rat left ventricle was used to deduce passive wall stiffness from diastolic pressure-volume data. This was done for rats in three age classes: young (1 mo), adult (17 mo) and old (17 mo). The model was based on finite deformation elasticity theory consistent with the magnitude of observed deformation. A least-squares procedure was used to determine elastic constants in postulated nonlinear stress-stretch relations for the myocardium. It was found that at a given level of stress, wall stiffness for ventricles in the young age class was consistently greater than wall stiffness in the other two classes. In addition, the difference in wall stiffness between rats in the adult and old age classes was found to be approximately 10%.

Aging

The effect of alcohol on active and passive stiffness, and on isometric contractions of glycerinated heart muscle in rats.

The response of the contractile and the series elastic elements to ethanol was studied isometric contraction and quick release methods after measuring the passive length-tension relationship in glycerinated heart muscle fibers of rats at resting state. In rats consuming 30% ethanol for an average of 5 weeks, maximal developed tension (P0), the maximal rate of tension development (dp/dtmax) and Vmax were significantly depressed, the time to peak tension (t0) was not changed. As in fresh papillary muscle the modulus of elasticity of active glycerinated muscle increased in proportion to load. The stiffness of the series elastic element showed significant elevation in rats receiving ethanol. The passive stiffness in resting state revealed no significant difference between control and alcohol exposed rats. Therefore, increased stiffness of the series elastic element and diminished contractility are present following chronic alcohol consumption.

Animals

Influence of muscle cooling on the viscoelastic response of the human ankle to sinusoidal displacements.

The changes in passive mechanical muscle properties due to cooling of the calf in healthy human volunteers were investigated. The technique, using sinusoidal driving of the foot, permitted the separation of muscle stiffness response into its elastic and viscous components. Cooling the calf with ice for 30 minutes increases the rate of change of elastic stiffness with frequency, and it increases the frictional stiffness over a frequency range of 3 to 12Hz. Such cooling would produce an estimated 3% to 10% increase in total stiffness, on average, in a spastic person. This increase in stiffness would counteract reductions in total stiffness achieved during the application of cryotherapy to relieve spasticity. However, one could expect that for a clinically significant reduction of spasticity, the increase in passive stiffness of the muscle generated by cooling would be largely overshadowed by the decrease in reflex reactivity.

Adult

Separation of active and passive components of short-range stiffness of muscle.

The short-range stiffness of smoothly but submaximally contracting isometric soleus muscles of anesthetised cats was measured by applying small fast stretches. The ratio of isometric tension to stiffness was plotted against tension over a wide range of muscle lengths and stimulus rates. The results fitted a straight line well, as predicted from crossbridge theory, showing the stiffness to be a function of tension only, independent of the combination of length and stimulus rate used to generate the tension. The major deviation from this line was attributed to incomplete fusion at low frequencies of stimulation. Values believed to be tendon compliance and crossbridge tension per unit of stiffness were found from the graph, and the tendon compliance correlated with the maximum muscle tension. Shortening the tendon by attaching nearer to the muscle changed the results in a manner consistent with the theory, provided that appropriate precautions were taken against slippage.

Animals

Dynamic cardiomyoplasty acutely impairs left ventricular diastolic function.

In patients with congestive heart failure, medical treatment has a high rate of mortality and morbidity, and transplantation is limited by the availability of donor hearts. Dynamic cardiomyoplasty is being investigated as surgical therapy to improve left ventricular function in these patients. To evaluate the early postoperative effects of this procedure on left ventricular diastolic function, we studied seven dogs through the use of sonomicrometry and micromanometry in a canine model of dynamic cardiomyoplasty. Left ventricular diastolic parameters were determined before wrapping the latissimus dorsi muscle (baseline), after latissimus dorsi muscle wrap but without stimulation, and with synchronous left ventricular contraction-latissimus dorsi muscle stimulation. End-diastolic pressure was increased in both conditions after latissimus dorsi muscle wrap (without stimulation, 5 +/- 1; with stimulation, 6 +/- 2 mm Hg; p < 0.05) compared with baseline (3 +/- 2 mm Hg). The peak rate of diastolic pressure decay was greater at baseline (1560 +/- 370 mm Hg/sec) than after latissimus dorsi muscle wrap, both without (1260 +/- 330 mm Hg/sec, p < 0.01) and with (1120 +/- 420 mm Hg/sec, p < 0.01) stimulation. The constant of pressure decay was prolonged both without (53 +/- 10 seconds, p < 0.05) and with (62 +/- 11 seconds, p < 0.01) latissimus dorsi muscle stimulation compared with the baseline (38 +/- 5 seconds). Compared with baseline (0.2 +/- 0.2 cm-2), the constant of passive chamber stiffness increased after the latissimus dorsi muscle was wrapped around the heart (1.6 +/- 0.7 cm-2, p < 0.05) and with stimulation (2.1 +/- 1.0 cm-2, p < 0.01). The maximal diastolic filling rate (baseline, 18.1 +/- 6.7; without stimulation, 16.6 +/- 8.9; with stimulation, 16.6 +/- 4.1 cm2/sec, not significant) and end-diastolic short-axis area (baseline, 7.3 +/- 2.3; without stimulation, 7.4 +/- 2.1; with stimulation, 7.5 +/- 2.3 cm2, not significant) were similar among the three conditions. The latissimus dorsi muscle wrap prolonged relaxation and increased left ventricular passive stiffness. Synchronous latissimus dorsi muscle stimulation with left ventricular contraction did not improve diastolic function in this model. The results suggest that in the early postoperative period, dynamic cardiomyoplasty impairs diastolic function.

Animals

Unstimulated force during hypoxia of rat cardiac muscle: stiffness and calcium dependence.

The stiffness of rat cardiac trabeculae was measured in vitro to distinguish between an increase in unstimulated force (Fu) caused by rapid cycling of cross bridges or caused by rigor bridges during hypoxia. The force was measured with a strain gauge, the sarcomere length was determined by laser diffraction techniques, and muscle length was controlled by means of a motor. Stiffness was analyzed by using small (less than 1% of muscle length) sinusoidal length perturbations of 1 and 100 Hz. The stiffness at 100 Hz increased linearly with force during tetani at a varied [Sr2+] (0.25-10 mM) in the Krebs-Henseleit (K-H) buffer, but remained virtually unchanged at 1 Hz. In contrast, the stiffness of both the passive muscle and the muscle exposed to either CN- or to PO2 less than 1.5 mmHg up to development of maximal Fu (Fumax) was similar at 1- and 100-Hz perturbations. Less profound hypoxia (PO2 6-10 mmHg) resulted in spontaneous sarcomere activity during the rise in Fu, and an increase in the ratio of stiffness at 100 Hz to stiffness at 1 Hz was detected. When oxidative phosphorylation was inhibited by CN- (2 mM) while the muscle was stimulated in the absence of both Ca2+ and Na+ (choline+substituted), the addition of Na+ at the time at which Fu had reached 30-40% of Fumax did not affect the rate of rise of Fu. These results show that the development of Fu during more complete anoxia in rat trabeculae is completely due to the formation of rigor links and that Ca2(+)-dependent cross-bridge activation can contribute to the rise in Fu during less severe hypoxia.

Animals

Osmotic compression and stiffness changes in relaxed skinned cardiac myocytes in PVP-40 and dextran T-500.

Sarcomere lengths, cell widths, indices of stiffness, and striation pattern uniformity were determined from radially compressed isolated adult cardiac myocytes from the rat. Single cells were bathed in a series of relaxing solutions containing 0-15% concentrations of nonpenetrating long chain polymers PVP-40 and dextran T-500. There were no significant changes observed in average sarcomere lengths or in striation pattern uniformity at any concentration. But cell widths decreased and stiffness increased in both polymers in a concentration-osmotic pressure-dependent relationship. Changes in cell width and stiffness were repeatable in either polymer, but only after an initial compression with a 10 or 15% concentration solution. The observed reduction in cell width after initial compression correlates well with known myofilament lattice spacing compression in rat cardiac muscle and is qualitatively similar to compressions seen in skeletal muscle preparations. But the cardiac myofilament lattice may not be as compressible as the skeletal lattice. Like skeletal muscle, stiffness exhibits a two-phase relationship where most of the increase occurs at solution osmotic pressures greater than 20 Torr. Finally, the inherently greater passive stiffness-length relationship of cardiac muscle is maintained at higher osmotic pressures such that the passive elastic modulus is strongly length dependent.

Actin Cytoskeleton

The effect of aging on ventricular contractile performance.

The effects of aging on mechanical performance of isolated canine right ventricular trabeculae were studied in two age groups. The first group was comprised of nine dogs, about 9 months of age. The second group was composed of seven dogs over 8 years of age. Aging had no significant effect on developed force. Extent of shortening tended to decrease. There was a significant decrease in both the rate of rise of tension and the velocity of shortening (20%). THis reduction was primarily due to an increased duration of contraction. Twitch duration increased by as much as 40% during aging but most of this prolongation was due to a profound slowing of relaxation. Aging caused a significant increase in passive stiffness since equivalent changes in muscle length brought about twice as much increase in resting tension in the aged muscle as in the young muscle. On the other hand, aging caused a significant shift of Lo to the right. Taken collectively, these results indicate that aging is associated with increased passive stiffness and decreased speed of contraction without changes in strength.

Aging

Effects of tetanic contraction of motor units of similar type on the initial stiffness to ramp stretch of the cat peroneus longus muscle.

1. The stiffness during the initial portion of a ramp stretch was measured in cat peroneus longus muscle at rest and during maximal tetanic contractions produced by increasing numbers of motor units of the same type [slow (S), fast fatigue resistant (FR), or fast fatigable (FF)]. 2. This initial ramp stiffness was defined as the ratio between tension and length change over the limited range of constant velocity extension during which tension rose linearly with length change. This stiffness was reduced by tetanic contraction of a number of motor units while other units remained inactive. The reduction had different characteristics in contractions produced by S, FR, or FF units. 3. Two brief ramp (triangular) stretches were applied at short intervals to evaluate the contribution of stable cross bridges to the changes in ramp stiffness. When the amplitude of the first stretch exceeded the presumed elastic limits of the stable cross bridges, the second ramp stretch showed a reduction of 20-60% in initial stiffness. This was seen both in passive muscles and in muscles in which several motor units were contracting. 4. When increasing numbers of motor units of the same type were activated, the initial ramp stiffness to the second of a pair of triangular stretches delivered during contraction increased almost linearly with the developed tension. The slope of this increase was 2.5 times steeper for S units than for FR units. This reflects the fact that contraction produced by S units causes a proportionally greater resistance to stretch than that of fast units.

Animals

The effects of neuromuscular stimulation on muscle tone at the knee in paraplegia.

Resting muscle tone of the leg was measured in terms of thigh muscle stiffness and knee resonant frequency in muscles of spinal cord injured subjects who had been involved in an electrical neuromuscular stimulation training programme of the thigh muscles over at least 2 months. The thigh circumference of these patients was 6.6% larger than before training commenced (P less than 0.001) and showed increased muscle stiffness and resonant frequency compared to a similar group of paralysed subjects who had not used any neuromuscular stimulation. Resonant frequency and stiffness after the long-term training were similar to those of non-injured controls and therefore the stimulation programme seemed to reverse the effects of paralysis on muscle tone. Short periods of rest (30 min) caused increased muscle stiffness in non-injured controls and paralysed muscles trained by neuromuscular stimulation. Additional 15 min periods of neuromuscular stimulation further increased muscle stiffness in the trained muscles but also in the muscles of paralysed subjects who had no long-term neuromuscular training. In contrast, 15 min sessions of passive movement of the knee decreased muscle stiffness in long-term trained paralysed muscles and untrained paralysed muscles. Knee resonant frequency was also significantly decreased in the trained paralysed muscles. Results show that muscle tone varies depending on the amount of previous movement or rest and that although neuromuscular stimulation of paralysed muscles increases muscle stiffness and knee resonant frequency, it is in fact restoring such properties of the muscle to a state approaching that of non-injured controls.

Adult

The effects of geometry, elasticity, and external pressures on the diastolic pressure-volume and stiffness-stress relations. How important is the pericardium?

The concept of an incremental elastic modulus is applied in the quantification of passive elastic stiffness-stress relations of intact heart muscle, and a transmural pressure-volume relation for the left ventricle is subsequently derived in terms of geometry, muscle elasticity, and external pressures to assess their importance. Physiological and clinical applications of this method indicate that: (1) stiffness-stress relations obtained on the basis of pressure-volume data from dog hearts are not significantly different from those obtained from muscle strips excised from these same hearts; (2) shape and the presence of right ventricular, pericardial, or pleural pressures are of secondary importance in an assessment of passive elastic stiffness; and (3) dramatic shifts in the left ventricular intracavity pressure-volume relations following drug interventions are primarily due to the presence of substantial pericardial pressures; however, the transmural pressure-volume relations are not markedly altered, implying no alteration in the intrinsic ventricular compliance.

Animals

The role of collagen crosslinking in the increased stiffness of avian dystrophic muscle.

The resting tension and stiffness in the range of sarcomere lengths 2.4-3.6 microns were studied in highly inbred normal and dystrophic chicken pectoral muscle bundles, and the results were compared with the collagen content and the extent of crosslinkage of the collagen. All parameters increased in the order normal homozygote (003/003) less than heterozygote (003/433) less than dystrophic homozygote (433/433) chickens, with the data from the heterozygotes being halfway between the two homozygotes, thus exhibiting a semi-dominant inheritance pattern. In separate experiments, lathyrism was induced by treating normal (412/412) and dystrophic (413/413) chickens with alpha-acetoaminonitrile, an inhibitor of lysyl oxydase, the enzyme responsible for the initiation of collagen crosslinkage formation. These experiments showed that the tension and stiffness in response to passive stretch did not change with lathyrism in normal muscles, whereas the tension and stiffness decreased significantly with lathyrism in dystrophic muscles. The collagen content did not change with lathryrism in both normal and dystrophic muscles. These results indicate that the increased content of collagen crosslinkages is the basis for the increased resting tension and stiffness in the dystrophic muscles of the chicken, and that the effects can be reversed by treatment with an inhibitor of collagen crosslinkage formation.

Animals

Passive interaction between sliding filaments in the osmotically compressed skinned muscle fibers of the frog.

Shortening and lengthening velocities, instantaneous stiffness, and tension transients after stretch were measured in compressed muscle fibers from the frog in the presence or absence of polyvinylpyrrolidone (PVP K30) or Dextran T70. Both shortening and lengthening velocities clearly decreased with the concentration of polymer. In the presence of polymer, "passive" stiffness was observed in relaxing solution depending on fiber diameter, and stiffness increased further by activation. This increase by activation above "passive" stiffness was nearly constant in the wide range of polymer concentrations. These active and "passive" stiffnesses were found to be dependent on sarcomere length. The stiffness of a compressed rigor fiber was indicated to be composed of constant rigor stiffness and a variable "passive" one. The tension transient after stretch in a compressed active or rigor fiber was also indicated to be composed of two kinds of transients. The above results suggest that (a) there exist two kinds of interactions in parallel in a compressed active or rigor fiber: one active or rigor and another "passive" between sliding filaments, and (b) the decrease in shortening velocity in a compressed fiber may be brought about by this "passive" interaction.

Actin Cytoskeleton