The functional and histological effects of clenbuterol on the canine skeletal muscle ventricle

BACKGROUND: We investigated the anabolic effects of the sympatho-mimetic drug clenbuterol upon pumping chambers constructed from latissimus dorsi muscle (LDM). METHODS AND RESULTS: In control and treatment groups (n = 4 dogs each), skeletal muscle ventricles (SMVs) were constructed followed by a 3-week recuperative delay and 6-7 weeks of electrical conditioning at 2 Hz to induce phenotypic expression of fatigue resistant slow muscle fibers. The treatment group received oral administration of clenbuterol (8 microg/kg, 2x/day) during this period. The clenbuterol group increased significantly in body weight as compared with the control group (P < 0.05). In a terminal experiment, the SMVs were assessed with a mock circulation device to determine pumping performance and also were examined with regard to fiber type distribution and area in the SMVs and their contralateral in situ LDMs. Initially the clenbuterol group performed better than the control group, but by the end of a 60-min fatigue test, there were no significant differences. With regard to fiber type distribution and areas, the SMVs of the clenbuterol group exhibited a fast fiber distribution similar to unconditioned muscles (28% +/- 4%), whereas the control group showed complete transformation (100%) to slow fibers. The fast fibers of the clenbuterol group were larger than control (P < 0.05), but the slow fibers were not significantly different. CONCLUSIONS: At the dose given, clenbuterol does induce hypertrophy and preserves the normal percentages of fiber types, possibly by hyperplasia, but it does not affect chronic pumping performance of skeletal muscle ventricles in the canine model.     

Conversion of the rabbit gracilis muscle for transposition as a neoanal sphincter by electrical stimulation

To re-establish anal function in fecally incontinent patients it may be feasible to transpose the gracilis muscle around the anal canal, using electrical stimulation to trigger contraction. However, because the fast-twitching gracilis muscle is incapable of prolonged contraction without fatigue, it is necessary to convert it to a slow-twitching, fatigue-resistant muscle. We demonstrated this conversion by long-term electrical stimulation at low frequencies using a rabbit model. The nerve to the gracilis muscle was continuously stimulated at 2 Hz, 5 Hz, and 10 Hz for 2, 4, or 6 weeks. In the 6-week conditioning group, the percentage of type I fibers, identified by ATPase staining, increased as the conditioning frequency became higher, but the twitch contraction speed reduced with conditioning at a frequency of more than 5 Hz. The fatigue resistance improved by conditioning at 10 Hz, and conversion occurred in 6 weeks. Thus, we concluded that conditioning at 10 Hz for 6 weeks can convert rabbit gracilis muscle to a slow-twitching, fatigue-resistant muscle suitable for use as a neoanal sphincter.     

Striated muscle fiber compositions of human male urethral rhabdosphincter and levator ani

PURPOSE: We clarified the contractile properties of human male periurethral striated muscle fibers to better understand how the rhabdosphincter and the levator ani maintain urinary continence. MATERIALS AND METHODS: Muscle specimens were obtained from 52 male patients who underwent radical prostatectomy or radical cystectomy. The specimens were frozen in liquid nitrogen. Frozen sections (10 microm thick) were stained with myofibrillar ATPase at different pH values (pH 4.2, 4.6 and 10.6), and evaluated for quantitative parameters and fiber type distribution. Myosin heavy chain analysis was performed using SDS-PAGE. RESULTS: Of all 52 cases 37 provided specimens that could be divided into the 2 major fiber types, type 1 (slow twitch) and type 2 (fast twitch). Although type 1 muscle fibers were predominant in RS and LA muscle groups (RS 69.6 +/- 2.7%, LA 67.0 +/- 2.0%), mean muscle fiber size was significantly smaller in RS (mean area 906 +/- 86 microm(2)) than in LA (mean area 2,967 +/- 170 microm(2)) (p <0.0001). In 11 specimens type 2 muscle fibers could be subdivided into types 2A (fast fatigue resistant) and 2B (fast fatigable). Type 2A fibers were significantly more prevalent than type 2B fibers (p <0.05). Likewise, MHC analysis of these 11 specimens found a significantly higher percentage of fiber type 2A expression products (MHC 2A) than of fiber type 2B expression products (MHC 2X) (p <0.05). CONCLUSIONS: RS and LA contribute to urinary continence mechanism by slow contraction. Moreover, the smaller mean size of muscle fibers in RS suggests more fatigue resistance compared with muscle fibers in LA because small fibers have a shorter diffusion distance for metabolic substrates. These results should help contribute to a more detailed understanding of the function of periurethral striated muscles in the human male.     

Cardiomyoplasty: the benefits of electrical prestimulation of the latissimus dorsi muscle in situ.

BACKGROUND: Ischemic damage in the latissimus dorsi muscle may limit the success of cardiomyoplasty. Electrical prestimulation of the muscle in situ is known to enhance thoracodorsal perfusion to the distal latissimus dorsi muscle immediately after grafting. In this study we asked whether prestimulation was also beneficial under typical postoperative conditions. METHODS: Ten sheep were randomly assigned to two equal groups. In one group the latissimus dorsi muscle was stimulated continuously in situ at 2 Hz for 2 weeks; in the other group the muscle was not stimulated. Regional blood flows in the muscle were determined sequentially (1) under baseline conditions, (2) immediately after surgical mobilization, handling, and reattachment at 80% of the resting length, and (3) after 5 days. RESULTS: Manipulation of the unstimulated muscle resulted in an acute global reduction in blood flow with no improvement after 5 days. The distal region was most severely affected (26.2%+/-4.2% of baseline blood flow). Electrical prestimulation significantly reduced regional blood flow under baseline conditions but rendered the whole muscle more resistant to the surgical manipulations; blood flow was significantly better-preserved immediately afterwards, and there was complete recovery to baseline levels after 5 days. CONCLUSIONS: Electrical prestimulation of the latissimus dorsi muscle in situ reduces the acute distal ischemia caused by surgical manipulations, and promotes subsequent recovery of blood flow to baseline levels after a few days. Use of a prestimulated graft may therefore improve the outcome of skeletal muscle cardiac assistance.     

A Technique for Sequential Segmental Neuromuscular Stimulation with Closed Loop Feedback Control

In dynamic myoplasty, dysfunctional muscle is assisted or replaced with skeletal muscle from a donor site. Electrical stimulation is commonly used to train and animate the skeletal muscle to perform its new task. Due to simultaneous tetanic contractions of the entire myoplasty, muscles are deprived of perfusion and fatigue rapidly, causing long-term problems such as excessive scarring and muscle ischemia. Sequential stimulation contracts part of the muscle while other parts rest, thus significantly improving blood perfusion. However, the muscle still fatigues. In this article, we report a test of the feasibility of using closed-loop control to economize the contractions of the sequentially stimulated myoplasty. A simple stimulation algorithm was developed and tested on a sequentially stimulated neo-sphincter designed from a canine gracilis muscle. Pressure generated in the lumen of the myoplasty neo-sphincter was used as feedback to regulate the stimulation signal via three control parameters, thereby optimizing the performance of the myoplasty. Additionally, we investigated and compared the efficiency of amplitude and frequency modulation techniques. Closed-loop feedback enabled us to maintain target pressures within 10% deviation using amplitude modulation and optimized control parameters (correction frequency = 4 Hz, correction threshold = 4%, and transition time = 0.3 s). The large-scale stimulation/feedback setup was unfit for chronic experimentation, but can be used as a blueprint for a small-scale version to unveil the theoretical benefits of closed-loop control in chronic experimentation.     

A technique for sequential segmental neuromuscular stimulation with closed loop feedback control.

In dynamic myoplasty, dysfunctional muscle is assisted or replaced with skeletal muscle from a donor site. Electrical stimulation is commonly used to train and animate the skeletal muscle to perform its new task. Due to simultaneous tetanic contractions of the entire myoplasty, muscles are deprived of perfusion and fatigue rapidly, causing long-term problems such as excessive scarring and muscle ischemia. Sequential stimulation contracts part of the muscle while other parts rest, thus significantly improving blood perfusion. However, the muscle still fatigues. In this article, we report a test of the feasibility of using closed-loop control to economize the contractions of the sequentially stimulated myoplasty. A simple stimulation algorithm was developed and tested on a sequentially stimulated neo-sphincter designed from a canine gracilis muscle. Pressure generated in the lumen of the myoplasty neo-sphincter was used as feedback to regulate the stimulation signal via three control parameters, thereby optimizing the performance of the myoplasty. Additionally, we investigated and compared the efficiency of amplitude and frequency modulation techniques. Closed-loop feedback enabled us to maintain target pressures within 10% deviation using amplitude modulation and optimized control parameters (correction frequency = 4 Hz, correction threshold = 4%, and transition time = 0.3 s). The large-scale stimulation/feedback setup was unfit for chronic experimentation, but can be used as a blueprint for a small-scale version to unveil the theoretical benefits of closed-loop control in chronic experimentation.     

Comparison of the histochemical and contractile properties of human gastrocnemius muscle

Contractile and histochemical properties of the human gastrocnemius muscle were compared in 21 male athletes ranging from 20 to 29 years of age. Surface electrical stimulation was used to determine muscle twitch parameters. The contractile variables of the muscle twitch were: latency (L), time to peak force (TPF1 and TPF2), peak force (Pf1 and Pf2), half-contraction time (1/2 CT), and half-relaxation time (1/2 RT). Muscle samples from the belly of the medial gastrocnemius muscle were obtained using the needle biopsy technique. The samples were treated histochemically for myosin ATPase in order to classify the fibers as slow twitch (ST) or fast twitch (FT) and to determine fiber areas. Multiple correlations were performed between the contractile and histochemical variables. The results demonstrated significant (p < 0.05) positive relationships between the calculated twitch recordings and percentage of FT fibers (R = 0.85), percentage of ST fibers (R = 0.85), relative percent area of FT fibers (R = 0.84), and relative percent area of ST fibers (R = 0.84). The results suggest that fiber type percentage and relative percent area determined using myosin ATPase are related to electrically stimulated isometric contractile properties. J Orthop Sports Phys Ther 1991;13(6):322-328.     

Skeletal muscle fiber composition, nutritional status and subjective fatigue during surgical convalescence

Samples from the lateral vastus femoris muscle were obtained by needle biopsy from 12 patients before and 20 days after elective abdominal surgery. The content of fast and slow twitch muscle fibers and their lesser diameter were determined and related to subjective degree of fatigue and changes in nutritional parameters. Fatigue increased from a mean preoperative level of 2.2 +/- 0.4 arbitrary units to 4.3 +/- 0.5 (p less than 0.001) on postoperative day 20. Body weight, triceps skin fold and slow twitch fiber diameter decreased postoperatively (p less than 0.05). There was no significant postoperative change in the distribution of muscle fiber types. Fatigue increase correlated to weight loss and reduction of triceps skin fold (r = 0.50 and 0.54, p = 0.08 and 0.06, respectively). Postoperative changes in slow and fast twitch fiber diameter correlated to loss of body weight (r = 0.60 and 0.74, p less than 0.05 and less than 0.01, respectively), but correlation was not found between postoperative fatigue and changes in fiber-type distribution (r = 0.34 and 0.29).     

Morphometric analysis of rabbit skeletal muscle and peripheral nerve tissue after electrical stimulation

Transcutaneous medium frequency electrical stimulation is used extensively to improve muscle strength in people who encounter difficulty in improving strength voluntarily. The purpose of this study was to describe some morphometric effects of electrical stimulation applied to rabbit skeletal muscle and peripheral nerve tissue (N = 5 control and 7 experimental rabbits). Intermittent electrical current (4000 Hz pulse modulated at 50 Hz) was applied transcutaneously to adult female rabbit thigh muscle 3 times/week for 3 months. Muscle ATPase histochemical staining, followed by morphometric analysis, demonstrated that type IIB fibers in stimulated muscles (sartorius and vastus lateralis) had larger cross-sectional areas in comparison to nonstimulated muscle fibers. Type IIA fibers of the stimulated sartorius muscle also were hypertrophied in comparison to nonstimulated muscle fibers. The percentage distribution of muscle fiber types did not change significantly as a result of stimulation. The femoral nerves of these rabbits were fixed and stained. Morphometric analysis did not detect any significant change in myelin cross-sectional area or thickness. Also, nerve axoplasmic cross-sectional area in stimulated femoral nerves was not significantly different from controls. These data suggest that electrical stimulation can increase the size of skeletal muscle fibers if applied consistently for an extended period of time. Further research is needed to further characterize this effect and to determine whether the same effect can be observed in humans after prolonged stimulation. J Orthop Sports Phys Ther 1990;11(7):313-320.     

Effects of Muscle Length, Frequency of Stimulation, and Fatigue on the Isometric Tension in Canine Latissimus Dorsi

Abstract: An experimental protocol was designed to study the mechanical response of the canine latissimus dorsi muscle stimulated to contract isometrically. Active and passive tensions were measured with the muscle's initial length varying within 10% of its physiologic length in situ. The force-frequency relationship was obtained at frequencies of stimulation of 1, 10, 15, 30, and 60 Hz. Muscle fatigability was assessed during 3 min of successive contractions. Tests were performed in pedicled muscles of anesthetized mongrel dogs (n = 10). Force-length characteristics were found to affect evoked tension markedly. Maximal active tension was generated near the muscle length in situ. Specific isometric tension measured with unit pulse stimulation was 2 ± 0.4 N/cm², and the time to peak twitch was 92.8 ± 2.67 ms. With 60 Hz of stimulation frequency, the tension was 9.31 ± 0.32 N/cm², and the time to peak tension was 216.05 ± 16.28 ms. After the fatigue test, the tension generated decreased to 62.5% of its initial value, and this decline was paralleled by the rate of tension development and tension relaxation.     

Isoform-dependent Effects of Halothane in Human Skinned Striated Fibers

Background: Reports of the effects of halothane on isoform contractile proteins of striated muscles are conflicting. To determine whether halothane affects cardiac and skeletal contractile proteins differently, the authors examined the effects of two doses of halothane (0.44 and 1.26 mM, equivalent to 0.75 and 2.25 vol%, respectively) on the Calcium sup ++ sensitivity and maximal force in human skinned cardiac, type I (slow twitch), and type II (fast twitch) skeletal muscle fibers.

Methods: Left ventricular muscle strips and skeletal muscle biopsy specimens were obtained from eight and ten patients undergoing cardiac and orthopedic surgery, respectively. Sarcolemma and sarcoplasmic reticulum were destroyed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus Brij 58. Calcium sup ++ sensitivity was studied by observing the isometric tension developed by skinned fibers challenged with increasing concentrations of Calcium sup ++. Muscle fiber type was determined in each skeletal fiber by the difference in strontium-induced tension measurements.

Results: Halothane shifted the Calcium sup ++ tension curves toward higher Calcium sup ++ concentrations and increased the Calcium sup ++ concentration for half-maximal activation in both cardiac and type I skeletal muscle fibers (from 1.96 micro Meter and 1.06 micro Meter under control conditions to 2.92 micro Meter and 1.71 micro Meter in presence of 0.75 vol% halothane, respectively) without changing the slope of this relationship (Hill coefficient). In contrast, no significant effect was observed in type II fibers. Halothane also decreased the maximal activated tension in the three groups of fibers with a lesser effect in type II fibers.     

Fatigue rate of the external anal sphincter

Aim Studies of skeletal muscle show that fatigue rate corresponds to the proportion of fast‐twitch and slow‐twitch fibres that are present in the muscle. Limited work has been done on the fatigue rate of the external anal sphincter. We have prospectively studied fatigability of the external anal sphincter in women with faecal incontinence and women with normal bowel control. Method Anorectal manometry was measured by a station‐pull technique using a water‐filled microballoon. Fatigue rate was calculated from anal pressure measurements taken every 0.1 s over a 20‐s squeeze. Results Women with faecal incontinence (n = 88, median ?12 cmH₂O/min) were less susceptible to fatigue than women with normal bowel control (n = 36, median ?43 cmH₂O/min) (P < 0.01). The external anal sphincter was less susceptible to fatigue with increasing age (P < 0.01, r = 0.499). In women with normal bowel control and in women with faecal incontinence fatigue rate was negatively correlated with maximum squeeze pressure (P < 0.01, r = ?0.287; P < 0.01, r = ?0.579). Conclusion The external anal sphincter was less susceptible to fatigue with increasing age. Women with faecal incontinence have a weaker but more fatigue‐resistant external anal sphincter. This might correspond to a higher proportion of slow‐twitch muscle fibres. Histological studies are needed to examine this hypothesis.     

Transforming skeletal muscle for myocardial assist: a feasibility study

Canine experiments were undertaken to determine the feasibility of transforming skeletal muscle for myocardial assistance. Initially, a rectus-muscle pedicle flap was used to power a ventricular assist conduit. A specially designed "pulse-train" stimulator produced skeletal muscle contractions capable of augmenting myocardial function. Statistically significant increases in either systolic or diastolic pressures were achieved by appropriate synchronization and signal delays of the stimulator. Then, the left rectus muscle was conditioned by stimulating it at low frequencies (2 to 10 Hz) for 6 to 12 weeks. The degree of transformation from type II (fast) to type I (slow) fibres was examined and the resultant tolerance to fatigue studied. Preliminary data show that a greater proportion of type I fibres is associated with increased resistance to fatigue. Such transformed skeletal muscle should therefore be more suited to power the cardiac assist devices.     

Skeletal muscle ventricle used for right ventricle assistance

There are a number of advantages in using an electrically stimulated autogenous skeletal muscle to construct an auxiliary ventricle to assist a heart. The purpose of this study was to determine the feasibility of biological right ventricular assistance using long-term electrically stimulated skeletal muscle grafts. In fourteen dogs, the latissimus dorsi muscles and the right thoracodorsal nerves were exposed and unipolar pulse generator was implanted. The initial rate of 70 cycle/min. was increased to a rate of 100 cycle/min. Six or 12 months later, the latissimus dorsi was wrapped around a latex pouch equipped with inflow and outflow valved conduit (skeletal muscle ventricles; SMVs). The SMVs were connected to main pulmonary artery and right atrium. These SMVs were stimulated 20 Hz for 200 msec at a fixed rate of 90 cycle/min, the hemodynamic changes with or without skeletal muscle ventricular assistance (SMVA) were measured. In as animals the circulation failed after total right ventricular bypass without SMVA. But the SMVA increased aortic blood pressure, aortic blood flow, left atrial pressure and peak pulmonary pressure significantly. There was a linear correlation between central venous pressure and skeletal muscle ventricular assist flow. Histologic studies showed the conditioned muscles had a greater percentage of slow-twitch, fatigue resistant fibers on ATPase stain. These results suggested the long-term electrical conditioning skeletal muscle could be possible to use SMVs in humans to provide support in children with some types of congenital heart disease.     

Skeletal muscle ventricles: improved performance at physiologic preloads

We attempted to construct skeletal muscle ventricles (SMVs) so that they would develop optimal stroke work at physiologic preloads of 5 to 15 mm Hg. Thirty-one SMVs were constructed in mongrel dogs. The effects of electrical preconditioning with 2 Hz continuous and 25 Hz burst patterns were evaluated, as well as the application of passive stretch to the muscle fibers during preconditioning. We evaluated the stroke work developed by these SMVs at afterloads of 30 mm Hg and 80 mm Hg in vivo, using a mock circulation device. During mock circulation studies, the SMVs were stimulated via the thoracodorsal nerve with either a 25 Hz or 85 Hz burst pattern. SMVs with 2 Hz preconditioning developed significantly higher stroke work than SMVs with 25 Hz preconditioning under all conditions of afterload, preload, and stimulation frequency (p less than or equal to 0.001). Under these conditions, for the 2 Hz preconditioned SMVs, passive stretch during preconditioning resulted in a further significant increase in developed stroke work (p less than 0.05). For these SMVs, with an 85 Hz stimulation frequency, stroke work averaged 410% of canine RV stroke work, and 59% of canine LV stroke work at physiologic preloads and afterloads.     

Effects of Succinylcholine Chloride on the Response of Fast and Slow Muscle in the Cat

Fast and slow muscle fibres differ histochemically, electrophysiologically and pharmacologically. In vivo, the effect of 0.05, 0.075 and 0.1 mg X kg-1 succinylcholine on the response of the indirectly stimulated gastrocnemius (fast) and soleus muscle (slow) to single, train-of-four and tetanic stimuli was studied. There was a significant difference between the response of the two muscles. Maximum twitch suppression occurred 2 min after injection and was more significant in the gastrocnemius than the soleus. The duration of the neuromuscular blockade was 9 min or more in both groups. The train-of-four ratio dropped below 0.7 in the gastrocnemius preparation at all doses and in the soleus when 0.1 mg X kg-1 succinylcholine was used. Sustained tetanus was achieved in both muscles at 5 and 10 min, respectively. There was evidence of fatigue in the soleus muscle at the 5-min observation suggestive of the presence of a phase II block, a phenomenon not observed in the gastrocnemius.     

Isometric contractile function recovery following tourniquet ischemia

The purpose of this study was to document the recovery of isometric contractile function following tourniquet ischemia. Male Wistar rats (N = 27) were subjected to unilateral hindlimb tourniquet ischemia of 0 hr (control, N = 6), 1 hr (N = 5), 2 hr (N = 5), 3 hr (N = 5) and 4 hr (N = 3). Following a 2-week recovery period, isometric force measurements were made from both gastrocnemii of each rat with the contralateral limb acting as the control side. Each muscle was analyzed for maximal twitch (Pt, N/g), maximal rate of rise of twitch tension (DP/dt, N/sec), time to peak tension (TPT, msec), half relaxation time (RT 1/2, msec), maximal tetanus (P0, N/g, at 100 Hz), and fatigue (Burke Fatigue Protocol). Pt, P0, and DP/dt were significantly different from control values (P less than 0.05) for all hours of tourniquet ischemia. A strong negative correlation (P less than 0.001) was found for twitch (R = -0.84), tetanus (R = -0.78), and maximal rate of force development (R = -0.83) with respect to increasing hours of ischemia. The recovery of isometric twitch and tetanic function following tourniquet ischemia is inversely related to the ischemic interval. This study quantified the relationship between muscle ischemia and recovery of function following a 2-week interval and stresses the functional physiological changes which occur in skeletal muscle following tourniquet ischemia.     

The chronically stimulated muscle as an energy source for artificial organs. Preliminary results of a basic study in sheep

In 5 adult sheep the psoas muscle of one side was electrically stimulated through the muscle nerves with an implantable stimulation unit for more than 5 weeks. In the final experiments the isometric tetanic tension of the stimulated muscles was reduced to 50-70% of the contralateral normal muscle. The use of only 15 Hz as a stimulation frequency led to a transformation of the originally fast muscle into a slow muscle with more resistance to fatigue. Future application of the chronically stimulated psoas muscle for driving artificial organs is discussed.     

A ventricular assist device powered by conditioned skeletal muscle.

BACKGROUND: We are developing and testing a new ventricular assist device (VAD) to be powered by conditioned skeletal muscle. METHODS: To evaluate the VAD hardware and to develop a muscle training regimen, 8 calves have been used in studies in which the right latissimus dorsi muscle was employed. The experiments were carried out to an approximately 4-month duration. RESULTS: There was significant conversion of type II (fast twitch) to type I (slow twitch) muscle fibers. This did not correlate well, however, with device performance. The device stroke volumes ranged from approximately 17 to 90 cc. This variability of outcome occurred despite the fact that identical hardware, surgical procedures, and training regimens were employed. CONCLUSIONS: The results from the first eight studies lead us to speculate that perfusion may be important even when the muscle is working at pressures much lower than systemic blood pressure levels. In an attempt to augment tissue perfusion, we plan to investigate thermally induced angiogenesis as a possible mechanism for increasing blood flow to the tissue.     

Histochemical study of human external urethral sphincter]

Human external urethral sphincters (n = 13) were studied histochemically, using ATP-ase staining. The proportions of constituent muscle fiber types (slow twitch type 1 fibers: 35.6-97.7%, mean 65.7%, SD 16.6) were different among the individuals. There were no significant relationships between the percentage of type 1 fibers and the sex or the age. As for the mean muscle fiber diameter of 13 cases, the sizes of type 2 fibers (19.2-42.4 um, mean 27.8 um) were significantly (p less than 0.05, t-test) larger than those of type 1 fibers (15.7-30.3 um, mean 22.3 um). In analyses of individual cases, 10 male cases had significantly larger type 2 fibers than type 1. Two of 3 female cases had no significant difference in diameter between the two fiber types and the remaining 1 female case, in contrast with male cases, had significantly larger type 1 fibers than type 2. There was no significant relationship between the proportions and the diameters of constituent muscle fiber types. Our study thus showed extreme interindividual variation and implicated the presence of sexual difference in human external urethral sphincter muscle.