Effects of aging and resistance training in rat tendon remodeling.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Neuromuscular electrical stimulation alters gene expression and delays quadriceps muscle atrophy of rats after anterior cruciate ligament transection.

INTRODUCTION: Neuromuscular electrical stimulation (NMES) is used to improve quadriceps mass after anterior cruciate ligament (ACL) injury. We studied the effect of NMES on mRNA levels of atrophy genes in the quadriceps muscle of rats after ACL transection. METHODS: mRNA levels of atrogin-1, MuRF-1, and myostatin were assessed by quantitative PCR and the polyubiquitinated proteins by Western blot at 1, 2, 3, 7, and 15 days postinjury. RESULTS: NMES minimized the accumulation of atrogenes and myostatin according to time period. NMES also prevented reduction in muscle mass in all muscles of the ACLES group at 3 days. CONCLUSIONS: Use of NMES decreased the accumulation of atrogenes and myostatin mRNA in the quadriceps muscles, inhibiting early atrophy at 3 days, although it did not prevent atrophy at 7 and 15 days after ACL transection. This study highlights the importance of therapeutic NMES interventions in the acute phase after ACL transection.

Stretching and electrical stimulation reduce the accumulation of MyoD, myostatin and atrogin-1 in denervated rat skeletal muscle.

Denervation causes muscle atrophy and incapacity in humans. Although electrical stimulation (ES) and stretching (St) are commonly used in rehabilitation, it is still unclear whether they stimulate or impair muscle recovery and reinnervation. The purpose of this study was to evaluate the effects of ES and St, alone and combined (ES + St), on the expression of genes that regulate muscle mass (MyoD, Runx1, atrogin-1, MuRF1 and myostatin), on muscle fibre cross-sectional area and excitability, and on the expression of the neural cell adhesion molecule (N-CAM) in denervated rat muscle. ES, St and ES + St reduced the accumulation of MyoD, atrogin-1 and MuRF1 and maintained Runx1 and myostatin expressions at normal levels in denervated muscles. None of the physical interventions prevented muscle fibre atrophy or N-CAM expression in denervated muscles. In conclusion, although ES, St and ES + St changed gene expression, they were insufficient to avoid muscle fibre atrophy due to denervation.

Acute Static Stretching Results in Muscle-Specific Alterations amongst the Hamstring Muscles.

This study aimed to explore the acute effects of static stretching on the musculotendinous properties of two hamstring muscles. Twelve male volunteers underwent two testing sessions. One session was dedicated to the evaluation of the semitendinosus muscle before (PRE) and after (POST) static stretching (five sets of 30-s stretching), and the other session similarly explored the long head of biceps femoris muscle. In addition to the displacement of the myotendinous junction (MTJ), passive torque and maximal voluntary isometric torque (MVIT) were evaluated. MVIT (-8.3 ± 10.2%, p = 0.0036, d = 0.497) and passive torque (-28.4 ± 16.9%, p = 0.0003, d = 1.017) were significantly decreased POST stretching. PRE stretching, MTJ displacement was significantly greater for semitendinosus muscle than biceps femoris muscle (27.0 ± 5.2 vs. 18.6 ± 3.6, p = 0.0011, d = 1.975). After the stretching procedure, greater MTJ displacement relative changes were observed for biceps femoris muscle as compared to semitendinosus muscle (22.4 ± 31.6 vs. -8.4 ± 17.9, p = 0.0167, d = 1.252). Because of the smaller MTJ displacement PRE stretching and greater alteration POST stretching in biceps femoris muscles, the present study demonstrated muscle-specific acute responses of hamstring muscles during stretching. Although stretching acutely impairs torque production, the passive torque reduction and alteration of MTJ displacement might impact hamstring injury prevention.

Regulation of extracellular matrix elements and sarcomerogenesis in response to different periods of passive stretching in the soleus muscle of rats.

Stretching is a common method used to prevent muscle shortening and improve limited mobility. However, the effect of different time periods on stretching-induced adaptation of the extracellular matrix and its regulatory elements have yet to be investigated. We aimed to evaluate the expression of fibrillar collagens, sarcomerogenesis, metalloproteinase (MMP) activity and gene expression of the extracellular matrix (ECM) regulators in the soleus (SOL) muscle of rats submitted to different stretching periods. The soleus muscles were submitted to 10 sets of passive stretching over 10 (St 10d) or 15 days (St 15d) (1 min per set, with 30 seconds' rest between sets). Sarcomerogenesis, muscle cross-sectional area (CSA), and MMP activity and mRNA levels in collagen (type I, III and IV), connective tissue growth factor (CTGF), growth factor-beta (TGF-ß), and lysyl oxidase (LOX) were analyzed. Passive stretching over both time periods mitigated COL-I deposition in the SOL muscle of rats. Paradoxically, 10 days of passive stretching induced COL-I and COL-III synthesis, with concomitant upregulation of TGF-ß1 and CTGF at a transcriptional level. These responses may be associated with lower LOX mRNA levels in SOL muscles submitted to 10 passive stretching sessions. Moreover, sarcomerogenesis was observed after 15 days of stretching, suggesting that stretching-induced muscle adaptations are time-dependent responses.

Effect of Resistance Training on Extracellular Matrix Adaptations in Skeletal Muscle of Older Rats.

Accumulation of connective tissue, particularly extracellular matrix (ECM) proteins, has been observed in skeletal muscles with advancing age. Resistance training (RT) has been widely recommended to attenuate age-induced sarcopenia, even though its effects on the components that control ECM turnover in skeletal muscles remain to be elucidated. Thus, the aim of this study was to determine the effects of RT on connective tissue content and gene expression of key components of ECM in the skeletal muscles of aged rats. Young (3 mo.) and older (21 mo.) adult male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), 3 times a week for 12 weeks. Forty-eight hours post-training, the soleus (SOL) and gastrocnemius (GAS) muscles were dissected for histological and mRNA analysis. RT mitigated the age-associated increase of connective tissue content in both muscles, even though mRNA levels of COL-1 and-3 were elevated in older trained rats. Overall, RT significantly elevated the gene expression of key components of connective tissue deposition (TGFß and CTGF; MMP-2 and-9; TIMP-1 and-2) in the GAS and SOL muscles of older rats. In conclusion, RT blunted the age-induced accumulation of connective tissue concomitant to the upregulation of genes related to synthesis and degradation of the ECM network in the SOL and GAS muscles of older rats. Although our findings indicate that RT plays a crucial role reducing connective tissue accumulation in aged hindlimb muscles, key components of ECM turnover were paradoxically elevated. The phenotypic responses induced by RT were not accompanied by the gene expression of those components related to ECM turnover.

Effects of Resistance Training on Matrix Metalloproteinase Activity in Skeletal Muscles and Blood Circulation During Aging.

Aging is a complex, multifactorial process characterized by the accumulation of deleterious effects, including biochemical adaptations of the extracellular matrix (ECM). The purpose of this study was to investigate the effects of 12 weeks of resistance training (RT) on metalloproteinase 2 (MMP-2) activity in skeletal muscles and, MMP-2 and MMP-9 activity in the blood circulation of young and old rats. Twenty-eight Wistar rats were randomly divided into four groups (n = 7 per group): young sedentary (YS); young trained (YT), old sedentary (OS), and old trained (OT). The stair climbing RT consisted of one training session every 2 other day, with 8-12 dynamic movements per climb. The animals were euthanized 48 h after the end of the experimental period. MMP-2 and MMP-9 activity was measured by zymography. There was higher active MMP-2 activity in the lateral gastrocnemius and flexor digitorum profundus muscles in the OT group when compared to the OS, YS, and YT groups (p ≤ 0.001). Moreover, there was higher active MMP-2 activity in the medial gastrocnemius muscle in the OT group when compared to the YS and YT groups (p ≤ 0.001). The YS group presented lower active MMP-2 activity in the soleus muscle than the YT, OS, OT groups (p ≤ 0.001). With respect to active MMP-2/9 activity in the bloodstream, the OT group displayed significantly reduced activity (p ≤ 0.001) when compared to YS and YT groups. In conclusion, RT up-regulates MMP-2 activity in aging muscles, while down-regulating MMP-2 and MMP-9 in the blood circulation, suggesting that it may be a useful tool for the maintenance of ECM remodeling.

The Effects of Resistance Training Volume on Skeletal Muscle Proteome.

Studies are conflicting to whether low volume resistance training (RT) is as effective as high-volume RT protocols with respect to promoting morphological and molecular adaptations. Thus, the aim of the present study was to compare, using a climbing a vertical ladder, the effects of 8 weeks, 3 times per week, resistance training with 4 sets (RT4), resistance training with 8 sets (RT8) and without resistance training control (CON) on gastrocnemius muscle proteome using liquid chromatography mass spectrometry (LC-MS/MS) and cross sectional area (CSA) of rats. Fifty-two proteins were identified by LC-MS/MS, with 39 in common between the three groups, two in common between RT8 and CON, one in common between RT8 and RT4, four exclusive in the CON, one in the RT8, and four in the RT4. The RT8 group had a reduced abundance of 12 proteins, mostly involved in muscle protein synthesis, carbohydrate metabolism, tricarboxylic acid cycle, anti-oxidant defense, and oxygen transport. Otherwise one protein involved with energy transduction as compared with CON group showed high abundance. There was no qualitative protein abundance difference between RT4 and CON groups. These results revealed that high volume RT induced undesirable disturbances on skeletal muscle proteins, while lower volume RT resulted in similar gains in skeletal muscle hypertrophy without impairment of proteome. The CSA was significantly higher in RT8 group when compared to RT4 group, which was significantly higher than CON group. However, no differences were found between trained groups when the gastrocnemius CSA were normalized by the total body weight.

Skinfold thickness affects the isometric knee extension torque evoked by Neuromuscular Electrical Stimulation.

BACKGROUND: Subcutaneous adipose tissue may influence the transmission of electrical stimuli through to the skin, thus affecting both evoked torque and comfort perception associated with neuromuscular electrical stimulation (NMES). This could seriously affect the effectiveness of NMES for either rehabilitation or sports purposes. OBJECTIVE: To investigate the effects of skinfold thickness (SFT) on maximal NMES current intensity, NMES-evoked torque, and NMES-induced discomfort. METHOD: First, we compared NMES current intensity, NMES-induced discomfort, and NMES-evoked torque between two subgroups of subjects with thicker (n=10; 20.7 mm) vs. thinner (n=10; 29.4 mm) SFT. Second, we correlated SFT to NMES current intensity, NMES-induced discomfort, and NMES-evoked knee extension torque in 20 healthy women. The NMES-evoked torque was normalized to the maximal voluntary contraction (MVC) torque. The discomfort induced by NMES was assessed with a visual analog scale (VAS). RESULTS: NMES-evoked torque was 27.5% lower in subjects with thicker SFT (p=0.01) while maximal current intensity was 24.2% lower in subjects with thinner SFT (p=0.01). A positive correlation was found between current intensity and SFT (r=0.540, p=0.017). A negative correlation was found between NMES-evoked torque and SFT (r=-0.563, p=0.012). No significant correlation was observed between discomfort scores and SFT (rs=0.15, p=0.53). CONCLUSION: These results suggest that the amount of subcutaneous adipose tissue (as reflected by skinfold thickness) affected NMES current intensity and NMES-evoked torque, but had no effect on discomfort perception. Our findings may help physical therapists to better understand the impact of SFT on NMES and to design more rational stimulation strategies.

Leucine minimizes denervation-induced skeletal muscle atrophy of rats through akt/mtor signaling pathways.

The aim of the present study was to evaluate the effect of leucine treatment (0.30 mM) on muscle weight and signaling of myoproteins related to synthesis and degradation pathways of soleus muscle following seven days of complete sciatic nerve lesion. Wistar rats (n = 24) of 3-4 months of age (192 ± 23 g) were used. The animals were randomly distributed into four experimental groups (n = 6/group): control, treated with leucine (L), denervated (D) and denervated treated with leucine (DL). Dependent measures were proteins levels of AKT, AMPK, mTOR, and ACC performed by Western blot. Leucine induced a reduction in the phosphorylation of AMPK (p < 0.05) by 16% in the L and by 68% in the DL groups as compared with control group. Denervation increased AMPK by 24% in the D group as compared with the control group (p < 0.05). AKT was also modulated by denervation and leucine treatment, highlighted by the elevation of AKT phosphorylation in the D (65%), L (98%) and DL (146%) groups as compared with the control group (p < 0.05). AKT phosphorylation was 49% higher in the D group as compared with the DL group. Furthermore, denervation decreased mTOR phosphorylation by 29% in the D group as compared with the control group. However, leucine treatment induced an increase of 49% in the phosphorylation of mTOR in the L group as compared with the control group, and an increase of 154% in the DL as compared with the D group (p < 0.05). ACC phosphorylation was 20% greater in the D group than the control group. Furthermore, ACC in the soleus was 22% lower in the in the L group and 50% lower in the DL group than the respective control group (p < 0.05). In conclusion, leucine treatment minimized the deleterious effects of denervation on rat soleus muscle by increasing anabolic (AKT and mTOR) and decreasing catabolic (AMPK) pathways. These results may be interesting for muscle recovery following acute denervation, which may contribute to musculoskeletal rehabilitation after denervation.

Quadriceps muscle atrophy after anterior cruciate ligament transection involves increased mRNA levels of atrogin-1, muscle ring finger 1, and myostatin.

OBJECTIVE: The aim of this study was to assess the mRNA levels of atrogin-1, muscle ring finger 1, and myostatin in rat quadriceps after anterior cruciate ligament (ACL) transection. DESIGN: Wistar rats were randomized into three different groups: ACL (surgery and ACL transection), sham (surgery without ACL transection), and control. Vastus medialis, rectus femoris, and vastus lateralis muscles were harvested at 1, 2, 3, 7, and 15 days after ACL transection. The mRNA levels of atrogin-1, muscle ring finger 1, and myostatin, as well as the ubiquitinated protein content, muscle mass, and cross-sectional area of the muscle fibers, were evaluated. RESULTS: Elevated levels of atrogin-1, muscle ring finger 1, and myostatin mRNA were detected in all tested muscles at most time points. The ubiquitinated protein content was increased at 3 days in the ACL and sham groups. The muscle mass of the ACL group was reduced at 3, 7, and 15 days (vastus lateralis and vastus medialis) and at 7 and 15 days (rectus femoris), whereas it was reduced in the sham group at 3 and 7 days (vastus lateralis and vastus medialis) and at 7 days (rectus femoris). The cross-sectional area of vastus medialis was reduced at 3, 7, and 15 days in the ACL group and at 3 and 7 days in the sham group. The cross-sectional area of the vastus lateralis was reduced at 7 and 15 days in the ACL group and at 7 days in the sham group. Whereas muscle mass and cross-sectional area recovery was noted in the sham group, no recovery was observed in the ACL group. CONCLUSIONS: Quadriceps atrophy after ACL transection involves increased levels of myostatin, atrogin-1, and muscle ring finger 1 mRNA and the accumulation of ubiquitinated protein.

Effects of electrical stimulation and stretching on the adaptation of denervated skeletal muscle: implications for physical therapy.

BACKGROUND: This review will describe the main cellular mechanisms involved in the reduction and increase of myoproteins synthesis commonly associated with muscle atrophy and hypertrophy, respectively. OBJECTIVE: We analyzed the effects of electrical stimulation (ES) and stretching exercise on the molecular pathways involved in muscle atrophy and hypertrophy. We also described the main effects and limits of these resources in the skeletal muscle, particularly on the denervated muscle. DISCUSSION: Recently, our studies showed that the ES applied in a similar manner as performed in clinical practice is able to attenuate the increase of genes expression involved in muscle atrophy. However, ES was not effective to prevent the loss of muscle mass caused by denervation. Regarding to stretching exercises, their mechanisms of action on the denervated muscle are not fully understood and studies on this area are scarce. Studies from our laboratory have found that stretching exercise increased the extracellular matrix remodeling and decreased genes expression related to atrophy in denervated muscle. Nevertheless, it was not enough to prevent muscle atrophy after denervation. CONCLUSIONS: In spite of the use of stretching exercise and ES in clinical practice in order to minimize the atrophy of denervated muscle, there is still lack of scientific evidence to justify the effectiveness of these resources to prevent muscle atrophy in denervated muscle.

Skinfold thickness affects the isometric knee extension torque evoked by Neuromuscular Electrical Stimulation

BACKGROUND: Subcutaneous adipose tissue may influence the transmission of electrical stimuli through to the skin, thus affecting both evoked torque and comfort perception associated with neuromuscular electrical stimulation (NMES). This could seriously affect the effectiveness of NMES for either rehabilitation or sports purposes. OBJECTIVE: To investigate the effects of skinfold thickness (SFT) on maximal NMES current intensity, NMES-evoked torque, and NMES-induced discomfort. METHOD: First, we compared NMES current intensity, NMES-induced discomfort, and NMES-evoked torque between two subgroups of subjects with thicker (n=10; 20.7 mm) vs. thinner (n=10; 29.4 mm) SFT. Second, we correlated SFT to NMES current intensity, NMES-induced discomfort, and NMES-evoked knee extension torque in 20 healthy women. The NMES-evoked torque was normalized to the maximal voluntary contraction (MVC) torque. The discomfort induced by NMES was assessed with a visual analog scale (VAS). RESULTS: NMES-evoked torque was 27.5% lower in subjects with thicker SFT (p=0.01) while maximal current intensity was 24.2% lower in subjects with thinner SFT (p=0.01). A positive correlation was found between current intensity and SFT (r=0.540, p=0.017). A negative correlation was found between NMES-evoked torque and SFT (r=-0.563, p=0.012). No significant correlation was observed between discomfort scores and SFT (rs=0.15, p=0.53). CONCLUSION: These results suggest that the amount of subcutaneous adipose tissue (as reflected by skinfold thickness) affected NMES current intensity and NMES-evoked torque, but had no effect on discomfort perception. Our findings may help physical therapists to better understand the impact of SFT on NMES and to design more rational stimulation strategies.

Stretching and electrical stimulation regulate the metalloproteinase-2 in rat denervated skeletal muscle.

OBJECTIVE: To evaluate the effects of electrical stimulation (ES), stretching and their combined effects in matrix metalloproteinases (MMPs) gene expression and activity during early denervation of rat tibialis anterior (TA) muscle by axonotmesis. METHODS: Thirty adult male Wistar rats were divided into five groups: normal (N), denervated TA (D), denervated TA submitted to daily ES (D + ES), denervated TA submitted to daily stretching (D + St) and denervated TA submitted daily to both ES and stretching concurrently (D + ES + St). Both zimographic analysis and real time polymerase chain reaction of MMPs were used to muscular evaluation. In addition, muscle fiber cross-section area (CSA) was also evaluated. RESULTS: Stretching increased MMP-2 activity in denervated muscle when performed alone as well as in association with ES (p<0·01). Both stretching and ES, individually and in association, increased MMP-2 gene expression in denervated muscle compared to N and D (p<0·05). All denervated groups decreased muscle fiber CSA compared to N (p<0·05). DISCUSSION: While stretching is the main stimulus leading to the activation of MMP-2, both ES and stretching are able to increase MMP-2 gene expression in rat denervated muscle suggesting ECM remodeling.

Effects of electrical stimulation and stretching on the adaptation of denervated skeletal muscle: implications for physical therapy / Efeitos da eletroestimulação e do alongamento muscular sobre a adaptação do músculo desnervado: implicações para a fisioterapia

BACKGROUND: This review will describe the main cellular mechanisms involved in the reduction and increase of myoproteins synthesis commonly associated with muscle atrophy and hypertrophy, respectively. OBJECTIVE: We analyzed the effects of electrical stimulation (ES) and stretching exercise on the molecular pathways involved in muscle atrophy and hypertrophy. We also described the main effects and limits of these resources in the skeletal muscle, particularly on the denervated muscle. DISCUSSION: Recently, our studies showed that the ES applied in a similar manner as performed in clinical practice is able to attenuate the increase of genes expression involved in muscle atrophy. However, ES was not effective to prevent the loss of muscle mass caused by denervation. Regarding to stretching exercises, their mechanisms of action on the denervated muscle are not fully understood and studies on this area are scarce. Studies from our laboratory have found that stretching exercise increased the extracellular matrix remodeling and decreased genes expression related to atrophy in denervated muscle. Nevertheless, it was not enough to prevent muscle atrophy after denervation. CONCLUSIONS: In spite of the use of stretching exercise and ES in clinical practice in order to minimize the atrophy of denervated muscle, there is still lack of scientific evidence to justify the effectiveness of these resources to prevent muscle atrophy in denervated muscle.

CONTEXTUALIZAÇÃO: Esta revisão abordará os principais mecanismos celulares envolvidos na redução e aumento da síntese de mioproteínas comumente associadas às situações de atrofia e hipertrofia muscular, respectivamente. OBJETIVO: Analisaremos os efeitos da estimulação elétrica (EE) e do exercício de alongamento sobre as vias moleculares envolvidas na atrofia e hipertrofia muscular. Serão descritos os principais efeitos e os limites desses recursos no músculo esquelético, particularmente sobre o músculo desnervado. DISCUSSÃO: Recentemente, nossos estudos mostraram que a EE, aplicada de modo semelhante ao realizado na prática clínica, é capaz de amenizar o aumento da expressão de genes envolvidos na atrofia muscular. Entretanto, a EE não foi efetiva para deter a perda de massa muscular decorrente da desnervação. Em relação ao alongamento, seus mecanismos de ação sobre o músculo desnervado não são totalmente conhecidos, e os trabalhos nessa área são escassos. Estudos do nosso laboratório identificaram que o alongamento aumentou o remodelamento da matriz extracelular e diminuiu a expressão de genes relacionados à atrofia no músculo desnervado. Porém, também não foi suficiente para impedir a atrofia muscular após a desnervação. CONCLUSÕES: Apesar do uso da EE e do alongamento muscular na prática clínica, com objetivo de minimizar a atrofia do músculo desnervado, ainda há carência de informações científicas que justifiquem a eficácia desses recursos para prevenir a atrofia no músculo desnervado.