Sabrina Messa Peviani

Electrical stimulation based on chronaxie reduces atrogin-1 and myoD gene expressions in denervated rat muscle.

Denervation induces muscle fiber atrophy and changes in the gene expression rates of skeletal muscle. Electrical stimulation (ES) is a procedure generally used to treat denervated muscles in humans. This study evaluated the effect of ES based on chronaxie and rheobase on the expression of the myoD and atrogin‐1 genes in denervated tibialis anterior (TA) muscle of Wistar rats. Five groups were examined: (1) denervated (D); (2) D+ES; (3) sham denervation; (4) normal (N); and (5) N+ES. Twenty muscle contractions were stimulated every 48 h using surface electrodes. After 28 days, ES significantly decreased the expression of myoD and atrogin‐1 in D+ES compared to the D group. However, ES did not prevent muscle‐fiber atrophy after denervation. Thus, ES based on chronaxie values and applied to denervated muscles using surface electrodes, as normally used in human rehabilitation, was able to reduce the myoD and atrogin‐1 gene expressions, which are related to muscular growth and atrophy, respectively. The results of this study provide new information for the treatment of denervated skeletal muscle using surface ES. Muscle Nerve, 2006

Short bouts of stretching increase myo-D, myostatin and atrogin-1 in rat soleus muscle

Stretching is widely used in rehabilitation and sports activities to improve joint range‐of‐motion and flexibility in humans, but the effect of stretching on the gene expression of skeletal muscle is poorly understood. We evaluated the effect of short bouts of passive stretching of rat soleus muscle on myo‐D, myostatin, and atrogin‐1 gene expressions. Six groups of animals were submitted to a single session of stretching (10 stretches of 1 minute with 30 seconds of rest between them, performed manually) and were evaluated immediately (I), and 8, 24, 48, 72, and 168 hours after the session. To evaluate the effect of repetitive sessions of stretching on the soleus muscle over 1 week, three groups of animals received a single session per day of stretching and the muscle was evaluated immediately after 2, 3, and 7 sessions. The mRNA levels of myo‐D, myostatin, and atrogin‐1 were determined by real‐time polymerase chain reaction. A single session of stretching increased the mRNA levels of myo‐D (after 24 h), myostatin (I, and 168 h later), and atrogin‐1 (after 48 h). Repeated daily session of stretching over 1 week increased myostatin (after 7 sessions) and atrogin‐1 expression (after 2, 3, and 7 sessions). Thus, short bouts of passive stretching are able to increase the gene expression of factors associated with muscle growth (myo‐D), negative regulation of muscle mass (myostatin), and atrophy (atrogin‐1), indicating muscle remodeling through different pathways. Muscle Nerve, 2006

Electrical stimulation increases matrix metalloproteinase‐2 gene expression but does not change its activity in denervated rat muscle

Muscle‐fiber atrophy occurs concomitantly with intramuscular connective tissue proliferation following denervation. These events contribute to the impairment of mechanical and functional properties of denervated muscles and compromise their recovery. Electrical stimulation (ES) is used in human rehabilitation to treat denervated muscles. However, the effects of this therapy on the intramuscular extracellular matrix (ECM) remain uncertain. Metalloproteinases (MMPs) are responsible by remodeling ECM in many neuromuscular disorders. This study evaluates the effect of ES on the activity of two important MMPs, MMP‐2 and MMP‐9, both involved in ECM remodeling of rat denervated muscles. Thirty‐four Wistar rats (3 months old, 356 ± 38.7 g) were divided into five groups: denervated (D); D+ES; sham denervation; normal (N); and N+ES. Twenty maximal muscle contractions were stimulated every 48 h using surface electrodes, as generally used in the rehabilitation of human denervated muscle. Both zymographic analysis and real‐time polymerase chain reaction (PCR) of MMPs were used to evaluate muscle after denervation for 28 days. Both the D and D+ES groups showed increased MMP‐2 activity compared with the N group (P < 0.05). Furthermore, only the D+ES had increased MMP‐2 gene expression compared with the N group (P < 0.05). MMP‐9 activity was not detected in any of the groups. The results of this study indicate that denervation increases MMP‐2 activity, and ES regulates MMP‐2 gene expression in rat denervated skeletal muscle. These findings clarify the effects of ES on the ECM of denervated muscle and may be helpful in designing new therapeutic strategies for rehabilitation in patients with denervation of muscle. Muscle Nerve, 2008

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

Abstract 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

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.

Muscle and Nerve Responses After Different Intervals of Electrical Stimulation Sessions on Denervated Rat Muscle

Lima SC, Caierão QM, Peviani SM, Russo TL, Somazz MC, Salvini TF, Teodori RM, Minamoto VB: Muscle and nerve responses after different intervals of electrical stimulation sessions on denervated rat muscle. Objective:Electrical stimulation is a procedure used to treat denervated muscles. The number of electrical stimulation sessions varies across muscle rehabilitation protocols, from daily to certain days throughout the week. The purpose of this study was to evaluate how muscle and nerve respond to different intervals of electrical stimulation applied to denervated muscle. Design:Denervation of rat gastrocnemius muscle was imposed via nerve crush, and electrical stimulation was applied to the muscle either daily (Monday through Friday) or on alternate days (Monday, Wednesday, and Friday). Four experimental groups were studied: denervated, denervated plus daily electrical stimulation, denervated plus alternate-day electrical stimulation, and control. Results:On the 12th day after nerve crush, levels of MyoD, myostatin, and atrogin-1 gene expression, as well as muscle fiber and nerve morphometry, were evaluated. Expression levels of all three genes were higher in the denervated group when compared with control. Also, expression levels of MyoD and myostatin were higher in denervated plus alternate-day electrical stimulation and denervated plus daily electrical stimulation groups when compared with denervated. The denervated plus daily electrical stimulation group had lower atrogin-1 expression, lower density of intramuscular connective tissue, and better morphometric nerve characteristics when compared with the denervated and denervated plus alternate-day electrical stimulation. Conclusions:These results indicate that the responses of both muscle and nerve to electrical stimulation after muscle denervation depend on the intervals of electrical stimulation application.

Effects of alternagin-C from Bothrops alternatus on gene expression and activity of metalloproteinases in regenerating skeletal muscle

This study evaluated the effects of alternagin-C (ALT-C) on mRNA levels of VEGF, MyoD and matrix metalloproteinase 2 (MMP-2) and on activity of MMPs in injured tibialis anterior (TA) muscle induced by cryolesioning in rats. Thirty-six Wistar rats (3 months old, 258.9+/-27 g) were divided into five groups: (1) control group; (2) injured TA and analyzed 3 days later; (3) injured TA treated with ALT-C and analyzed 3 days later; (4) injured TA and analyzed 7 days later and (5) injured TA treated with ALT-C and analyzed 7 days later. The injured muscle received 25 microl of ALT-C (50 nM). The injured and uninjured muscle areas were quantified by light microscopy. The MMP activity was evaluated through zymography, and mRNA of MyoD, VEGF and MMP-2 was assessed by quantitative polymerase chain reaction. ALT-C neither reduced the muscle injury area nor altered the pattern of MyoD and VEGF expression in injured muscles. However, ALT-C reduces both MMP-2 mRNA and gelatinolytic activity in injured muscles. The study indicates that ALT-C, at the tested concentrations, did not improve muscle regeneration process in rats. The effect on MMP-2 mRNA and gelatinolytic activity suggests that ALT-C changes the overall balance of ECM protein turnover during muscle regeneration.

Quadriceps Muscle Atrophy After Anterior Cruciate Ligament Transection Involves Increased mRNA Levels of Atrogin-1, Muscle Ring Finger 1, and Myostatin

ObjectiveThe 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. DesignWistar 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. ResultsElevated 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. ConclusionsQuadriceps atrophy after ACL transection involves increased levels of myostatin, atrogin-1, and muscle ring finger 1 mRNA and the accumulation of ubiquitinated protein.

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. Muscle Nerve 49: 120–128, 2014

Neuromuscular electrical stimulation induces beneficial adaptations in the extracellular matrix of quadriceps muscle after anterior cruciate ligament transection of rats.

ObjectiveThe aim of this study was to assess the effect of neuromuscular electrical stimulation (NMES) on the extracellular matrix remodeling of the quadriceps muscle after anterior cruciate ligament (ACL) transection in rats. The hypothesis of this study was that ACL transection would induce maladaptive modifications in the extracellular matrix through the increase in connective tissue (CT) accumulation and net degradation of type IV collagen of the quadriceps muscle. In addition, clinical-like NMES, applied to the quadriceps muscle immediately after the ACL transection, would reduce the accumulation of the CT content and net degradation of type IV collagen. DesignWistar male rats were randomized into five different groups: ACL (surgery and ACL transection), Sham (surgery without ACL transection), ACLES (surgery, ACL transection, and NMES), ShamES (surgery without ACL transection, but NMES), and Control (intact animals). The vastus medialis, rectus femoris, and vastus lateralis muscles of the quadriceps were harvested 1, 2, 3, 7, and 15 days after surgery. Matrix metalloproteinase–2 (MMP-2) (messenger RNA [mRNA] levels and activity), collagen IV (mRNA and protein levels), and CT density were assessed. ResultsThe ACL transection increased the CT content and MMP-2 mRNA levels and decreased collagen IV mRNA and protein levels. NMES minimized the CT density in all muscles and reduced the MMP-2 mRNA levels mainly in the vastus lateralis muscle at 7 days. Moreover, type IV collagen mRNA levels were increased in all muscles at 7 days, as was the protein level only at 15 days, in the NMES groups. ConclusionsThis study showed that ACL transection increases CT content and MMP-2 mRNA levels and induces rapid changes in basement membranes, causing net degradation of type IV collagen during the first 2 wks after ACL injury. Furthermore, clinical-like NMES minimized the accumulation of CT density, regulated the MMP-2 mRNA levels, and increased both type IV collagen mRNA and protein levels.