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Eur J Cardiothorac Surg 1998;13:588-598
© 1998 Elsevier Science NL
a Department of Cardiac Surgery, Royal Brompton Hospital and NHLI, London, SW3 6NP, UK
b Physiological Flow Studies Group, Imperial College, London, UK
c Department of Human Anatomy and Cell Biology; University of Liverpool, Liverpool, UK
Received 29 September 1997; received in revised form 9 February 1998; accepted 16 February 1998.
Corresponding author. Tel./fax: +44 171 3518530; e-mail: d.barron@rbh.nthames.nhs.uk
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Abstract |
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 Top Abstract Introduction
MethodsResults
Discussion
Appendix A. Conference...References |
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Key Words: Cardiomyoplasty • Muscle transformation • Latissimus dorsi muscle • Microelectrodes
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Introduction |
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TopAbstractIntroduction
MethodsResults
Discussion
Appendix A. Conference...References |
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Studies of cardiomyoplasty over the past few years include reports of the late development of atrophy and fibrosis in the LDM associated with a decrease in the contractile function of the muscle wrap in both clinical [5] [16] and experimental contexts [9] [15]. The contributory factors are thought to be ischaemia of the muscle flap as a consequence of mobilisation and, possibly, sustained activity. A further consequence of fast-to-slow transformation is slower relaxation of the muscle wrap, which may interfere with the diastolic performance of the heart [15] [17] [18].
These fundamental concerns about the viability and performance of the LDM can be answered only if more is understood about the changes in nutrition and perfusion that occur within muscles in terms of conditioning and surgical mobilisation. In the present experiments we used a microelectrode technique to study the perfusion and tissue oxygenation of the LDM of the rabbit following mobilisation and electrical stimulation. We used two different conditioning regimes in order to study the significance of different degrees of fibre type transformation.
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Methods |
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TopAbstractIntroduction
MethodsResults
Discussion
Appendix A. Conference...References |
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Recovery procedures
Implantable stimulators were made to a published design
[19]. The stimulators deliver pulses of 0.2 ms duration and 3.2 V amplitude at a frequency of either 2.5 or 10 Hz as required and are controlled by a light-sensitive switch triggered by flashes of light which are transmitted through the skin by an electronic flash-gun. The electrode leads consisted of a pair of polyvinylchloride-insulated multistranded stainless steel wires ending in a bared loop which could be fixed in position with an attached Dacron velour pad. The stimulators were gas sterilised in ethylene oxide and all recovery procedures were carried out under aseptic conditions. A single incision about 25 mm long was made parallel to the anterior border of the LDM close to the axilla, and the muscle border was dissected free to expose the thoracodorsal vessels as they entered the costal surface. One electrode loop was then fixed close to the nerve with two 5/0 Vicryl® (Ethicon, UK) sutures, one through the loop itself and one through the Dacron patch. The second electrode was placed on the subcutaneous surface of the muscle immediately overlying the first. The stimulator was implanted underneath the skin by creating a pouch inferior to the incision, and was switched on for a few seconds to confirm satisfactory operation. Wounds were closed with 4/0 Prolene® (Ethicon, UK) to the subcutaneous tissue and subcuticular 3/0 Vicryl® to the skin. The animals were left to recover for 48 h, after which the stimulators were switched on. Stimulation was confirmed daily by palpation over the LDM.
Muscle testing procedures
After completion of a predetermined stimulation period the animals were anaesthetised by the technique already described, a tracheostomy was performed, and the animals were ventilated by a Harvard rodent ventilator. Blood pressure was monitored via a carotid artery line, which also allowed access for blood-gas sampling. Ventilation was controlled to maintain pH in the range 7.36–7.44 and pCO2 at 3.5–5.0 kPa. Body temperature was maintained between 36.5 and 38°C by means of a heating blanket, and saline was given at 5 ml/kg per h intravenously to replace insensible loss.
The implanted stimulator was switched off after inducing anaesthesia. A longitudinal incision was made immediately below the point of the scapula to expose the LDM, and two microelectrode needles were placed intramuscularly, one distally and one proximally. The microelectrodes were purpose built to our own design [20] and consisted of a 125 µm-diameter insulated silver wire embedded in epoxy resin within a 23G needle. The epimysium was removed with a scalpel blade and the microelectrode introduced along the plane of the muscle fibres to a point where it rested 1–2 mm below the surface of the muscle. The exposed muscle was covered with a warm, saline-soaked swab to prevent it from drying out.
A calomel electrode (Russell, UK), acting as a combined counter and reference electrode, was placed subcutaneously in the groin and connected to a potentiostat (Energy Microsystems, Oxford, UK) interfaced by an A/D Converter (Strawberry Tree Graphics, USA) to a personal computer running Workbench software.
The microelectrodes measure tissue pO2 amperometrically at a voltage between -0.6 and -0.7 V, the precise value being determined for each electrode from the plateau region of the in-vivo current-voltage curve obtained at the beginning of each experiment. Perfusion was measured by an adaptation of the washout technique which we have reported previously [21] [22], using nitrous oxide as the gaseous tracer. Each electrode can measure the pO2 current and the pN2O current simultaneously by means of a voltage switching technique described elsewhere [20] [21]. Perfusion measurements were made by ventilating the animal with 20% N2O, 20% O2, 5%CO2 and the balance N2 for a wash-in phase until a constant current was reached, and then returning to air ventilation to allow wash-out. This process was repeated at different stages of the protocol to generate a series of wash-in and clearance curves from which tissue perfusion could be derived.
Baseline measurements of pO2 and perfusion were made with the muscle in situ. The LDM was then mobilised in a similar fashion to that used in cardiomyoplasty, by dividing the aponeurotic origin and raising the muscle as a pedicled flap [1] [2]. The deep surface of the muscle was reflected cranially, dividing and ligating the small intercostal perforators. The attachments between the medial edge of the muscle and the vertebral spines were divided to complete mobilisation of the flap. The mobilised muscle flap was then wrapped 1.25 times around a 9 mm diameter latex tube to simulate aortomyoplasty [23]. The wrap was fixed in place with a running 5/0 polypropylene suture no deeper than the epimysial layer, to minimise damage to the tissue. The latex tube was mounted on a custom-made jig which formed part of a compliant hydraulic system illustrated in Fig. 1 . The muscle preparation was allowed to stabilise for 30 min before any measurements were taken.
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The hydraulic system was kept at 37°C by means of a heating jacket and the pressure within the system was set at 60 mmHg to represent the diastolic pressure, which approximates to the preload on the muscle wrap in the clinical situation. The pressure developed within the system by contraction of the muscle wrap was recorded by a transducer and stored via the same data acquisition system as used for the amperometric measurements. The pressure-time plots were analysed to determine the pressure developed during contraction and the maximum rate and duration of relaxation.
All animals received humane care in accordance with the guidelines published by the National Society for Medical Research (Principles of Laboratory Animal Care) and by the National Institutes of Health (Guide for Care and Use of Laboratory Animals). The project was licensed and performed in accordance with the Animals (Scientific Procedures) Act, 1986 which governs animal experimentation in the UK.
Tissue samples and histochemistry
The stimulated muscles were dissected out, trimmed of superficial fat and weighed. In order to standardise for individual variation the contralateral muscle was dissected out in the same way and muscle weight was expressed as a ratio of the experimental muscle to the contralateral control. A further index of muscle bulk was derived from the weight of a full thickness muscle biopsy 10x10 mm in size from a point 20 mm from the tendinous insertion, where the muscle is thickest. In addition, biopsies were taken from each muscle for histological examination. The biopsies were 3–4 mm wide and 10 mm long, cut parallel to the fibre orientation. Two biopsies were taken from the proximal region and two from the distal region.
Each biopsy sample was wrapped in aluminium foil and frozen in isopentane suspended in a bath of liquid nitrogen. The samples were then stored at -40°C until required for sectioning. Two separate tissue blocks were prepared from each biopsy. Frozen sections 20 µm thick were stained with haematoxylin and eosin, for succinate dehydrogenase (SDH) and for the myosin heavy chain isoforms characteristic of slow and fast muscles (MHCs and MHCf).
Histochemical demonstration of SDH
This was based on the method of Nachlas et al.
[25] using nitroblue tetrazolium (NBT). The air-dried 20 µm sections were incubated in a medium of 0.2 M sodium succinate and 25 mM NBT in phosphate buffer (disodium hydrogen phosphate and potassium dihydrogen phosphate) at pH 7.0 for 1 h at 37°C. Sections were then fixed in 10% phosphate buffered formalin, washed, dehydrated, cleared and mounted.
Immunohistochemical demonstration of fast and slow myosin heavy chains
Sections were air dried and first incubated with normal serum at room temperature. After washing with saline they were incubated with the primary antibody to rabbit MHCs and MHCf at a 1:50 dilution for 1 h at room temperature followed by biotinylated rabbit anti-mouse IgG for a further 1 h. Slides were then washed in saline and incubated with the ABC complex for 1 h. Peroxidase activity was developed with diaminobenzidine (DAB) solution containing 8.8mM H2O2. Sections were counterstained with Carazzi's haematoxylin for 4 min, dehydrated, cleared and mounted
[26].
Type I and IIA fibres stain darkly for SDH whereas type IIB and IID stain lightly. Since types IIB and D cannot be distinguished by the techniques used, they are referred to here as a single class, type IIB. All type II fibres stain darkly with the antibody to fast myosin whereas type I fibres stain darkly with the antibody to slow myosin. Sections from each biopsy were examined under light microscopy at 250x magnification and the proportions of fibre types were measured in five representative areas containing 100–150 fibres. Four sections were prepared from each animal and the mean fibre counts from all four biopsies were used to derive the mean fibre distribution for that animal.
Statistical methods
Differences between the three groups in fibre type distribution, perfusion, oxygenation and muscle pressure data were examined using ANOVA with equal variance. Results were taken to have statistical significance if P<0.05. All results were expressed as the mean±SE.
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Results |
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TopAbstractIntroduction
MethodsResults
Discussion
Appendix A. Conference...References |
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Histology
Two distinct muscle phenotypes were generated by the stimulation regimes and the composition of the control and conditioned muscles is shown in
Fig. 2
. The control muscles were of typical fast muscle composition, with 17±2% type I, 10±2% type IIA and 73±2% type IIB fibres. Six weeks of stimulation at 10 Hz (Group A) produced a muscle that consisted of 66±6% type I and 19±6% type IIA fibres, leaving only 16±3% type IIB fibres. This represented a significant increase in the type I fibres (P<0.001) and a significant decrease in the type IIB fibres (P<0.001), a fibre-type pattern more typical of a slow muscle.
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Muscle weight
Muscle weights and the weights of standard biopsies are shown in Table 1. In Group A the 6-week conditioning resulted in a significant decrease in both indices compared to the contralateral control (P<0.01). However, in Group B neither the muscle weight nor the biopsy weight differed from control.
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Muscle perfusion and oxygenation
The effects of mobilisation on perfusion are shown in
Fig. 4 a,c. The resting perfusion of the conditioned muscles (of both Groups A and B) was greater than that of control muscles both before (P<0.05) and after (P<0.05) mobilisation in all regions of the muscle. In the proximal region, mobilisation did not cause any significant change in perfusion in any of the groups. In the distal region, however, there was a significant decrease in the perfusion of the control muscles (P<0.05) whereas in both conditioned muscle groups the perfusion was more effectively maintained. The effect of mobilisation on tissue pO2 is shown in
Fig. 4b,d. The mean pO2 at rest was lower in conditioned muscles than in the control muscles and there was no difference between the two conditioned groups. Mobilisation did not alter the tissue pO2 significantly in either the proximal or distal regions of any of the groups (
Fig. 4b,d).
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Discussion |
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TopAbstractIntroduction
MethodsResults
Discussion
Appendix A. Conference...References |
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The rabbit has been used extensively in studies of muscle transformation although less information is available about the LDM in this species. This may seem surprising, as the LDM is the single most used muscle in clinical applications of muscle conditioning. The muscle is, however, less well suited to the recording of linear force. In this study, the force-generating capacity of this muscle was assessed by coupling it to a hydraulic measurement system; this had the additional merit of testing the muscle under conditions that approximate clinical use.
Conditioning regimes have been developed to produce different phenotypes in the tibialis anterior muscle [9] [11] [28] and we have adapted these regimes in the present work. Six weeks of CLFS at 10 Hz (Group A) produced a `slow' phenotype consisting of predominantly type I fibres, typical of the fibre transformation reported with cardiomyoplasty regimes of stimulation. As in previous studies, the muscles had excellent fatigue resistance but reduced pressure development and speed of contraction. They also showed the reduction in bulk that is a well-documented accompaniment of fast-to-slow transformation [29] [30] [31]. The 20% decrease in wet weight is similar to that observed in other animal studies [15] [16] [31] [32] but is less than the changes reported in clinical series [5] [33], which suggests that the latter may be due to additional factors such as ischaemia.
Two weeks of CLFS at 2.5 Hz produced a muscle consisting mainly of type IIA fibres, in agreement with what has been found in the tibialis anterior muscle [11] [28] [34]. These muscles had a fatigue resistance similar to that of the more completely transformed muscles but could generate greater pressure and showed more rapid rates of contraction and relaxation. This is a highly advantageous combination of characteristics for cardiomyoplasty, in which slow relaxation of a fully transformed muscle may impair diastolic filling and prevent the achievement of maximum end-diastolic fibre length [15] [17]. These muscles showed no evidence of fibre degeneration and no significant loss of muscle mass. Longer term experiments have shown that the muscle can be maintained in this intermediate state without the transformation process continuing further [11] [28]. To create similar conditions in cardiomyoplasty it would be necessary to avoid unnecessarily demanding conditioning regimes and 1:1 systolic augmentation.
In the present study, micro-amperometric measurements provided new insight into changes in muscle perfusion and tissue oxygenation in the conditioned muscles. The resting perfusion of the conditioned muscles was significantly elevated over controls. This is significant in the context of cardiomyoplasty, since current protocols require the LDM to be mobilised (a process which, as demonstrated here, results in a decline in distal perfusion) and the onset of conditioning then demands greater perfusion than the muscle had before being mobilised. Our findings suggest that muscles conditioned prior to mobilisation would be able to maintain higher levels of resting perfusion after mobilisation. This suggestion is supported by changes in tissue oxygenation: in the unconditioned muscles pO2 fell significantly during stimulation and the muscles fatigued rapidly, whereas in the conditioned muscles, pO2 did not fall and the muscles were more resistant to fatigue. The perfusion profiles of the muscles transformed to an intermediate state were very similar to those of the 6-week-conditioned muscles and the improved ability to maintain pO2 was also clearly seen, suggesting that there was also less risk of distal ischaemia in this group.
Micro-amperometric measurements also provided a new insight into the effects of mobilising the flap. Control muscles showed a significant decrease in resting perfusion in the distal areas of the LDM flap as a consequence of mobilisation, although they were able to maintain tissue pO2. However, when the metabolic load upon the muscle was increased by stimulation, the perfusion-oxygenation pattern changed. Although all regions of the muscle showed active and reactive hyperaemia, pO2 in the distal part of the muscle fell by a mean of 9 mmHg whereas in conditioned muscles the pO2 fell by less than 3 mmHg. These observations support the view that ischaemia of the distal LDM can occur in cardiomyoplasty if the muscle is not sufficiently fatigue-resistant when contractions begin.
In conclusion, our results strengthen the case for conditioning the LDM prior to mobilisation, and show that the necessary changes can be achieved with a conditioning regime that is less demanding than that currently used clinically, with better preservation of power and contractile speed, and over a shorter time period.
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Acknowledgments |
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Footnotes |
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Appendix A. Conference discussion |
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TopAbstractIntroduction
MethodsResults
Discussion
Appendix A. Conference...References |
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Dr Barron: In terms of the in situ conditioning, the muscles were not mobilised in any way prior to the in situ conditioning, so original muscle length was left unchanged. They were acute experiments. The muscles were allowed to settle for a period of 30 min after the muscle had been raised. But I appreciate your comment that these are relatively acute experiments performed under the same anaesthetic. So in terms of the final results, all the testing was performed after a period of 30 min for the muscle to be ready to restart contracting again.
Dr Ali: I strongly agree with your approach of a new stimulation protocol of an intermediate muscle type. We have observed similar results in our experiments.
Dr G. Bolotin (Tel Aviv, Israel): Usually we have a 2-week vascular delay before starting the conditioning period, and if you do stimulation training in situ, it means that you will probably have to wait 2 weeks without any working of the muscles. Do you have any idea of this intermediate change whether they will stay or they would be different after 2 weeks without any working of the muscle?
Dr Barron: That is a very good point, and I think that the 2-week vascular delay may not in fact be so necessary if we are able to undertake a protocol of in situ conditioning because the muscles have already got better perfusion after mobilisation of the muscle. As you say, if you then do nothing for 2 weeks, the muscle will start to regress back to being a fast-twitch muscle again and will lose that benefit of having an intermediate phenotype. But it may be possible by using these continuous low-frequency stimulation protocols to continue stimulating the muscle even during those first 2 weeks. They are not tetanic twitches. They are single twitches that would probably not affect the performance of the ventricle.
Dr L. Von Segesser (Lausanne, Switzerland): Knowing all of this now, did you change your clinical protocol?
Dr Barron: No, we have not, but I think it is something that we would like to undertake.
Dr Von Segesser: What is your proposal, as a next step?
Dr Barron: We would like to do in situ conditioning and we would also like to consider a two-stage mobilisation where the muscle is partially elevated and then undergoes an in situ conditioning before the final procedure is performed.
Dr Von Segesser: Of course, one might wonder if one could use your protocol after wrapping.
Dr Barron: Indeed, but I think there are two benefits here, and one part of the benefit is a degree of in situ conditioning prior to raising the muscle because you are less likely to get distal ischemia.
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References |
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TopAbstractIntroduction
MethodsResults
Discussion
Appendix A. Conference...References |
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This article has been cited by other articles:
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  A. Lopez-Guajardo, H. Sutherland, J. C. Jarvis, and S. Salmons Induction of a fatigue-resistant phenotype in rabbit fast muscle by small daily amounts of stimulation J Appl Physiol, May 1, 2001; 90(5): 1909 - 1918. [Abstract] [Full Text] [PDF]
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 D. J. Barron, P. J. Etherington, C. P. Winlove, J. C. Jarvis, S. Salmons, and J. R. Pepper Combination of preconditioning and delayed flap elevation: evidence for improved perfusion and oxygenation of the latissimus dorsi muscle for cardiomyoplasty Ann. Thorac. Surg., March 1, 2001; 71(3): 852 - 861. [Abstract] [Full Text] [PDF]
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