activity–rest stimulation of latissimus dorsi for cardiomyoplasty: 1-year results in sheep

1998;66:1983-1990 Ann Thorac SurgAngelo Pierangeli

Giuseppe Marinelli, Sandra Zampieri, Abdul H. El Messlemani, Katia Rossini and Giorgio Arpesella, Ugo Carraro, Piero M. Mikus, Franco Dozza, Pierloca Lombardi,

sheepActivity–rest stimulation of latissimus dorsi for cardiomyoplasty: 1-year results in

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Activity–Rest Stimulation of Latissimus Dorsi forCardiomyoplasty: 1-Year Results in SheepGiorgio Arpesella, MD, Ugo Carraro, MD, Piero M. Mikus, MD, Franco Dozza, MD,Pierluca Lombardi, MD, Giuseppe Marinelli, Sandra Zampieri, SciBiol,Abdul H. El Messlemani, Katia Rossini, SciBiol, and Angelo Pierangeli, MDCardiovascular Surgery, The S. Orsola Hospital, University of Bologna, Bologna, and C.N.R. Unit for Muscle Biology andPhysiopathology and Department of Biomedical Sciences, University of Padova, Padova, Italy

Background. In dynamic cardiomyoplasty electro-stimulation achieves full transformation of the latissi-mus dorsi (LD); therefore, its slowness limits the systolicsupport. Daily activity–rest could maintain partial trans-formation of the LD.

Methods. Sheep LD were burst-stimulated either 10 or24 hours/day. Before and 2, 4, 6, and 12 months afterstimulation, LD power output, fatigue resistance, andtetanic fusion frequency were assessed. Latissimus dorsiwere biopsied at 6 months, and sheep sacrificed at 12months.

Results. After 1 year of 10 hours/day stimulation LD

was substantially conserved and contained large amountsof fast type myosin. From 2 months to 1 year of stimulationthe power per muscle of the daily rested LD was greaterthan that of the left ventricle, being three to four timeshigher than in the 24-hour/day stimulation.

Conclusions. If extended to humans, these results couldbe the rationale for the need of a cardiomyostimulator,whose discontinuous activity could offer to patients thelong-standing advantage of a faster and powerful musclecontraction.

(Ann Thorac Surg 1998;66:1983–90)© 1998 by The Society of Thoracic Surgeons

Experiments in sheep, aimed at investigating the useof latissimus dorsi (LD) as an energy source for a

skeletal muscle ventricle, allowed us to conclude that thepower generated by a fully conditioned LD could provideno better than partial assistance for a failing heart [1–3].On the other hand, those results are useful for a criticalevaluation of the power of the LD in dynamic cardiomy-oplasty, a procedure in which the patient’s own left LD iswrapped circumferentially around the failing heart, con-ditioned and stimulated to augment cardiac contractility[4, 5].

The limiting factors of LD–heart interactions in cardio-myoplasty are (1) loss of resting tension attributable toLD mobilization; (2) circumferential wrapping aroundthe failing heart; and (3) muscle performance after fullconditioning. Both the mobilization of the muscle and theneed not to interfere with heart diastole reduce LDresting tension, thereby decreasing its work potential.Latissimus dorsi may contribute to the hemodynamicwork of the heart if its power is at least equal to theinstant power of the ventricle during its own contraction–relaxation cycle. In cardiomyoplasty only a portion of theLD is circumferentially wrapped around the heart, andbecause, according to Laplace’s law, by doubling theradius of the heart the muscle mass must be four timesgreater to maintain the same pressure, it is conceivablethat in the dilated heart the contribution of a fully

conditioned muscle to systolic work is difficult to dem-onstrate by beat-to-beat analysis [6].

After a few weeks of chronic stimulation, LD mitochon-drial content and capillary/myofiber ratio increase, butintracellular calcium handling becomes less efficient and,therefore, the contraction–relaxation cycle significantlyslows; finally slow myosin substitutes for fast myosins,and thus a fast, powerful anaerobic (but early fatiguable)LD is transformed into an aerobic slow contracting mus-cle that is fatigue resistant at moderate power [7].

Because maximum instant power of a fully conditionedLD is smaller than the peak power of the left ventricle[1–3], we suggest that the grafted muscle could assist theheart only during late end-systole, just before closure ofthe aortic valve. Of course, such a short window asks fora fast, powerful contraction that is not delivered by a fullytransformed LD. Therefore, we reevaluated the conceptof “muscle conditioning” and its goal in cardiomyoplasty.

Actual clinical protocol makes the LD very resistant tofatigue, but meanwhile its dynamic characteristics aresuboptimal; with a stimulation train of six impulses, thecontraction–relaxation cycle of a fully conditioned LDcould last longer than the heart systole [10].

We are testing whether an intermediate state of muscletransformation could be maintained long term, to havethe advantages of a fatigue-resistant muscle that main-tains fast dynamic contractile characteristics. We present1-year results of a pilot study based on the hypothesisthat resting the LD several hours per day allows it tomaintain an intermediate state of transformation as aresult of the daily training–detraining effect. Further-more, a daily intermittent stimulation of the LD could

Accepted for publication June 3, 1998.

Address reprint requests to Dr Carraro, Department of BiomedicalSciences, University of Padova, Viale Colombo, 3, I-35121 Padova, Italy.

© 1998 by The Society of Thoracic Surgeons 0003-4975/98/$19.00Published by Elsevier Science Inc PII S0003-4975(98)00906-0



also be less detrimental for the muscle tissue as such aprotocol gives the muscle time to recover in betweenactivity periods.

Material and Methods

Surgical procedures and analyses of dynamic character-istics of sheep LD were performed at the ExperimentalSurgery Unit, The S. Orsola Hospital, University of Bo-logna; gross anatomy and histochemical and molecularanalyses on muscle specimens were accomplished at theDepartment of Biomedical Sciences, University ofPadova. After a series of preliminary experiments thepilot study was conducted in six adult sheep. Animalsreceived proper care in compliance with “Guide for theCare and Use of Laboratory Animals” formulated by theNational Academy of Science and published by theNational Institutes of Health (NIH publication 85-23,revised 1985). Sheep were medicated with intramuscularketamine (10 mg/kg) and diazepam (0.5 mg/kg), thenanesthetized with 2% isoflurane. During operation, ani-mals were ventilated with 400 mL of 88% oxygen perbreath at a rate of 12 cycles/min; body temperature,blood pressure, and electrocardiogram were monitored.Operation was accomplished using sterile procedure. Tomimic flap transposition effects of cardiomyoplasty,which result in LD distal devascularization and de-creased resting tension, an incision was made that ex-tended along the lateral and posterior borders from theaxillary fold to the costal margin of the 11th rib and theLD. The incision along the aponeurosis origin completelydissected the experimental muscle from the surroundingtissue, therefore all the vessels were sacrificed (one tothree major collateral blood vessels). As indicated inFigure 1, from its natural insertions to ribs and spinalcolumn (interrupted line) the LD was resutured to ribs inthe shortened position it spontaneously attained. ITRELstimulator (Medtronic, Minneapolis, MN) and intramus-cular electrodes were implanted according to theMedtronic protocol [3], and the skin was sutured.

Two weeks after operation (to allow wound healing),the sheep were anesthetized as above, and tetanic fusionfrequency, power output, and fatigue tests were assessed

according to the Medtronic protocol [3]. In terms ofmuscle performance, endurance has a similar connota-tion to fatigue resistance. It can be measured directly asthe time in which a specified task can be performed, orindirectly as the magnitude of the power that can besustained over time (sustained power). To measure sus-tained power, the average force during the duty cycle wasmultiplied by the velocity of shortening, then the averagepower during the duty cycle was averaged over the totalduty–rest cycle and expressed per muscle (in watts). Themeasures were repeated using bursts at higher frequency(in-burst frequency of 10, 20, and 30 Hz) or more frequent(either 30 or 100 bursts per minute) until muscle fatigueappeared. The protocol is analogous to the graded exer-cise test commonly used in work physiology.

To allow repetitive measurements of the contractilecharacteristics of the shortened LD in the same sheep,the leg but not the muscle tendon was secured to a forcetransducer. Because in these conditions isometric testscould not be performed at optimal muscle length, tetanicfusion frequency was used as an index of duration of thecontraction–relaxation cycle of the surgically shortenedLD. Then stimulators were programmed to settings thatjust elicited fatiguing contractions in the shortened LD.Four sheep were stimulated 10 hours/day, and two sheepwere stimulated 24 hours/day. In both cases LD wasstimulated about 30 times per minute with bursts of threeimpulses lasting about 140 ms at 20 Hz.

Tetanic fusion frequency, fatigue tests, and poweroutput were reassessed after 2, 4, 6, and 12 months ofstimulation. Because fatigue appeared just above theconditioning settings (but of course at higher sustainedpower outputs), stimulation parameters were notchanged during the experiment. In sheep stimulated 24hours/day stimulation was suspended after 6 monthswhen biopsies were taken from the distal part of all theexperimental LD muscles. At 12 months the sheep weresacrificed by excessive anesthesia. The LD were dis-sected, perimysial fat and connective tissue carefullyremoved, and the muscles weighed and photographed.Three muscle specimens were cut out from proximal,intermediate, and distal portions of the LD, quenched inliquid nitrogen and stored at 280°C until use.

Morphometry of myofibers and interstitial tissue, my-osin ATPase, and isomyosin profile by sodium dodecylsulfate–polyacrylamide gel electrophoresis of the myosinheavy chains were performed as described by Rizzi andcolleagues [11] on serial cryostat sections of the 12-monthspecimens and on distal biopsies performed only after 6months of stimulation to limit the surgical muscle dam-age during the experiment [3]. Molecular markers ofmuscle damage/repair/regeneration (contents of totallipids, total protein, myosin/actin ratio, and sodium do-decyl sulfate–polyacrylamide gel electrophoresis of my-osin heavy chains) were determined in whole musclehomogenate of each experimental LD [11].

Results

Figure 2 shows the gross anatomy of experimental mus-cles removed after 12 months of 10-hour/day stimulationor 6 months of 24-hour/day stimulation followed by a

Fig 1. Cardiomyoplasty-like mobilization of sheep latissimus dorsiand daily continuous or activity–rest stimulation. From its naturalinsertions to ribs and spinal column (interrupted line) the latissimusdorsi was resutured to ribs in the shortened position it spontaneouslyattained.

1984 ARPESELLA ET AL Ann Thorac SurgACTIVITY–REST STIMULATION REGIMEN FOR CARDIOMYOPLASTY 1998;66:1983–90



6-month rest. The largest muscles are the contralateralnormal LD (Fig 2B, D), and the muscles in Figure 2A, C,E, and F are the LD muscles stimulated for 12 months for10 hours/day; although slightly diminished in size andweight the chronically stimulated muscle appears trophicand as red as the normal muscle. The muscles in Figure2G and H are sheep LD that had been stimulated for 6months for 24 hours/day and then rested for additional 6months; they appear pale and fibrotic. In Figure 2I thecross section of the muscles demonstrate the differentialcontent of fat and fibrotic tissue in the different experi-mental muscles. Table 1, which shows wet weight, con-tent of contractile and soluble proteins, and the fat of the

contralateral and experimental muscles, confirms thedifferential effects of half-day and daily LD stimulation.Muscles stimulated for 12 months for 10 hours/day showonly about 10% atrophy in comparison with normalcontralaterals, whereas the LD stimulated for 6 monthsfor 24 hours/day and then rested for an additional 6months present about 40% atrophy.

Figure 3 shows hematoxylin and eosin and ATPaseappearances of the experimental muscles after 6 monthsof stimulation. Normal LD muscle of adult sheep isdivided by numerous perimysial septa and containsnarrow endomysial spaces between the polygonal mus-cle fibers. The mean diameter of the muscle fibers is

Fig 2. Cardiomyoplasty-like mobiliza-tion of sheep latissimus dorsi and dailycontinuous or activity–rest stimulation.Anatomic records. Normal latissimusdorsi from sheep: (B) 5/95; (D) 6/95;latissimus dorsi after 12 months of 10-hour/day-stimulation from sheep: (A)5/95; (C) 6/95; (E) 4/95; (F) 1/95; latis-simus dorsi after 6 months of 24-hour/day stimulation followed by a 6-monthrest: (G) 2/95; (H) 3/95. (I) Cross sec-tion of the muscles. Although slightlydiminished in size and weight, after 1year of daily activity–rest stimulationthe latissimus dorsi appear trophic andas red as the normal contralateralmuscles.

Table 1. Cardiomyoplasty-Like Mobilization of Sheep Latissimus Dorsi and Daily Continuous or Activity–Rest Stimulation:Wet Weight, Contractile Protein, Soluble Protein and Fat Content, and Myosin/Actin Ratio of Sheep Latissimus Dorsi AfterEither 12 Months of 10 Hours/Day Stimulation or 6 Months of 24 Hours/Day Stimulation Followed by 6-Month Rest

Variables

ContralateralNormal

(2)

Sheep 95-2, 95-36 mo 24-h Stimulation and

6 mo Rest (2)

Sheep 95-1, 95-5, 95-612 mo 10-h Stimulation

(3)

Latissimus dorsi wet weight (g) 235 (220–250) 138 (122–155) 215 (170–250)Contractile proteins (mg/g ww) 21 (20–22) 4 (3–4) 10 (6–14)Soluble proteins (mg/g ww) 15 (14–16) 14 (14–14) 15 (12–19)Fat (% ww) 0.4 (0.3–0.4) 4 (3–5) 2 (1–3)Myosin/actin ratio 2.2 2.1 (2.0–2.1) 2.1 (1.9–2.2)

Data are mean (range) (observations).

1985Ann Thorac Surg ARPESELLA ET AL1998;66:1983–90 ACTIVITY–REST STIMULATION REGIMEN FOR CARDIOMYOPLASTY



0.39 6 0.14 mm. Myonuclei are in a subsarcolemmalposition. Fat tissue is rarely seen and confined to perimy-sial septa (Fig 3E). Histochemically, the normal LD showsthe properties of fast and fatiguing muscle containingonly 20% fibers of slow, fatigue-resistant type 1 (Fig 3F).Although 6 months of daily stimulation transform allmyofibers (Fig 3B), the 10-hour/day stimulated LD wereincompletely transformed (Fig 3D).

Figures 4 and 5 show hematoxylin and eosin andATPase appearances of the muscles at the end of theexperiment. Results are in full agreement with the mo-lecular characterization described by both bioptic andtotal muscle analyses. Histology of distal muscle samplesshows that the LD stimulated 10 hours/day are either ofalmost normal appearance or slightly fibrotic and infil-trated of fat (Fig 4C–F). Overall mean diameter of themuscle fibers is 0.30 6 0.10 mm. On the other hand, theLD stimulated for 24 hours/day are heavily fat infiltrated,the muscle fibers being scarce and atrophic (Figs 4A, 4B).

The mean diameter of the measurable muscle fibers inproximal LD samples (data not shown) is 0.29 6 0.14 mm,suggesting that they are innervated and activated bypostural reflexes. Indeed, the myosin-to-actin ratio in allof the experimental LD are near to the normal value of 2.2(Table 1).

Figure 5C–F shows that 1 year of 10-hour/day stimula-tion still results in an incomplete transformation of themuscle, which maintains a chessboard appearance in allexperimental LD. The surviving muscle fibers which areof almost normal size in LD stimulated for 6 months for24 hours/day and then rested for the additional 6 monthsalso show an intermediate state of muscle transformation(Figs 5A, 5B), which is in keeping with the detrainingeffect of ceased stimulation in several animal models andhumans [7].

Table 2 shows the results of the isomyosin analysisperformed on LD at 6 and 12 months. After 12 monthsmyosin heavy chains of the experimental LD are verydifferent from normal LD owing to their high content ofmyosin heavy chain 1, the isoform peculiar of slow

Fig 3. Cardiomyoplasty-like mobilization of sheep latissimus dorsiand daily continuous or activity–rest stimulation. Hematoxylin andeosin stain (A, C, E) and pH 4.35 ATPase (B, D, F) of distal samplesof latissimus dorsi. Six months of 24-hour/day stimulation: (A)sheep 2/95 (hematoxylin and eosin); (B) sheep 2/95 (ATPase); 6months of 10-hour/day stimulation: (C) sheep 6/95 (hematoxylinand eosin); (D) sheep 6/95 (ATPase); (E) normal ATPase latissimusdorsi (hematoxylin and eosin); (F) normal latissimus dorsi (ATPase).(Hematoxylin and eosin; 3100 before 14% reduction, ATPase; 380before 14% reduction.)

Fig 4. Cardiomyoplasty-like mobilization of sheep latissimus dorsiand daily continuous or activity–rest stimulation. (Hematoxylin andeosin; 3100 before 8% reduction.) Distal samples of sheep latissi-mus dorsi after 6 months of 24-hour/day stimulation followed by 6months of rest: (A) sheep 2/95; (B) sheep 3/95; latissimus dorsi after12 months of 10-hour/day stimulation: (C) sheep 1/95; (D) sheep4/95; (E) sheep 5/95; (F) sheep 6/95.




myofibers, but again they are not fully transformed byactivity–rest regimens of stimulation either discontinuedevery day or after 6 months of daily stimulation. Tointerpret these results it is essential to remember that at6 months of stimulation LD biopsies of the all-day stim-ulated muscles contained only myosin heavy chain 1 (theslow type isoform peculiar of slow, fatigue-resistant mus-cle fibers), whereas the muscles stimulated 10 hours/daycontained large amounts of fast type myosins, in partic-ular myosin heavy chain 2A, the isoform peculiar of fastoxidative fibers, less prone to fatigue than the type 2Bisoforms, of which normal LD of adult sheep is abundant[3, 7].

Accordingly, Table 3 shows that the tetanic fusionfrequency at 1 year is similar in the two groups ofmuscles, whereas at 6 months the daily stimulated LDwere slower than the half-day stimulated LD. It is worthstressing that the tetanic fusion frequency (that is, thecontraction–relaxation cycle) of the 10-hour/day stimu-lated muscles is similar after 6 and 12 months ofstimulation.

Finally, Table 4 shows power outputs and fatigue tests

of LD muscles 6 and 12 months after operation. Up to 6months sheep LD stimulated 24 hours/day sustain about0.5 W of external power, whereas the 10-hour/day stim-ulated muscles deliver without fatigue about 2 W ofexternal power up to 12 months of stimulation.

Comment

The first notion we would like to stress is that fatigueresistance of skeletal muscle is a relative concept. Mus-cles are fatiguable or fatigue resistant to a given workloadand for a given period of time; normal muscles are able tosustain indefinitely a task if it is energetically low de-manding. Indeed our diaphragm, a muscle of mixed typeas are the majority of human skeletal muscles, sustainsventilation 24 hours/day.

Previous experiments that we performed in a series ofsheep to investigate the use of LD as an energy source fora skeletal muscle ventricle allowed us to conclude thatthe power generated by a fully conditioned LD couldprovide no better than partial assistance for a failingheart [1–3]. On the other hand, those results are useful fora critical evaluation of the energetic contribution of theLD to dynamic cardiomyoplasty [4, 5]. To many investi-gators, cardiomyoplasty is a clinical reality, whose basis

Fig 5. Cardiomyoplasty-like mobilization of sheep latissimus dorsiand daily continuous or activity–rest stimulation. (ATPase pH 10.4380 before 14% reduction.) Distal samples of latissimus dorsi after6 months of 24-hour/day stimulation followed by 6 months of rest:(A) sheep 2/95; (B) sheep 3/95; latissimus dorsi after 12 months of10-hour/day stimulation: (C) sheep 1/95; (D) sheep 4/95; (E) sheep5/95; (F) sheep 6/95.

Table 2. Cardiomyoplasty-Like Mobilization of SheepLatissimus Dorsi and Daily Continuous or Activity–RestStimulation: Myosin Heavy Chains Complement of SheepLatissimus Dorsi After Either 12 Months of 10 Hours/DayStimulation or 6 Months of 24 Hours/Day StimulationFollowed by 6 Month Rest

Variables

Fatigue-Resistant FastType Fibers

Fatigue-ResistantSlow Type Fiber

MHB2B MHC2A MHC1

Normal sheep (15) 73 15 126 mo of 24-h

stimulation (2)100 (100–100)

6 mo of 10-hstimulation (4)

30 (0–60) 40 (18–66) 32 (21–55)

6 mo of 24-hstimulation and6-mo rest (2)

35 (32–38) 22 (22–23) 43 (40–45)

12 mo of 10-hrstimulation (4)

22 (0–42) 22 (18–25) 57 (35–77)


Table 3. Cardiomyoplasty-Like Mobilization of SheepLatissimus Dorsi and Daily Continuous or Activity–RestStimulation: Tetanic Fusion Frequency of Sheep LD AfterEither 12 Months of 10 Hours/Day Stimulation or 6 Monthsof 24 Hours/Day Stimulation Followed by 6 Month Rest

VariablesTetanic FusionFrequency (Hz)

2 weeks after LD mobilization (6) .30 (.30–.30)6 mo of 24-h stimulation (2) ,5 (,5–,5)6 mo of 10-h stimulation (4) 10 (10–10)6 mo of 24-h stimulation and 6-mo rest (2) 20 (20–20)12 mo of 10-h stimulation (4) 15 (10–20)





is founded more on a girdle effect that limits or evenreverses the progressive dilatation of a failing heart, thanon an active systolic assist; indeed a very critical ap-proach is needed to demonstrate beat-to-beat assistance.On the other hand, load-independent measurementsdemonstrate a real amelioration of the heart energeticwhen analyses are compared before and after cardiomy-oplasty [6].

The factors that limit LD–heart interactions in cardio-myoplasty are (1) loss of resting tension due to LDmobilization; (2) circumferential wrapping around thefailing heart; and (3) muscle performance after full con-ditioning. Both the mobilization of the muscle and theneed not to interfere with heart diastole reduce LDresting tension, thereby decreasing its work potential.

The LD may contribute to hemodynamic work of theheart if its power is equal to the instant power of theventricle during its own contraction–relaxation cycle. Incardiomyoplasty only a portion of LD is circumferentiallywrapped around the heart, and using Laplace’s lawdoubling the radius of the heart, the muscle mass mustbe four times greater to maintain the same pressure. It isconceivable that in dilated heart the contribution of afully conditioned muscle to systolic work is difficult todemonstrate by beat-to-beat analysis.

After a few weeks of chronic stimulation, LD mitochon-drial content and capillary-to-myofiber ratio increase,but intracellular calcium handling becomes less efficientand therefore, the contraction–relaxation cycle signifi-cantly slows; finally slow myosins substitute for fastmyosins, thereby a fast, powerful anaerobic (but earlyfatiguable) LD is transformed into an aerobic slow-contracting muscle that is fatigue resistant at moderatepower [7]. Because the heart delivers 1.3 W of power tomaintain basal metabolism, altogether the above men-

tioned factors explain why the systolic contribution of afully transformed LD is low in cardiomyoplasty [8].

Clinically, the LD benefits the patient’s quality of lifeonly if its activation is critically delayed after sensed QRSto avoid mitral regurgitation [9]. Because maximum in-stant power of a fully conditioned LD is smaller than thepeak power of the left ventricle [1–3], we suggest that thegrafted muscle could assist the heart only during lateend-systole, just before closure of the aortic valve. Ofcourse, such a small window demands a fast, powerfulcontraction that is not delivered by a fully transformedLD. Therefore, we reevaluated the concept of “muscleconditioning” and its goal in cardiomyoplasty.

Actual clinical protocol makes the LD very resistant tofatigue, but its dynamic characteristics are suboptimal.Indeed with a 160-ms stimulation train of six impulses(six impulses delivered every 32 ms), the contraction–relaxation cycle of a conditioned LD could last longerthan the heart systole [10]. We are testing whether anintermediate state of muscle transformation could bemaintained long term to better sustain faster contractions(cardiac-like amount of averaged external power, .1 Wof power per LD).

Previously [3] we showed that after shortening butbefore conditioning the sheep LD was able to deliverabout 0.1 W of extracted power by stimulating it withsingle impulses at 2 Hz (120 events/minute), which is thehigher frequency of a heart pacemaker. Of course whentetanic contractions were elicited by bursts of impulses,the shortened LD delivered 0.2 to 0.3 W of external powerwithout signs of fatigue. After increasing the frequency oftetani or inducing more powerful tetani by bursts athigher frequency, the external power reached 0.5 to 1.0 Wper muscle, but the muscles fatigued in a few minutesand then external power either leveled off at about 0.2 Wper muscle, or even ceased when workloads were main-tained near maximal values. An implication of our obser-vations is that after cardiomyoplasty LD could deliversustainable power immediately after the healing periodby setting the stimulator at very low muscle demands [3].

After chronic stimulation the working capacity of theLD increases, but only if the LD is rested several hoursevery day when the sustained power exceeds the value ofthe left ventricle at rest, as it seems possible to maintainan intermediate state of myofiber transformation in thesheep LD by a daily activity–rest regimen.

The biological basis of such an approach is that “inter-mediate” myofibers do exist in nature; several differenttypes of myofibers with intermediate characteristics be-tween very fast and very slow contracting fibers exist inskeletal muscles of mammals, humans included, theircharacteristics being induced and maintained by differ-ent levels of activity against load [7]. By imposing theproper workload, it is possible to regulate gene expres-sion and to transform all the fibers to a desired type.

To allow repetitive measurements of the contractilecharacteristics of the shortened LD in the same sheep,the leg but not the muscle tendon was secured to theforce transducer. Because under these conditions isomet-ric tests could not be performed at optimal musclelength, tetanic fusion frequency was used as an index ofduration of the contraction–relaxation cycle of the short-

Table 4. Cardiomyoplasty-Like Mobilization of SheepLatissimus Dorsi and Daily Activity–Rest or ContinuousStimulation: Sustained External Power and Fatigue of SheepLD After Either 12 Months of 10 Hours/Day Stimulation or6 Months of 24 Hours/Day Stimulation Followed by 6-Month Rest

Variables

24 h/dayStimulation

10 h/dayStimulation

Sheep 95-2and 95-3

Sheep 95-1, 95-4,95-5, and 95-6

36 Events per min(3 pulses/burst)

at 30 Hz

28 Events per min(3 pulses/burst)

at 30 Hz

6 mo of stimulationMean external power (W) 0.5 (0.4–0.6) (2) 2.7 (2.2–3.9) (4)Fatigue (D%) 0 (2) 0 (4)6 mo of stimulation and

6-mo restMean external power (W) 0.8 (0.4–1.2) (2)Fatigue (D%) 0 (2)12 mo of stimulationMean external power (W) 1.7 (0.5–3.2) (4)Fatigue (D%) 0 (4)





ened LD. As the values are lower than those expected forsheep LD under optimal length [3], we have indirectevidence that the LD are really shortened by ourprocedure.

Furthermore, it is worth stressing that after 6 months ofstimulation the frequency of tetanic fusion was higher (ie,the contraction–relaxation cycle was faster) in the 10-hour/day than in the 24-hour/day stimulated LD, andthat this difference disappeared at 1 year as the fusionfrequency of the 6-month rested muscle recovered tovalues of the 1-year 10-hour/day stimulated LD. Al-though demonstrated under suboptimal conditions,changes in frequency of tetanic fusion are evidence of atraining–detraining effect of our stimulation regimen.

Convincing results are collected by analysis of isomyo-sins in the experimental muscles. Analyses of myosinheavy chain isoforms by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in serial sections ofmuscle biopsies taken 6 months after stimulation showedthat the 24-hour/day stimulated LD contain only myosinheavy chain 1 (ie, they are fully transformed), whereasthe muscles stimulated 10 hours/day still contain largeamounts of fast type myosin heavy chain, in particulartype 2A. Furthermore we show that after 12 months ofstimulation, the 10-hour/day stimulated LD contain sub-stantial amounts of fast type myosin heavy chains, andthat after 6 months of resting the fast-type myosin heavychain are reexpressed in the 24-hour/day stimulated LD.These observations explain the different dynamic char-acteristics of the two groups of LD, as calcium uptake/release is faster in type 2A than in type 1 myofibers.

At 6-month stimulation, frequency of tetanic fusionwas higher in 10-hour/day stimulated muscles than in LDfully conditioned by electrostimulation for 24 hours/day.At 1 year the contraction–relaxation cycle of the 10-hour/day stimulated LD is as fast as at 6 months. The 6-monthdiscontinued stimulation retrodifferentiates the LD,which was fully transformed by 6 months of 24-hour/dayactivity. Interestingly the complement of myosin heavychains is very similar in the two groups of muscles,suggesting that the total amount of muscle contractionshave a main role in driving gene expression in themyofibers. These results are in full agreement withresults of long-term training and detraining experimentsin rodents, rabbit, goat, sheep, and humans [2, 7, 12–14].

Comparison between various regimens of stimulation,such as daily amount of treatment or frequency, are rare.It is likely that 10 hours of stimulation per day willproduce different results than 24 hours/day. First, 10hours of stimulation cover about one-third of the 24-hourstimulation period and, second, this protocol gives themuscle time to recover in between. Although the possi-bility exists that final outcome of changes using eithermethod may be, ultimately, similar after long-term peri-ods of stimulation (ie, after several years), it is wellestablished that in animals stimulated 12 hours/day,mRNA of myosin heavy chain 1 becomes detectablewhen stimulation periods exceed 20 days, whereas con-tinuous stimulation (24 hours/day) leads to an earlierappearance of the mRNA (9 days). Furthermore, cessa-tion of stimulation has pronounced effects on the mRNA

pattern leading to a rapid reversal (hours) of the stimu-lation-induced changes [3].

Biochemical changes (eg, acidosis, AMP, inorganicphosphate that accumulate during muscle fatigue, orcytosolic calcium) are probably the intracellular messen-gers of muscle plasticity. The actual clinical stimulationprotocol of cardiomyoplasty is very demanding, thereforeit is not surprising that the LD is transformed in a pureslow-type muscle by long-term continuous stimulation.Indeed either 9 months or 2 years after cardiomyoplastyhistochemical analyses revealed only type 1 fibers in theLD flap stimulated every cardiac cycle with 30-Hz burstslasting 160 ms [13].

In summary, it is conceivable that in our pilot experi-ment in sheep an intermediate state of LD transformationis maintained at least up to 1 year by daily modulation ofthe working periods.

A second issue in dynamic cardiomyoplasty is whethermuscle damage is induced by the chronic abnormalstimulation, in particular when a muscle-to-heart con-traction ratio of 1:1 is applied. Exercise may inducemuscle damage, and physiologists, sports scientists, andphysiatrists are well aware that spontaneous exercise perse could be a trauma to muscle fibers [15, 16]. Cardiomy-oplasty is a complex procedure and it is difficult to evenidentify the relevant variables [2, 3, 14]. Because thecontrolled environments of a physiologist’s experimentare not applicable, we have variable results in our fewsheep.

In any case the biopsies of 10-hour/day stimulated LDpresent a well-preserved muscle structure with moderateand nonspecific changes; myofiber size is much largerand interstitial tissue is smaller than in biopsies ofall-day-stimulated LD. This result is in agreement withdata previously reported on goat LD surgically dissectedand stimulated for 2 months either 24 hours/day or 16hours/day [14], and long-term studies in rabbit androdents whose continuous stimulation is known to de-crease the surface-to-diameter and muscle mass-to-blood perfusion ratios to favor oxidative metabolism ofthe myofibers [12]. If the muscle is rested daily homeosta-sis seems to be near normal values. Indeed after 1 year of10-hour/day stimulation wet weight of muscle is only10% lower than that of normal contralateral LD, whereasthe LD stimulated 6 months for 24 hours/day and thenrested for 6 months shows a 40% decrease in wet weight.

Taking into account the fat and collagen content in themuscles, it is evident that gross anatomy underestimatesthe extent of ongoing damage in chronically stimulatedmuscles. Results of the histologic analyses performed onbiopsies taken after 6 months of stimulation stronglysuggest that the decreased weight of the LD stimulatedfor 6 months for 24 hours/day and then rested for anadditional 6 months is more likely the consequence of the6 months of daily stimulation than of the 6 months of rest.Indeed the myofibers of those LD were atrophic and thetissue was heavily infiltrated by fat and connective tissueafter 6 months of 24 hours/day of stimulation [3]. Fur-thermore, although shortened, the LD are properly in-nervated and therefore possibly activated by standingand walking activity as with the normal contralateral LD.

On the other hand, the true question is whether the




unusual work performed in cardiomyoplasty by the graftdamages human LD. There are reports explaining long-term ceased effect of the procedure with indirect evi-dence of major muscle atrophy, fibrosis, and fat infiltra-tion; furthermore, direct histologic evidence of muscledamage had been collected in sheep and goat experi-ments [9, 14, 17]. On the contrary, two autoptic casesdirectly show that this is not an obligatory event; 15months or even 8 years after cardiomyoplasty morpho-logic and molecular analyses of the pedicled LD showedpreserved muscle mass and patent vessels with normalendothelial and smooth muscle walls. Interestingly, inthese two cases LD graft was activated every second orfourth sensed QRS, and clinical results were excellent[18].

Several independent factors may damage the musclebesides the pattern of activation (ie, lesions of nerves,arteries, or veins during or after operation, loss of restingtension) [10, 14]. Histopathologic observations of graftedLD up to 8 years after cardiomyoplasty demonstrate thatdamage is not a mandatory consequence of the unusualactivity the muscle performs to assist the failing heart[18].

Eleven years after the first clinical case, we may hopethat cardiomyoplasty is at the stage heart transplantationwas after immunopharmacologists solved the problem ofrejection of autologous transplant by immunosuppres-sive drugs, which are now accepted clinical practice. Alsoin heart transplantation, the surgical problems weresolved several years earlier than the rejection problem.Carpentier and Chachques [4] established the basic sur-gical procedure 12 years ago; now the knowledge existsto overcome some of the remaining problems of car-diomyoplasty. Several investigators are collecting scien-tific evidence on the mechanisms and effectiveness ofcardiomyoplasty [6, 9]. Risks of “damage” of LD may bereduced and muscle performance increased by (1) usingpre- and post-cardiomyoplasty different work–rest stim-ulation regimens; (2) testing nerve versus intramuscularelectrostimulation; (3) optimizing the surgical procedure;and (4) administrating local anabolic agents to the LDflap [2, 3, 19].

We are confident that our pilot experiment will attractattention, and reinforcing the concept of a lighter anddemand stimulation of the grafted LD, it will contributeto a larger acceptance of the procedure, to a bettermanagement of pharmacologically intractable heart fail-ure with an acceptable quality of life for the subjects.Preliminary results in patients are more than encourag-ing [20].

This study was supported by the Italian Ministero Universita eRicerca Scientifica e Tecnologica (MURST) funds to GiorgioArpesella and in part by funds from the Italian ConsiglioNazionale delle Ricerche (CNR) to the Unit for Muscle Biologyand Physiopathology, and MURST to Ugo Carraro. The financialsupport of TELETHON—ITALY to the project “Role of apoptosisof myofibers, satellite cells and endothelia in exercise-inducedmuscle damage and in progression of muscular dystrophies (n.968)” is gratefully acknowledged.

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1998;66:1983-1990 Ann Thorac SurgAngelo Pierangeli

Giuseppe Marinelli, Sandra Zampieri, Abdul H. El Messlemani, Katia Rossini and Giorgio Arpesella, Ugo Carraro, Piero M. Mikus, Franco Dozza, Pierloca Lombardi,

sheepActivity–rest stimulation of latissimus dorsi for cardiomyoplasty: 1-year results in

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