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Salbutamol and the Conditioning of Latissimus Dorsi for Cardiomyoplasty
Journal of Surgical Research 81, 209 –215 (1999) Article ID jsre.1998.5507, available online at http://www.idealibrary.com on
Salbutamol and the Conditioning of Latissimus Dorsi for Cardiomyoplasty 1 L. D. Wright, M.D.,* K-M. Zhang, M.D., Ph.D.,† L. C. McClain, M.D.,* P. W. E. Hsia, Ph.D.,* F. N. Briggs, Ph.D.,† ,2 and J. A. Spratt, M.D.* *Department of Surgery and †Department of Physiology, Medical College of Virginia Branch of Virginia Commonwealth University, Richmond, Virginia 23298-0551 Submitted for publication May 26, 1998
Background. Cardiomyoplasty is a new surgical alternative therapy for CHF. Although conditioning of muscle for cardiomyoplasty has a positive effect on fatigue resistance it also produces negative effects. In this study we assessed the effect of salbutamol, a b 2agonist, on both the positive and the negative effects of conditioning. Methods. In a control group of six animals one latissimus dorsi was subject to chronic, 1 Hz, lowfrequency stimulation (CLFS) while the other served as a control. The experimental group of seven dogs received a continuous SC infusion of salbutamol and one latissimus dorsi was subjected to CLFS. The other muscle demonstrated the effects of salbutamol per se. After 42 days the animals were anesthetized and fatigue resistance, muscle mass, and mechanical properties of the muscles were evaluated. Results. Salbutamol increased muscle mass, tetanic tension, and rate of rise and fall of tetanic tension. It diminished fatigue resistance and had no effect on shortening velocity. Chronic stimulation decreased muscle mass, tetanic tension, rate of rise and fall of tetanic tension, and muscle shortening velocity in both groups of dogs. Salbutamol diminished the declines in muscle mass, rate of tension development, and rate of muscle shortening due to CLFS, but did not change the effects of CLFS on tetanic tension and the rate of fall of tetanic tension. Salbutamol did not alter the increase in fatigue resistance induced by CLFS. Conclusions. The favorable effect of CLFS on fatigue resistance was unaffected by salbutamol. The unfavorable effects of CLFS on loss of muscle mass, rate of 1
This research was supported in part by NIH Grant HL45957 to F.N.B., a MCV Foundation grant to J.A.S., and a NIH Minorities Fellowship to L.D.W. 2 To whom correspondence should be addressed at P.O. Box 1337, White Stone, VA 22578.
tension development, and decline in shortening velocity were partially blocked by salbutamol, improving the ability of the latissimus dorsi to augment cardiac systole. © 1999 Academic Press Key Words: cardiomyoplasty; skeletal muscle transformation; salbutamol; b 2-agonist; force-velocity; muscle fatigue; muscle mechanics. INTRODUCTION
Dynamic cardiomyoplasty was introduced in 1985 as an experimental treatment for chronic heart failure refractory to medical treatment [1]. In dynamic cardiomyoplasty, the latissimus dorsi (LD) muscle is wrapped around the heart and electrically stimulated to augment cardiac performance. The LD, a predominantly fast-twitch skeletal muscle, is fatigue prone. It must therefore be transformed to a fatigue-resistant state before it can perform long-term cardiac augmentation. Chronic low-frequency electrical stimulation (CLFS) produces this desirable change by transforming the fast-twitch, fatigue-prone muscle into a slowtwitch, fatigue-resistant muscle [2]. Unfortunately, CLFS also produces undesirable changes. These changes include decreases in force generation and increases in half-relaxation time [3, 4] and decreases in shortening velocity [3]. Prevention of these undesirable effects while maintaining the desirable fatigue resistance produced by CLFS could improve cardiac augmentation by skeletal muscle. Androgenic steroids have been shown to prevent the loss of force generation that accompanies CLFS [5]. They also increase the rate of conversion of fast-twitch fibers to slow-twitch fibers and allow fatigue resistance to develop. This change in fiber type leaves unsolved the decrease in shortening velocity and the increase in
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0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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half-relaxation time that accompanies fiber transformation. Chronic administration of b 2-agonists produces skeletal muscle hypertrophy [6, 7] and increases muscle strength [7, 8]. These effects have the potential to modify the decreases in force generation produced by CLFS. The b 2-agonist, clenbuterol, has been shown to decrease half-relaxation time in slow-twitch muscles [7], suggesting that it promotes the fast-twitch phenotype. Salbutamol, also a b 2-agonist, has effects similar to those of clenbuterol. Salbutamol has been shown to promote the fast-twitch phenotype. When administered to dogs, salbutamol decreases MHC-I (slowtwitch myosin heavy chain I isoform) and increases MHC-IIx (fast-twitch myosin heavy chain IIx isoform) and decreases the expression of MHC-IIa (fast-twitch myosin heavy chain IIa isoform) levels in muscle fibers of the canine LD [9]. The expression of sarcoplasmic reticulum protein isoforms also change toward that of fast-twitch fibers. The levels of phospholamban and SERCA2b (slow-twitch isoforms) decrease and the level of SERCA1 (fast-twitch isoform) increases [9]. In contrast to the androgenic steroids, Hu et al. [10] found that salbutamol did not promote the slow-twitch phenotype in muscles subjected to CLFS. It partially blocked the switch to the slow-twitch phenotype. The purpose of the present study was to discover if the undesirable loss of muscle mass, force generation, shortening velocity, and increase in half-relaxation time produced by CLFS could be reduced by salbutamol while allowing fatigue resistance to develop. MATERIALS AND METHODS Experimental preparation. All studies were conducted in accordance with the guidelines of the National Research Council and all protocols were approved by the Institutional Animal Care and Use Committee. A total of 13 adult male mongrels dogs were used, 6 in the control group and 7 in the experimental group. All animals underwent implantation of a Teletronix (Denver, CO). Myostimulator 2772 as described by Wright et al. [3]. The electrodes were threaded through the left LD to excite the thoracodorsal nerve. After a 1-week recovery period the myostimulator was set to deliver single 10-V pulses of 25-ms duration at a frequency of 1 Hz. The period of CLFS was 42 days. At the beginning of the stimulation period Alzet osmotic pumps from Alzo Corp. (Palo Alto, CA) were implanted into 7 of the animals. The osmotic pumps, designed to operate for 4 weeks, were set to deliver Salbutamol hemisulfate (Sigma Chemical CO., St. Louis, MO) at a rate of 1.43 mg/kg body weight/day. After 4 weeks these pumps were replaced by pumps that would deliver the drug for 2 additional weeks. Data collection. After completion of the stimulation period the animals underwent a terminal data collection study. They were premedicated with acepromazine maleate (0.55–1.10 mg/kg im) and anesthetized with sodium pentobarbital (30 –35 mg/kg iv). Supplemental doses of pentobarbital (5– 8 mg/kg iv) were given as needed. Both LD were dissected free from their origins over the iliac crest and lumbar spine but were left attached at the humeral insertions. All perforating vessels were left intact to preserve blood flow. The mobilized end of each muscle was securely sutured to a metal yoke. The myostimulator leads were dissected free from surrounding ad-
hesions and disconnected from the myostimulator. The leads were then attached to a Grass Model SD9 stimulator (Grass instrument Inc., Quincy, MA) and tested for function. For measurement of isometric contractions the yoke was connected to an isometric force transducer (Omega LCL-040, Omega Group, Stamford, CN) mounted on a metal rod oriented parallel to the long axis of the muscle. The transducer could be secured at different lengths along the rod to stretch the muscle and set the preload. The voltage required to produce maximum twitch tension was determined. The position of the force transducer was adjusted to set the muscle length for maximum twitch tension. Muscle length was recorded and later used to calculate muscle cross-sectional area and muscle shortening velocity. The preload varied from 800 –1000 g. The stimulus pulse width was set at 0.2 ms. Isometric twitches were generated with a stimulus voltage 110% of that required to produce a maximal twitch. Fusion frequency was determined by varying the rate of repetitive stimuli. The lowest stimulation frequency that generated a fused tetanus was termed the fusion frequency. A frequency of 10 Hz above fusion frequency was used to record tetanic tension. To perform isotonic contractions the yoke was detached from the force transducer and connected to a light chain used to measure length displacement. The chain was draped over a 10-turn potentiometer and connected to a pan used to hold weights which served as afterloads. Muscle shortening caused a rotation of the potentiometer. A series of afterloads ranging from 5 to 80% of tetanic tension was then applied to the muscle. An average of six afterloads was used for each study. Fatigue resistance was tested by stimulating the thoracodorsal nerve for 20 min with a burst stimulator set to operate at 0.71 Hz. The burst was a pulse train of 600-ms duration followed by an off period of 800 ms. Within the pulse train there were twenty 10-V square-wave pulses of 20-ms duration. The conditioned (CLFS) muscle was studied first, followed by the noncondtioned muscle. Muscle temperature was measured and kept close to 37°C by application of warm blankets when necessary. Upon completion of data collection the animals were sacrificed by an overdose of sodium pentobarbital and both LD were excised, cleaned of connective tissue, and weighed. A sample of muscle was quickly frozen in liquid nitrogen for future determination of muscle fiber cross-sectional areas. Data analysis. All data were digitized and recorded on magnetic disk using commercially available software (CODAS, DATAQ Instrument Inc., Akron, OH). Custom-designed programs were used to calculate the mechanical parameters. Whole muscle cross-sectional area was calculated by dividing muscle mass by muscle length, divided by muscle density (muscle crosssectional area 5 [muscle mass/muscle length/muscle density]). A muscle density of 1 g/cm 3 was assumed. Muscle fiber cross-sectional areas were determined as described by Zhang et al. [9]. Crosssections were cut from muscle embedded in paraffin and stained with Masson trichrome. Cross-sectional areas were visualized microscopically and measured with the aid of the Bioquant System IV from R&M Biometrics Incorporated. For each muscle sample 20 muscle fibers were measured in each of five muscle fascicles. One hundred muscle fibers were measured in each muscle sample. The force-velocity data were fitted to the Hill equation by nonlinear regression using commercial software (SAS Institute, Inc., Cary, NC) to obtain values for a and b. The Hill equation is (P 1 a)(V 1 b) 5 b(P o 1 a), where P is the load on the muscle, P o is tetanic tension, V is velocity of shortening, and a and b are constants with units of force and velocity, respectively [11]. V max, the velocity of shortening at zero load, was calculated from the Hill equation, V max 5 bP o /a. The experimental dogs received salbutamol. The left LD of the control dogs and experimental dogs were subjected to CLFS for 42 days. The effects of salbutamol per se were tested on LD not subjected to CLFS. The significance of differences in means was tested by ANOVA and then with the Student-Newman-Keuls method
WRIGHT ET AL.: SALBUTAMOL AND CARDIOMYOPLASTY
FIG. 1. Effect of neuromuscular stimulation 6 salbutamol on muscle mass. Abbreviations used here and for subsequent figures: LD, untreated (control) latissimus dorsi; LD 1 S, latissimus dorsi from salbutamol-treated animals; LDE, chronic (6 weeks) neuromuscular stimulation of the latissimus dorsi; LDE 1 S, chronically stimulated latissimus dorsi from animals simultaneously receiving salbutamol. Ordinate, muscle weight expressed in grams per kilogram body weight. Columns indicate means, and error bars 11 SEM. Group means with different letters (a, b, c or d) are statistically significantly different (P , 0.05). There were 6 –7 muscles in each study.
(SigmaStat, Jandel Scientific, San Rafael, CA). Differences with a P , 0.05 were considered significant.
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FIG. 2. Effect of neuromuscular stimulation 6 salbutamol on muscle cross-sectional area. Ordinate, muscle cross-sectional area. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
cross-sectional area. CLFS produced a 36% decline. Salbutamol reduced this to a 14% decline in crosssectional area. Isometric force. Salbutamol increased tetanic isometric force (Fig. 4) by 28.6%, an increase comparable to that in muscle mass. The loss of tetanic tension due
RESULTS
All animals survived implantation and conditioning. At the time of study, muscle temperatures averaged 37°C and systemic temperatures averaged 39°C. Mass. Salbutamol increased the mass of the LD, Fig. 1, by 30%. The loss of muscle mass due to CLFS was 28% in the control dogs. This loss was prevented by salbutamol. The mass of the muscle subjected to CLFS plus salbutamol was not statistically different from that of the control dogs. Muscle cross-sectional area. The effects of CLFS, salbutamol, and the combination of CLFS plus salbutamol on muscle cross-sectional areas are shown in Fig. 2. As muscle length was not changed by these procedures the changes in muscle cross-sectional areas followed the changes in muscle mass. Salbutamol increased the muscle cross-sectional area of the LD, Fig. 2, by 27%. CLFS caused a significant decrease in muscle cross-sectional area in control dogs. This effect of CLFS was blocked by salbutamol treatment. Muscle fiber cross-sectional area. As shown in Fig. 3, salbutamol caused a 23% increase in muscle fiber
FIG. 3. Effect of neuromuscular stimulation 6 salbutamol on single muscle fiber cross-sectional area. Ordinate, the mean muscle fiber cross-sectional area in square micrometers. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
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FIG. 4. Effect of neuromuscular stimulation 6 salbutamol on tetanic tension. Ordinate, the maximal force (in newtons) generated during isometric contraction. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
to CLFS was 45.4%. This loss was reduced to 33% by salbutamol. Rate of rise and fall of tetanic tension. Salbutamol increased the rates of rise (1dT/dt) and fall (2dT/dt) of tetanic tension as shown in Figs. 5 and 6. CLFS decreased the rates of rise and fall of tetanic tensions by 68 and 57%, respectively. The decrease in the rate of rise of tetanic tension was partially blocked by salbutamol, Fig. 5, but did not affect the rate of fall of tetanic tension, Fig. 6. Force-velocity relationship. Salbutamol had no effect on V max, the rate of muscle shortening at zero load, Fig. 7. The 44% decrease in V max induced by CLFS was reduced to 25% by salbutamol. Fatigue resistance. Fatigue was evaluated from the loss of peak tension during repeated, 0.71 Hz, tetanic stimulation of the thoracodorsal nerve as described under Materials and Methods. The tetanic tensions achieved at the beginning of the fatigue tests were identical to those recorded during the first tests of tetanic tensions, Fig. 4. Relaxation tension was not compromised during the fatigue test. Salbutamol decreased fatigue resistance. This became apparent after 10 min of muscle stimulation, Fig. 8. Conversely CLFS increased fatigue resistance. Salbutamol had no effect on the increase in fatigue resistance induced by CLFS. DISCUSSION
Salbutamol increased the mass of the LD. This was in accord with reports that salbutamol produces mus-
FIG. 5. Effect of neuromuscular stimulation 6 salbutamol on the maximum rate of rise of tetanic tension (1dT/dt). Ordinate, the maximal rate of rise of tetanic tension expressed in newtons per millisecond. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
cle hypertrophy and increases muscle strength in rats [6] and humans [8]. Petrou [12] reported that clenbuterol, a b 2-agonist, increased rat LD mass by 20 to 29%, values similar to the 30% found in this study, Fig. 1. When skeletal muscle is transformed by CLFS a
FIG. 6. Effect of neuromuscular stimulation 6 salbutamol on the maximum rate of fall of tetanic tension (2dT/dt). Ordinate, the maximal rate of fall of tetanic tension in newtons per millisecond. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
WRIGHT ET AL.: SALBUTAMOL AND CARDIOMYOPLASTY
FIG. 7. Effect of neuromuscular stimulation 6 salbutamol on the maximum rate of muscle shortening (V max). Ordinate, the maximal rate of muscle shortening at zero load expressed in muscle length per millisecond. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
decrease in muscle mass occurs early during the process [3] and coincides with the reduction in fiber size associated with formation of slow-twitch fibers [13]. Salbutamol partially attenuated the loss of muscle mass induced by CLFS, Fig. 1. CLFS produced a 28% loss of muscle mass. Coadministration of salbutamol decreased this to a 6.4% loss. The data for changes in muscle cross-sectional areas, Fig. 2, were very similar. The changes in mean muscle fiber cross-sectional areas were also similar, Fig. 3, but not identical. CLFS produced a 36.1% decline in mean fiber cross-sectional area. Coadministration of salbutamol reduced this to a 14% decline. The data show that salbutamol partially antagonizes the change in muscle fiber size induced by CLFS. The change in mean muscle fiber size need not account exactly for changes in muscle cross-sectional area because fiber sizes are not normally distributed about mean fiber size [13]. The development of skeletal muscle tension is proportional to muscle cross-sectional area. The effects of salbutamol on tetanic tension can be explained by the increase it produced in cross-sectional area. Salbutamol increased muscle cross-sectional area by 26.1% and tetanic tension by 28.6%. CLFS decreased muscle cross-sectional area by 27% and tetanic tension by 45.4%. The loss of tetanic tension in excess of that expected from the loss of muscle cross-sectional area indicates that CLFS produces effects on muscle fibers beyond the loss of fiber cross-sectional area. One possibility is some loss of coupling of excitation to contraction. We [14] found that CLFS reduces the levels of the
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ryanodine and dihydropyridine receptors by 80 to 90%, a magnitude greater than expected on the basis of switching from fast-twitch to slow-twitch fibers. The excess loss of tetanic tension found in the present study suggests that this loss of components of the excitationcontraction coupling apparatus has significant physiological consequences. Salbutamol lowered the loss of cross-sectional area in the muscles subjected to CLFS from 27 to 7.6% and the loss of tetanic tension from 45.4 to 33.7%. This difference in the loss of tetanic tension between CLFS and CLFS 1 S was not, however, statistically significant. We believe that the difference in developed tension, 13 newtons between CLFS and CLFS 1 salbutamol, might achieve statistical significance if a greater number of animals were studied. The velocity of muscle shortening depends upon the ATPase activities of myosin isoforms [15] which vary with muscle fiber type. Sieck et al. [16] found that the relative actomyosin ATPase activities of rat muscle with type IIx, IIa, and I fibers were 100:71:57 respectively. Similar data for canine actomyosins do not exist. In the discussion which follows we assumed that canine actomyosin ATPases have similar relative velocities. Zhang et al. [13] observed the distribution of fiber types in the control canine latissimus dorsi to be 25%, type I; 35%, type IIa; and 40%, type IIx. Salbutamol [9] changed this distribution to 15%, type I; 25%, type IIa; and 60% type IIx fibers. Chronic stimulation produced muscles that have 90% type I and 10% type IIa fibers
FIG. 8. Effect of neuromuscular stimulation 6 salbutamol on the fatigue resistance. The loss of tension caused by 600 ms of tetanic stimulation repeated at 0.71 Hz was tested at 5-min intervals for a period of 20 min (for further details see Materials and Methods). Symbols: E, control; , CLFS; F, salbutamol; ■, CLFS 1 salbutamol. The tensions that are statistically different (P , 0.05) from the control dogs are identified by an asterisk. There were 6 –7 muscles in each study.
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[13]. The fiber-type distribution in muscles from salbutamol-treated dogs subjected to CLFS was 28%, type I; 60%, type IIa; and 12%, type IIx [10]. Chronic low-frequency stimulation increased the frequency of type I fibers from 25 to 90% and decreased V max as expected. Salbutamol decreased the level of type I fibers slightly and increased the frequency of type IIx fibers slightly but had no statistically significant affect on V max. When the LD was subjected to CLFS in dogs receiving salbutamol the frequency of type I fibers was similar to that of the untreated LD. The frequency of IIx fibers, 12%, in the LD muscle subjected to CLFS plus salbutamol was, however, much lower than the frequency in the control muscle, 40%, and yielded muscles with a V max less than in the control muscle but greater than in the CLFS muscle, as expected on the basis of fiber-type distribution. Both V max and 1dT/dt depend on the rate of contractile element shortening and should respond to changes in fiber type in the same direction. The magnitude of the changes are not, however, expected to be the same. Maximum 1dT/dt is measured under isometric conditions during a twitch and its value depends upon the tension which the muscle can develop as well as the velocity of sarcomere shortening. This tension is greatly reduced in muscle subjected to CLFS and is increased in muscle receiving salbutamol, Fig. 4, which had a large effect on 1dT/dt. V max was computed from velocities measured during isotonic muscle shortening. The after loads were adjusted to the maximum load, tetanic tension, and therefore are independent of muscle strength. Although the direction of changes was the same for 1dT/dt and V max, the magnitude of the changes was greater for 1dT/dt than for V max because of the dependence of 1dT/dt on muscle strength. Maintaining 1dT/dt and V max in muscle subjected to CLFS is advantageous for muscles used in cardiomyoplasty. It allows the muscle to develop tension sooner and to eject blood more rapidly and more completely. The rate of loss of tension, 2dT/dt, is dependent on calcium sequestration and the tension developed by the muscle, i.e., muscle strength. The changes observed in tetanic tension and 2dT/dt are similar, with the changes in 2dT/dt slightly greater. We believe that this magnification of changes is due to differences in the rate of calcium sequestration. The best-established system for calcium sequestration is the sarcoplasmic reticulum, although other sequestering systems may exist. Hu et al. [10] have reported that CLFS decreases the sarcoplasmic reticulum calcium-uptake rate from 22.2 mmol Ca 21/min/g muscle to 11.5 mmol Ca 21/min/g muscle. Zhang et al. [9] reported that salbutamol stimulated the uptake rate to 37.4 mmol Ca 21/min/g muscle, and Hu et al. [10] reported that concurrent CLFS and salbutamol treatment yielded a calcium-uptake rate of 19.5 mmol Ca 21/min/g muscle. Salbutamol increased
2dT/dt and CLFS decreased 2dT/dt as expected from the changes observed in calcium-uptake rates and muscle strength. The combined effects of CLFS and salbutamol reduced 2dT/dt more than calcium-uptake rate-indicating the importance of muscle strength as well as calcium-sequestering systems. The failure of salbutamol to prevent the decline in 2dT/dt induced by CLFS is disadvantageous to the use of skeletal muscle for cardiomyoplasty as it could reduce diastolic filling, a problem noted with cardiomyoplasty by Bellotti et al. [17]. Salbutamol decreased fatigue resistance as shown in previous studies with b 2-agonists [18, 19]. CLFS increased fatigue resistance and this increase was unaffected by salbutamol, Fig 8. The effect of CLFS and salbutamol on the distribution of fiber types may account for these findings. Muscle fatigue can be considered the result of an imbalance between energy supply and energy demand [20, 21]. Energy supply can be estimated by succinic dehydrogenase (SDH) activity and energy demand by myofibrillar ATPase activity. The ratio of supply to demand can be estimated by the relative levels of SDH and ATPase activity. Sieck et al. [16], in a study with rat diaphragm muscle, found relative (SDH) levels of 4.6, 7.5, and 6.6 for type IIx, IIa, and I fibers, respectively. Type IIx fibers have relatively low levels of SDH activity and would be fatigable since type IIx fibers also have high actomyosin ATPase activities [16]. The frequency of type IIx fibers would, thus, predict fatigability. In our study salbutamol increased the frequency of type IIx fibers from 35– 40% [9, 10, 13] to 58 – 60% [9, 10], thereby decreasing fatigue resistance. Stimulation decreased the level of type IIx fibers to 0% [13], thereby increasing fatigue resistance. Stimulation of muscles in dogs receiving salbutamol produces fibers with 8% type IIx fibers, not a sufficient percentage of fibers to measurably reduce fatigue resistance. CLFS produced both favorable and unfavorable changes in skeletal muscle intended for use in cardiomyoplasty. Most importantly salbutamol did not block the favorable development of fatigue resistance. Salbutamol partially blocked the unfavorable effects of CLFS on 1dT/dt and V max but failed to influence the effects of CLFS on 2dT/dt. These favorable effects on shortening velocity in the absence of effects on fatigue resistance suggest that b 2-agonists deserve consideration for use in cardiomyoplasty. Studies of longer duration are needed to determine if the effects observed with 6 weeks of administration will continue to be effective. REFERENCES 1.
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Salbutamol and the Conditioning of Latissimus Dorsi for Cardiomyoplasty 1 L. D. Wright, M.D.,* K-M. Zhang, M.D., Ph.D.,† L. C. McClain, M.D.,* P. W. E. Hsia, Ph.D.,* F. N. Briggs, Ph.D.,† ,2 and J. A. Spratt, M.D.* *Department of Surgery and †Department of Physiology, Medical College of Virginia Branch of Virginia Commonwealth University, Richmond, Virginia 23298-0551 Submitted for publication May 26, 1998
Background. Cardiomyoplasty is a new surgical alternative therapy for CHF. Although conditioning of muscle for cardiomyoplasty has a positive effect on fatigue resistance it also produces negative effects. In this study we assessed the effect of salbutamol, a b 2agonist, on both the positive and the negative effects of conditioning. Methods. In a control group of six animals one latissimus dorsi was subject to chronic, 1 Hz, lowfrequency stimulation (CLFS) while the other served as a control. The experimental group of seven dogs received a continuous SC infusion of salbutamol and one latissimus dorsi was subjected to CLFS. The other muscle demonstrated the effects of salbutamol per se. After 42 days the animals were anesthetized and fatigue resistance, muscle mass, and mechanical properties of the muscles were evaluated. Results. Salbutamol increased muscle mass, tetanic tension, and rate of rise and fall of tetanic tension. It diminished fatigue resistance and had no effect on shortening velocity. Chronic stimulation decreased muscle mass, tetanic tension, rate of rise and fall of tetanic tension, and muscle shortening velocity in both groups of dogs. Salbutamol diminished the declines in muscle mass, rate of tension development, and rate of muscle shortening due to CLFS, but did not change the effects of CLFS on tetanic tension and the rate of fall of tetanic tension. Salbutamol did not alter the increase in fatigue resistance induced by CLFS. Conclusions. The favorable effect of CLFS on fatigue resistance was unaffected by salbutamol. The unfavorable effects of CLFS on loss of muscle mass, rate of 1
This research was supported in part by NIH Grant HL45957 to F.N.B., a MCV Foundation grant to J.A.S., and a NIH Minorities Fellowship to L.D.W. 2 To whom correspondence should be addressed at P.O. Box 1337, White Stone, VA 22578.
tension development, and decline in shortening velocity were partially blocked by salbutamol, improving the ability of the latissimus dorsi to augment cardiac systole. © 1999 Academic Press Key Words: cardiomyoplasty; skeletal muscle transformation; salbutamol; b 2-agonist; force-velocity; muscle fatigue; muscle mechanics. INTRODUCTION
Dynamic cardiomyoplasty was introduced in 1985 as an experimental treatment for chronic heart failure refractory to medical treatment [1]. In dynamic cardiomyoplasty, the latissimus dorsi (LD) muscle is wrapped around the heart and electrically stimulated to augment cardiac performance. The LD, a predominantly fast-twitch skeletal muscle, is fatigue prone. It must therefore be transformed to a fatigue-resistant state before it can perform long-term cardiac augmentation. Chronic low-frequency electrical stimulation (CLFS) produces this desirable change by transforming the fast-twitch, fatigue-prone muscle into a slowtwitch, fatigue-resistant muscle [2]. Unfortunately, CLFS also produces undesirable changes. These changes include decreases in force generation and increases in half-relaxation time [3, 4] and decreases in shortening velocity [3]. Prevention of these undesirable effects while maintaining the desirable fatigue resistance produced by CLFS could improve cardiac augmentation by skeletal muscle. Androgenic steroids have been shown to prevent the loss of force generation that accompanies CLFS [5]. They also increase the rate of conversion of fast-twitch fibers to slow-twitch fibers and allow fatigue resistance to develop. This change in fiber type leaves unsolved the decrease in shortening velocity and the increase in
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half-relaxation time that accompanies fiber transformation. Chronic administration of b 2-agonists produces skeletal muscle hypertrophy [6, 7] and increases muscle strength [7, 8]. These effects have the potential to modify the decreases in force generation produced by CLFS. The b 2-agonist, clenbuterol, has been shown to decrease half-relaxation time in slow-twitch muscles [7], suggesting that it promotes the fast-twitch phenotype. Salbutamol, also a b 2-agonist, has effects similar to those of clenbuterol. Salbutamol has been shown to promote the fast-twitch phenotype. When administered to dogs, salbutamol decreases MHC-I (slowtwitch myosin heavy chain I isoform) and increases MHC-IIx (fast-twitch myosin heavy chain IIx isoform) and decreases the expression of MHC-IIa (fast-twitch myosin heavy chain IIa isoform) levels in muscle fibers of the canine LD [9]. The expression of sarcoplasmic reticulum protein isoforms also change toward that of fast-twitch fibers. The levels of phospholamban and SERCA2b (slow-twitch isoforms) decrease and the level of SERCA1 (fast-twitch isoform) increases [9]. In contrast to the androgenic steroids, Hu et al. [10] found that salbutamol did not promote the slow-twitch phenotype in muscles subjected to CLFS. It partially blocked the switch to the slow-twitch phenotype. The purpose of the present study was to discover if the undesirable loss of muscle mass, force generation, shortening velocity, and increase in half-relaxation time produced by CLFS could be reduced by salbutamol while allowing fatigue resistance to develop. MATERIALS AND METHODS Experimental preparation. All studies were conducted in accordance with the guidelines of the National Research Council and all protocols were approved by the Institutional Animal Care and Use Committee. A total of 13 adult male mongrels dogs were used, 6 in the control group and 7 in the experimental group. All animals underwent implantation of a Teletronix (Denver, CO). Myostimulator 2772 as described by Wright et al. [3]. The electrodes were threaded through the left LD to excite the thoracodorsal nerve. After a 1-week recovery period the myostimulator was set to deliver single 10-V pulses of 25-ms duration at a frequency of 1 Hz. The period of CLFS was 42 days. At the beginning of the stimulation period Alzet osmotic pumps from Alzo Corp. (Palo Alto, CA) were implanted into 7 of the animals. The osmotic pumps, designed to operate for 4 weeks, were set to deliver Salbutamol hemisulfate (Sigma Chemical CO., St. Louis, MO) at a rate of 1.43 mg/kg body weight/day. After 4 weeks these pumps were replaced by pumps that would deliver the drug for 2 additional weeks. Data collection. After completion of the stimulation period the animals underwent a terminal data collection study. They were premedicated with acepromazine maleate (0.55–1.10 mg/kg im) and anesthetized with sodium pentobarbital (30 –35 mg/kg iv). Supplemental doses of pentobarbital (5– 8 mg/kg iv) were given as needed. Both LD were dissected free from their origins over the iliac crest and lumbar spine but were left attached at the humeral insertions. All perforating vessels were left intact to preserve blood flow. The mobilized end of each muscle was securely sutured to a metal yoke. The myostimulator leads were dissected free from surrounding ad-
hesions and disconnected from the myostimulator. The leads were then attached to a Grass Model SD9 stimulator (Grass instrument Inc., Quincy, MA) and tested for function. For measurement of isometric contractions the yoke was connected to an isometric force transducer (Omega LCL-040, Omega Group, Stamford, CN) mounted on a metal rod oriented parallel to the long axis of the muscle. The transducer could be secured at different lengths along the rod to stretch the muscle and set the preload. The voltage required to produce maximum twitch tension was determined. The position of the force transducer was adjusted to set the muscle length for maximum twitch tension. Muscle length was recorded and later used to calculate muscle cross-sectional area and muscle shortening velocity. The preload varied from 800 –1000 g. The stimulus pulse width was set at 0.2 ms. Isometric twitches were generated with a stimulus voltage 110% of that required to produce a maximal twitch. Fusion frequency was determined by varying the rate of repetitive stimuli. The lowest stimulation frequency that generated a fused tetanus was termed the fusion frequency. A frequency of 10 Hz above fusion frequency was used to record tetanic tension. To perform isotonic contractions the yoke was detached from the force transducer and connected to a light chain used to measure length displacement. The chain was draped over a 10-turn potentiometer and connected to a pan used to hold weights which served as afterloads. Muscle shortening caused a rotation of the potentiometer. A series of afterloads ranging from 5 to 80% of tetanic tension was then applied to the muscle. An average of six afterloads was used for each study. Fatigue resistance was tested by stimulating the thoracodorsal nerve for 20 min with a burst stimulator set to operate at 0.71 Hz. The burst was a pulse train of 600-ms duration followed by an off period of 800 ms. Within the pulse train there were twenty 10-V square-wave pulses of 20-ms duration. The conditioned (CLFS) muscle was studied first, followed by the noncondtioned muscle. Muscle temperature was measured and kept close to 37°C by application of warm blankets when necessary. Upon completion of data collection the animals were sacrificed by an overdose of sodium pentobarbital and both LD were excised, cleaned of connective tissue, and weighed. A sample of muscle was quickly frozen in liquid nitrogen for future determination of muscle fiber cross-sectional areas. Data analysis. All data were digitized and recorded on magnetic disk using commercially available software (CODAS, DATAQ Instrument Inc., Akron, OH). Custom-designed programs were used to calculate the mechanical parameters. Whole muscle cross-sectional area was calculated by dividing muscle mass by muscle length, divided by muscle density (muscle crosssectional area 5 [muscle mass/muscle length/muscle density]). A muscle density of 1 g/cm 3 was assumed. Muscle fiber cross-sectional areas were determined as described by Zhang et al. [9]. Crosssections were cut from muscle embedded in paraffin and stained with Masson trichrome. Cross-sectional areas were visualized microscopically and measured with the aid of the Bioquant System IV from R&M Biometrics Incorporated. For each muscle sample 20 muscle fibers were measured in each of five muscle fascicles. One hundred muscle fibers were measured in each muscle sample. The force-velocity data were fitted to the Hill equation by nonlinear regression using commercial software (SAS Institute, Inc., Cary, NC) to obtain values for a and b. The Hill equation is (P 1 a)(V 1 b) 5 b(P o 1 a), where P is the load on the muscle, P o is tetanic tension, V is velocity of shortening, and a and b are constants with units of force and velocity, respectively [11]. V max, the velocity of shortening at zero load, was calculated from the Hill equation, V max 5 bP o /a. The experimental dogs received salbutamol. The left LD of the control dogs and experimental dogs were subjected to CLFS for 42 days. The effects of salbutamol per se were tested on LD not subjected to CLFS. The significance of differences in means was tested by ANOVA and then with the Student-Newman-Keuls method
WRIGHT ET AL.: SALBUTAMOL AND CARDIOMYOPLASTY
FIG. 1. Effect of neuromuscular stimulation 6 salbutamol on muscle mass. Abbreviations used here and for subsequent figures: LD, untreated (control) latissimus dorsi; LD 1 S, latissimus dorsi from salbutamol-treated animals; LDE, chronic (6 weeks) neuromuscular stimulation of the latissimus dorsi; LDE 1 S, chronically stimulated latissimus dorsi from animals simultaneously receiving salbutamol. Ordinate, muscle weight expressed in grams per kilogram body weight. Columns indicate means, and error bars 11 SEM. Group means with different letters (a, b, c or d) are statistically significantly different (P , 0.05). There were 6 –7 muscles in each study.
(SigmaStat, Jandel Scientific, San Rafael, CA). Differences with a P , 0.05 were considered significant.
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FIG. 2. Effect of neuromuscular stimulation 6 salbutamol on muscle cross-sectional area. Ordinate, muscle cross-sectional area. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
cross-sectional area. CLFS produced a 36% decline. Salbutamol reduced this to a 14% decline in crosssectional area. Isometric force. Salbutamol increased tetanic isometric force (Fig. 4) by 28.6%, an increase comparable to that in muscle mass. The loss of tetanic tension due
RESULTS
All animals survived implantation and conditioning. At the time of study, muscle temperatures averaged 37°C and systemic temperatures averaged 39°C. Mass. Salbutamol increased the mass of the LD, Fig. 1, by 30%. The loss of muscle mass due to CLFS was 28% in the control dogs. This loss was prevented by salbutamol. The mass of the muscle subjected to CLFS plus salbutamol was not statistically different from that of the control dogs. Muscle cross-sectional area. The effects of CLFS, salbutamol, and the combination of CLFS plus salbutamol on muscle cross-sectional areas are shown in Fig. 2. As muscle length was not changed by these procedures the changes in muscle cross-sectional areas followed the changes in muscle mass. Salbutamol increased the muscle cross-sectional area of the LD, Fig. 2, by 27%. CLFS caused a significant decrease in muscle cross-sectional area in control dogs. This effect of CLFS was blocked by salbutamol treatment. Muscle fiber cross-sectional area. As shown in Fig. 3, salbutamol caused a 23% increase in muscle fiber
FIG. 3. Effect of neuromuscular stimulation 6 salbutamol on single muscle fiber cross-sectional area. Ordinate, the mean muscle fiber cross-sectional area in square micrometers. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
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FIG. 4. Effect of neuromuscular stimulation 6 salbutamol on tetanic tension. Ordinate, the maximal force (in newtons) generated during isometric contraction. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
to CLFS was 45.4%. This loss was reduced to 33% by salbutamol. Rate of rise and fall of tetanic tension. Salbutamol increased the rates of rise (1dT/dt) and fall (2dT/dt) of tetanic tension as shown in Figs. 5 and 6. CLFS decreased the rates of rise and fall of tetanic tensions by 68 and 57%, respectively. The decrease in the rate of rise of tetanic tension was partially blocked by salbutamol, Fig. 5, but did not affect the rate of fall of tetanic tension, Fig. 6. Force-velocity relationship. Salbutamol had no effect on V max, the rate of muscle shortening at zero load, Fig. 7. The 44% decrease in V max induced by CLFS was reduced to 25% by salbutamol. Fatigue resistance. Fatigue was evaluated from the loss of peak tension during repeated, 0.71 Hz, tetanic stimulation of the thoracodorsal nerve as described under Materials and Methods. The tetanic tensions achieved at the beginning of the fatigue tests were identical to those recorded during the first tests of tetanic tensions, Fig. 4. Relaxation tension was not compromised during the fatigue test. Salbutamol decreased fatigue resistance. This became apparent after 10 min of muscle stimulation, Fig. 8. Conversely CLFS increased fatigue resistance. Salbutamol had no effect on the increase in fatigue resistance induced by CLFS. DISCUSSION
Salbutamol increased the mass of the LD. This was in accord with reports that salbutamol produces mus-
FIG. 5. Effect of neuromuscular stimulation 6 salbutamol on the maximum rate of rise of tetanic tension (1dT/dt). Ordinate, the maximal rate of rise of tetanic tension expressed in newtons per millisecond. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
cle hypertrophy and increases muscle strength in rats [6] and humans [8]. Petrou [12] reported that clenbuterol, a b 2-agonist, increased rat LD mass by 20 to 29%, values similar to the 30% found in this study, Fig. 1. When skeletal muscle is transformed by CLFS a
FIG. 6. Effect of neuromuscular stimulation 6 salbutamol on the maximum rate of fall of tetanic tension (2dT/dt). Ordinate, the maximal rate of fall of tetanic tension in newtons per millisecond. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
WRIGHT ET AL.: SALBUTAMOL AND CARDIOMYOPLASTY
FIG. 7. Effect of neuromuscular stimulation 6 salbutamol on the maximum rate of muscle shortening (V max). Ordinate, the maximal rate of muscle shortening at zero load expressed in muscle length per millisecond. Columns indicate means, and error bars 11 SEM. For abbreviations and statistical significance see the legend to Fig. 1. There were 6 –7 muscles in each study.
decrease in muscle mass occurs early during the process [3] and coincides with the reduction in fiber size associated with formation of slow-twitch fibers [13]. Salbutamol partially attenuated the loss of muscle mass induced by CLFS, Fig. 1. CLFS produced a 28% loss of muscle mass. Coadministration of salbutamol decreased this to a 6.4% loss. The data for changes in muscle cross-sectional areas, Fig. 2, were very similar. The changes in mean muscle fiber cross-sectional areas were also similar, Fig. 3, but not identical. CLFS produced a 36.1% decline in mean fiber cross-sectional area. Coadministration of salbutamol reduced this to a 14% decline. The data show that salbutamol partially antagonizes the change in muscle fiber size induced by CLFS. The change in mean muscle fiber size need not account exactly for changes in muscle cross-sectional area because fiber sizes are not normally distributed about mean fiber size [13]. The development of skeletal muscle tension is proportional to muscle cross-sectional area. The effects of salbutamol on tetanic tension can be explained by the increase it produced in cross-sectional area. Salbutamol increased muscle cross-sectional area by 26.1% and tetanic tension by 28.6%. CLFS decreased muscle cross-sectional area by 27% and tetanic tension by 45.4%. The loss of tetanic tension in excess of that expected from the loss of muscle cross-sectional area indicates that CLFS produces effects on muscle fibers beyond the loss of fiber cross-sectional area. One possibility is some loss of coupling of excitation to contraction. We [14] found that CLFS reduces the levels of the
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ryanodine and dihydropyridine receptors by 80 to 90%, a magnitude greater than expected on the basis of switching from fast-twitch to slow-twitch fibers. The excess loss of tetanic tension found in the present study suggests that this loss of components of the excitationcontraction coupling apparatus has significant physiological consequences. Salbutamol lowered the loss of cross-sectional area in the muscles subjected to CLFS from 27 to 7.6% and the loss of tetanic tension from 45.4 to 33.7%. This difference in the loss of tetanic tension between CLFS and CLFS 1 S was not, however, statistically significant. We believe that the difference in developed tension, 13 newtons between CLFS and CLFS 1 salbutamol, might achieve statistical significance if a greater number of animals were studied. The velocity of muscle shortening depends upon the ATPase activities of myosin isoforms [15] which vary with muscle fiber type. Sieck et al. [16] found that the relative actomyosin ATPase activities of rat muscle with type IIx, IIa, and I fibers were 100:71:57 respectively. Similar data for canine actomyosins do not exist. In the discussion which follows we assumed that canine actomyosin ATPases have similar relative velocities. Zhang et al. [13] observed the distribution of fiber types in the control canine latissimus dorsi to be 25%, type I; 35%, type IIa; and 40%, type IIx. Salbutamol [9] changed this distribution to 15%, type I; 25%, type IIa; and 60% type IIx fibers. Chronic stimulation produced muscles that have 90% type I and 10% type IIa fibers
FIG. 8. Effect of neuromuscular stimulation 6 salbutamol on the fatigue resistance. The loss of tension caused by 600 ms of tetanic stimulation repeated at 0.71 Hz was tested at 5-min intervals for a period of 20 min (for further details see Materials and Methods). Symbols: E, control; , CLFS; F, salbutamol; ■, CLFS 1 salbutamol. The tensions that are statistically different (P , 0.05) from the control dogs are identified by an asterisk. There were 6 –7 muscles in each study.
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[13]. The fiber-type distribution in muscles from salbutamol-treated dogs subjected to CLFS was 28%, type I; 60%, type IIa; and 12%, type IIx [10]. Chronic low-frequency stimulation increased the frequency of type I fibers from 25 to 90% and decreased V max as expected. Salbutamol decreased the level of type I fibers slightly and increased the frequency of type IIx fibers slightly but had no statistically significant affect on V max. When the LD was subjected to CLFS in dogs receiving salbutamol the frequency of type I fibers was similar to that of the untreated LD. The frequency of IIx fibers, 12%, in the LD muscle subjected to CLFS plus salbutamol was, however, much lower than the frequency in the control muscle, 40%, and yielded muscles with a V max less than in the control muscle but greater than in the CLFS muscle, as expected on the basis of fiber-type distribution. Both V max and 1dT/dt depend on the rate of contractile element shortening and should respond to changes in fiber type in the same direction. The magnitude of the changes are not, however, expected to be the same. Maximum 1dT/dt is measured under isometric conditions during a twitch and its value depends upon the tension which the muscle can develop as well as the velocity of sarcomere shortening. This tension is greatly reduced in muscle subjected to CLFS and is increased in muscle receiving salbutamol, Fig. 4, which had a large effect on 1dT/dt. V max was computed from velocities measured during isotonic muscle shortening. The after loads were adjusted to the maximum load, tetanic tension, and therefore are independent of muscle strength. Although the direction of changes was the same for 1dT/dt and V max, the magnitude of the changes was greater for 1dT/dt than for V max because of the dependence of 1dT/dt on muscle strength. Maintaining 1dT/dt and V max in muscle subjected to CLFS is advantageous for muscles used in cardiomyoplasty. It allows the muscle to develop tension sooner and to eject blood more rapidly and more completely. The rate of loss of tension, 2dT/dt, is dependent on calcium sequestration and the tension developed by the muscle, i.e., muscle strength. The changes observed in tetanic tension and 2dT/dt are similar, with the changes in 2dT/dt slightly greater. We believe that this magnification of changes is due to differences in the rate of calcium sequestration. The best-established system for calcium sequestration is the sarcoplasmic reticulum, although other sequestering systems may exist. Hu et al. [10] have reported that CLFS decreases the sarcoplasmic reticulum calcium-uptake rate from 22.2 mmol Ca 21/min/g muscle to 11.5 mmol Ca 21/min/g muscle. Zhang et al. [9] reported that salbutamol stimulated the uptake rate to 37.4 mmol Ca 21/min/g muscle, and Hu et al. [10] reported that concurrent CLFS and salbutamol treatment yielded a calcium-uptake rate of 19.5 mmol Ca 21/min/g muscle. Salbutamol increased
2dT/dt and CLFS decreased 2dT/dt as expected from the changes observed in calcium-uptake rates and muscle strength. The combined effects of CLFS and salbutamol reduced 2dT/dt more than calcium-uptake rate-indicating the importance of muscle strength as well as calcium-sequestering systems. The failure of salbutamol to prevent the decline in 2dT/dt induced by CLFS is disadvantageous to the use of skeletal muscle for cardiomyoplasty as it could reduce diastolic filling, a problem noted with cardiomyoplasty by Bellotti et al. [17]. Salbutamol decreased fatigue resistance as shown in previous studies with b 2-agonists [18, 19]. CLFS increased fatigue resistance and this increase was unaffected by salbutamol, Fig 8. The effect of CLFS and salbutamol on the distribution of fiber types may account for these findings. Muscle fatigue can be considered the result of an imbalance between energy supply and energy demand [20, 21]. Energy supply can be estimated by succinic dehydrogenase (SDH) activity and energy demand by myofibrillar ATPase activity. The ratio of supply to demand can be estimated by the relative levels of SDH and ATPase activity. Sieck et al. [16], in a study with rat diaphragm muscle, found relative (SDH) levels of 4.6, 7.5, and 6.6 for type IIx, IIa, and I fibers, respectively. Type IIx fibers have relatively low levels of SDH activity and would be fatigable since type IIx fibers also have high actomyosin ATPase activities [16]. The frequency of type IIx fibers would, thus, predict fatigability. In our study salbutamol increased the frequency of type IIx fibers from 35– 40% [9, 10, 13] to 58 – 60% [9, 10], thereby decreasing fatigue resistance. Stimulation decreased the level of type IIx fibers to 0% [13], thereby increasing fatigue resistance. Stimulation of muscles in dogs receiving salbutamol produces fibers with 8% type IIx fibers, not a sufficient percentage of fibers to measurably reduce fatigue resistance. CLFS produced both favorable and unfavorable changes in skeletal muscle intended for use in cardiomyoplasty. Most importantly salbutamol did not block the favorable development of fatigue resistance. Salbutamol partially blocked the unfavorable effects of CLFS on 1dT/dt and V max but failed to influence the effects of CLFS on 2dT/dt. These favorable effects on shortening velocity in the absence of effects on fatigue resistance suggest that b 2-agonists deserve consideration for use in cardiomyoplasty. Studies of longer duration are needed to determine if the effects observed with 6 weeks of administration will continue to be effective. REFERENCES 1.
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