Adaptation of energy metabolism of canine latissimus dorsi muscle in response to chronic electrical stimulation

Transformation of the latissimus dorsi (LD) muscle from a fast-twitch, fatigue-prone to a fatigue-resistant (heart-like) muscle, necessary to allow its application in cardiac assist devices, can be induced by chronic electrical stimulation. In adult dogs we studied the nature and time course of myofibrillar and metabolic adaptations in the LD muscle when exposed in situ to 24 weeks of continuous electrical stimulation. In addition, the metabolic properties of the stimulated muscle were compared with those of canine cardiac muscle. The proportion of immunohistochemically identified type I fibres increased on stimulation from 28% to 80%, while that of type II fibres decreased from 69% to 16%. Fibres of intermediate type (IIC and IC) appeared transiently; the highest levels were found between 4 and 8 weeks of stimulation. The activities of fructose-6-phosphate kinase and lactate dehydrogenase (LDH), which before stimulation were similar to those in heart, decreased to 18% and 34% of their initial values respectively. However, the LDH isozyme pattern changed towards that typical for cardiac muscle. These changes indicate a markedly decreased flux capacity through the glycolytic pathway which, however, is directed more towards the oxidative conversion of substrates. The mitochondrial capacity (maximal palmitate oxidation and pyruvate dehydrogenase complex activities) of the muscle did not change and remained at a level less than half of that of cardiac ventricular muscle. Contents of adenine nucleotides and endogenous substrates were maintained during stimulation. No further changes in the observed adaptations occurred after week 12 of stimulation. In conclusion, electrical stimulation of canine LD muscle induces a conversion to predominantly slow-twitch fibres, but the metabolic system of the stimulated muscle remains still markedly different from that of the heart.A preliminary report of this study was presented at the 3rd International Symposium on Transformed Skeletal Muscle for Cardiac Assist and Repair, Banff, Canada, October 1988 (see [11, 14])     

Histopathological findings in skeletal muscle used in human dynamic cardiomyoplasty

Patients submitted to dynamic cardiomyoplasty had an initial clinical improvement followed by a decrease in cardiac failure indices. A histopathological study of the skeletal muscle was undertaken to explain this. Latissimus dorsi fragments from 15 patients submitted to dynamic cardiomyoplasty in a 1:1 (heart beat:muscle stimulation) conditioning were analysed by light microscopy. The interval between surgery and obtaining the specimens (13 from necropsies, two from heart transplants) ranged from 37 days to 6 years. Nuclear clumps and internalization, the presence of round fibres, inflammation, and fibrosis were analysed semi-quantitatively; the thickness of muscle fibres and the percentage of tissue fat were measured by image analysis. The quantitative data were also compared, in 12 cases, with gender- and age-matched necropsy controls. The mean thickness of muscle fibres in cases and controls was 27.21+/-5.33 and 40.84+/-9.42 microm, respectively (p=0.001). The percentage of tissue fat in cases and controls was 12.04+/-12.66% and 0.93+/-0.91%, respectively (p=0.008). The duration of grafts correlated positively with the quantity of nuclear clumps (R=0.80, p<0.001) and round fibres (R=0.53, p=0.04), as well as with the percentage of tissue fat (R=0.68, p=0.005). Accordingly, a negative correlation was found between the duration of grafts and the mean diameter of fibres, characterizing muscle atrophy (R=-0.66, p=0.01). The longer the post-surgical period, the more intense the degenerative lesions. This study shows that skeletal muscle used in human dynamic cardiomyoplasty may atrophy and be replaced by fat when stimulation is synchronized to every cardiac beat. These findings could play a role in explaining the long-term results of this surgical procedure.     

The electrical stimulation of carbon nanotubes to provide a cardiomimetic cue to MSCs

Once damaged, cardiac muscle has little intrinsic repair capability due to the poor regeneration potential of remaining cardiomyocytes. One method of overcoming this issue is to deliver functional cells to the injured myocardium to promote repair. To address this limitation we sought to test the hypothesis that electroactive carbon nanotubes (CNT) could be employed to direct mesenchymal stem cell (MSC) differentiation towards a cardiomyocyte lineage. Using a two-pronged approach, MSCs exposed to medium containing CNT and MSCs seeded on CNT based polylactic acid scaffolds were electrically stimulated in an electrophysiological bioreactor. After electrical stimulation the cells reoriented perpendicular to the direction of the current and adopted an elongated morphology. Using qPCR, an upregulation in a range of cardiac markers was detected, the greatest of which was observed for cardiac myosin heavy chain (CMHC), where a 40-fold increase was observed for the electrically stimulated cells after 14 days, and a 12-fold increase was observed for the electrically stimulated cells seeded on the PLA scaffolds after 10 days. Differentiation towards a cardioprogenitor cell was more evident from the western blot analysis, where upregulation of Nkx2.5, GATA-4, cardiac troponin t (CTT) and connexin43 (C43) was seen to occur. This was echoed in immunofluorescent staining, where increased levels of CTT, CMHC and C43 protein expression were observed after electrical stimulation for both cells and cell-seeded scaffolds. More interestingly, there was evidence of increased cross talk between the cells as shown by the pattern of C43 staining after electrical stimulation. These results establish a paradigm for nanoscale biomimetic cues that can be readily translated to other electroactive tissue repair applications.     

Transmission from intramural inhibitory nerves to the smooth muscle of the guinea-pig taenia coli

1. Membrane potential changes of smooth muscle cells were recorded during stimulation of the intramural inhibitory nerves to the taenia coli.2. Stimulation across the taenia coli with single pulses of 200 musec duration excites the intramural nerves and not the muscle directly.3. The membrane potential changes due to stimulation of the intramural inhibitory nerves were different from those produced by perivascular inhibitory nerve stimulation in the following ways: hyperpolarizations (i.j.p.'s) of up to 25 mV were produced in response to single pulses; the latency, i.e. the time taken for the membrane to hyperpolarize after a stimulus of maximal strength, was as short as 80 msec; when the nerves were stimulated repetitively the membrane was hyperpolarized by up to 35 mV and all spontaneous activity was abolished; the mean hyperpolarization due to repetitive stimulation increased with the frequency of stimulation up to 10 pulses/sec and then remained constant; the hyperpolarization due to stimulation at frequencies greater than 5 pulses/sec was not maintained but decreased after 3-5 sec of stimulation; and finally when stimulation had ceased action potentials commenced firing at frequencies greater than normal.4. The amplitude and rate of hyperpolarization of the i.j.p. increased with increasing strength of stimulation until a maximum amplitude and rate of hyperpolarization was reached. The recovery or depolarizing phase of the i.j.p. was exponential with a time constant which varied from about 250 msec to 500 msec and could not therefore be due to the discharge of the membrane capacitance. In some cases there was an inflexion on this depolarizing phase and in these cases recovery led directly into an action potential.5. Spontaneous hyperpolarizations of the membrane were seen in some cells, and these hyperpolarizations were similar to those recorded on submaximal stimulation of the intramural nerves.6. There were no changes in the characteristics of the i.j.p. in the presence of guanethidine or bretylium.     

Properties of the inhibitory potential of smooth muscle as observed in the response to field stimulation of the guinea-pig taenia coli

1. The inhibitory potential evoked by field stimulation of the guineapig taenia coli was studied in hypertonic solution in which the spontaneous electrical and mechanical activity was slowed or abolished.2. There was no direct correlation between the inhibitory potential and the muscle membrane polarization in response to the stimulating current. The chronaxie for the inhibitory potential was less than 1 msec and that for the muscle spike 20-30 msec.3. The inhibitory potential decreased in size along the tissue when one end of the tissue was stimulated, but its spatial decay was slower than that of the electrotonic potential in the muscle bundle.4. Tetrodotoxin (5 x 10(-7) g/ml.) abolished the inhibitory potential without affecting the electrical properties of the muscle membrane. It was concluded that the inhibitory potential was the result of stimulation of intrinsic inhibitory nerves.5. The following properties of the intrinsic nerve fibres were deduced from the experimental results: The length of the nerve fibres is probably only a few mm and the space constant of the order of 0.1 mm. No repetitive firing is produced by a long current pulse. The absolute refractory period is 3-4 msec.     

Aspects of the Central Integration of Arterial Baroreceptor and Cardiac Ventricular Receptor Reflexes in the Cat

The possible central integrative mechanisms, responsible for the earlier reported, differentiated reflex engagement of the renal and muscle vessels and the heart from cardiac ventricular receptors and arterial baroreceptors, respectively, were analyzed in atropinized cats. The reflex renal vessel, muscle vessel and heart rate responses, expressed as per cent of maximum, to graded activations of arterial baroreceptors (sinus pressure variations) and stimulations of ventricular receptor afferents in the cardiac nerve were systematically compared. Cardiac nerve stimulation with low frequencies was found to elicit more pronounced reflex renal vessel responses than muscle vessel and heart rate responses. In contrast, elevations of sinus pressure induced equally pronounced renal and muscle vessel responses. High frequency cardiac nerve stimulation elicited maximal reflex renal vessel responses, but only submaximal effects on muscle vessels and heart rate, while intense baroreceptor stimulation induced maximal reflex effector responses throughout. The submaximal heart rate response to cardiac nerve stimulation is probably due to a simultaneous activation of excitatory afferents. On the other hand, the less pronounced muscle than renal vessel responses when the cardiac nerve was stimulated probably reflect a relatively sparse innervation of muscle vasomotor neurons by ventricular receptor afferents, which seem instead to be preferentially oriented towards renal vasomotor and, possibly, cardiac motor neurons.     

Aspects of the central integration of arterial baroreceptor and cardiac ventricular receptor reflexes in the cat.

The possible central integrative mechanisms, responsible for the earlier reported, differentiated reflex engagement of the renal and muscle vessels and the heart from cardiac ventricular receptors and arterial baroreceptors, respectively, were analyzed in atropinized cats. The reflux renal vessel, muscle vessel and heart rate responses, expressed as per cent of maximum, to graded activations of arterial baroreceptors (sinus pressure variations) and stimulations of ventricular receptor afferents in the cardiac nerve were systematically compared. Cardiac nerve stimulation with low frequencies was found to elicit more pronounced reflex renal vessel responses than muscle vessel and heart rate responses. In contrast, elevations of sinus pressure induced equally pronounced renal and muscle vessel responses. High frequency cardiac nerve stimulation elicited maximal reflex renal vessel responses, but only submaximal effects on muscle vessels and heart rate, while intense baroreceptor stimulation induced maximal reflex effector responses throughout. The submaximal heart rate response to cardiac nerve stimulation is probably due to a simultaneous activation of excitatory afferents. On the other hand, the less pronounced muscle than renal vessel responses when the cardiac nerve was stimulated probably reflect a relatively sparse innervation of muscle vasomotor neurons by ventricular receptor afferents, which seem instead to be preferentially oriented towards renal vasomotor and, possibly, cardiac motor neurons.     

The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds

One of the major challenges in engineering thick, complex tissues such as cardiac muscle, is the need to pre-vascularize the engineered tissue in vitro to enable its efficient integration with host tissue upon implantation. Herein, we explored new magnetic alginate composite scaffolds to provide means of physical stimulation to cells. Magnetite-impregnated alginate scaffolds seeded with aortic endothelial cells stimulated during the first 7 days out of a total 14 day experimental course showed significantly elevated metabolic activity during the stimulation period. Expression of proliferating cell nuclear antigen (PCNA) indicated that magnetically stimulated cells had a lower proliferation index as compared to the non-stimulated cells. This suggests that the elevated metabolic activity could instead be related to cell migration and re-organization. Immunostaining and confocal microscopy analyses supported this observation showing that on day 14 in magnetically stimulated scaffolds without supplementation of any growth factors, cellular vessel-like (loop) structures, known as indicators of vasculogenesis and angiogenesis were formed as compared to cell sheets or aggregates observed in the non-stimulated (control) scaffolds. This work is the first step in our understanding of how to accurately control cellular organization to form tissue engineered constructs, which together with additional molecular signals could lead to a creation of an efficient pre-vascularized tissue construct with potential applicability for transplantation.     

An Engineering Model of Dynamic Cardiomyoplasty. I.

Dynamic cardiomyoplasty (DCM) is an emerging surgical procedure for heart failure in which the patient's latissimus dorsi (LD) muscle is wrapped around the heart and stimulated to contract in synchrony with the heartbeat as a cardiac assist measure. A 6 week training protocol of progressive electrical stimulation renders the normally fatigueable skeletal muscle fatigue-resistant and suitable for chronic stimulation. To date, over 500 procedures have been performed in worldwide clinical trials. Investigators typically report symptomatic improvement and modest hemodynamic improvement in patients. Controversy exists regarding the exact mechanism of DCM. To test the hypothesis that DCM augments cardiac stroke volume through improvement in systolic function, we formulated an engineering model of dynamic cardiomyoplasty to predict stroke volume. The heart and the LD were modeled as nested (series) elastance chambers, and the vasculature was represented by a two-element Windkessel model. Using five healthy goats, we verified model predictions of stroke volume for both stimulator ON beats (y=1.00x–0.08, r=0.87, p < 0.0001) and OFF beats (y=1.01x+1.06, r=0.91, p < 0.0001), where x and y are the measured and predicted stroke volumes, respectively. The model confirms that using untrained latissimus dorsi applied to the normal myocardium produces only moderate increases in stroke volume and suggests that future research should focus on increasing LD strength after training.     

Model study of the spread of electrotonic potential in cardiac tissue

This model study describes the electrotonic response of a cable model of cardiac tissue stimulated at one point. The stimulus is applied intracellularly in the form of a 2ms pulse of current of near threshold amplitude. The attenuation of the electrotonic potential with distance and its mode of propagation along the cable are compared for equivalent passive, continuous and discontinuous cables. The three structures have the same basic physical and electrical characteristic and they differ either with respect to being active or passive or to the presence or absence of intercellular gap junctions. In the continuous cable a just subthreshold stimulus produces a local active response which propagates more slowly and is attenuated less rapidly with distance than in a passive cable. The spatial decrement of the local response in a discontinuous cable is faster than in a continuous cable of equal average resistivity. It is suggested that the larger time constant of the foot of the action potential observed in the longitudinal direction in cardiac muscle could be due in part to the electrotonic spread of the local response from the site of stimulation.     

Comparison of stimulation patterns for FES-cycling using measures of oxygen cost and stimulation cost

AIM: The energy efficiency of FES-cycling in spinal cord injured subjects is very much lower than that of normal cycling, and efficiency is dependent upon the parameters of muscle stimulation. We investigated measures which can be used to evaluate the effect on cycling performance of changes in stimulation parameters, and which might therefore be used to optimise them. We aimed to determine whether oxygen cost and stimulation cost measurements are sensitive enough to allow discrimination between the efficacy of different activation ranges for stimulation of each muscle group during constant-power cycling. METHODS: We employed a custom FES-cycling ergometer system, with accurate control of cadence and stimulated exercise workrate. Two sets of muscle activation angles ("stimulation patterns"), denoted "P1" and "P2", were applied repeatedly (eight times each) during constant-power cycling, in a repeated measures design with a single paraplegic subject. Pulmonary oxygen uptake was measured in real time and used to determine the oxygen cost of the exercise. A new measure of stimulation cost of the exercise is proposed, which represents the total rate of stimulation charge applied to the stimulated muscle groups during cycling. A number of energy-efficiency measures were also estimated. RESULTS: Average oxygen cost and stimulation cost of P1 were found to be significantly lower than those for P2 (paired t-test, p<0.05): oxygen costs were 0.56+/-0.03l min-1 and 0.61+/-0.04l min-1 (mean+/-S.D.), respectively; stimulation costs were 74.91+/-12.15 mC min-1 and 100.30+/-14.78 mC min-1 (mean+/-S.D.), respectively. Correspondingly, all efficiency estimates for P1 were greater than those for P2. CONCLUSION: Oxygen cost and stimulation cost measures both allow discrimination between the efficacy of different muscle activation patterns during constant-power FES-cycling. However, stimulation cost is more easily determined in real time, and responds more rapidly and with greatly improved signal-to-noise properties than the ventilatory oxygen uptake measurements required for estimation of oxygen cost. These measures may find utility in the adjustment of stimulation patterns for achievement of optimal cycling performance.     

Nonlinear cell response to strong electric fields

The response of living cells to externally applied electric fields is of widespread interest. In particular, the intensification of electric fields across cell membranes is believed to be responsible, through membrane rupture and reversible membrane breakdown processes, for certain types of tissue damage in electrical trauma cases which cannot be attributed to Joule heating. Large elongated cells such as skeletal muscle fibres are particularly vulnerable to such damage. Previous theoretical studies of field intensification across cell membranes in such cells have assumed the membrane current to be linear in the applied field (Ohmic membrane conductivity) and were limited to sinusoidal applied fields. In this paper, we investigate a simple model of a long cylindrical cell, corresponding to nerve or skeletal muscle cells. Employing the electroquasistatic approximation, a system of coupled first-order differential equations for the membrane electric field is derived which incorporates arbitrary time dependence in the external field and nonlinear membrane response (non-Ohmic conductivity). The behaviour of this model is investigated for a variety of applied fields in both the linear and highly nonlinear regimes. We find that peak membrane fields predicted by the nonlinear model are approximately twice as intense, for low-frequency electrical trauma conditions, as those of the linear theory.     

Effects of chronic low-frequency stimulation on Ca2+-regulatory membrane proteins in rabbit fast muscle

Since chronic low-frequency stimulation of fast-twitch muscle fibers has a profound effect on all major functional elements of skeletal muscle, we analyzed the potential changes in the levels of Ca2+-regulatory membrane proteins during fast-to-slow transformation. In this study we show that, in addition to isoform-switching in myosin heavy chains, electrostimulation triggers a decline in fast isoforms and an increase in slow/cardiac isoforms of Ca2+-ATPase and calsequestrin. The levels of excitation-contraction coupling elements, such as the ryanodine receptor, the dihydropyridine receptor, triadin and sarcalumenin, decreased sharply following stimulation. In contrast, levels of Na+/K+-ATPase and calreticulin increased in the microsomal fraction. Crosslinking studies have revealed that in normal and stimulated muscle the Ca2+-ATPase isoforms exist predominantly as oligomeric structures, and that the central elements of excitation-contraction coupling also form large triad complexes. Changes in the levels and pattern of isoform expression of the muscle membrane proteins studied here suggest that these biochemical alterations reflect molecular adaptations to changed demands in ion homeostasis and signal transduction in muscle that exhibits enhanced contractile activity. Overall, these findings support the physiological concept that there are muscle fiber-type specific differences in the fine-tuning of the excitation-contraction-relaxation cycle, as well as the idea that mature skeletal muscle fibers exhibit a high degree of plasticity.     

Effect of Carotid Sinus Stimulation on Cardiac Output and Peripheral Vascular Resistance during Changes in Blood Volume Distribution in Man

In ten healthy subjects (mean age 29.6 years) the hemodynamic response to carotid sinus stimulation (neck suction - 40 mmHg) was studied under control conditions and during peripheral pooling of blood (lower body negative pressure). Heart rate, arterial and central venous pressure, cardiac output and forearm blood flow were measured. The time sequence of the heart rate response was studied separately in six healthy subjects. During control conditions, carotid sinus stimulation induced a significant decrease in arterial pressure and heart rate. The blood pressure decrease mainly reflected a reduction in cardiac output, total peripheral vascular resistance being essentially unchanged. However, in the skeletal muscle, represented by a forearm segment, vascular resistance decreased significantly. During lower body negative pressure (LBNP) the same stimulation of the carotid sinus induced a significantly greater fall in mean arterial pressure even though the reduction in cardiac output was slightly smaller on the average than in the control condition. The heart rate increased, probably secondary to a time dependent increase in heart rate elicited by the continuous LBNP stimulus. Total peripheral vascular resistance decreased significantly during LBNP, the reaction likewise differing significantly from that in the control condition. Thus the augmented blood pressure response was due to a more pronounced vasodilatation when the carotid sinus was stimulated during lower body negative pressure. The results indicate that the hemodynamic changes elicited by carotid sinus stimulation are modified by changes in the distribution of blood volume and in the tone of resistance vessels.     

Termination of transmitter release by stimulation of sodium-potassium activated ATPase.

1. The release of acetylcholine (ACh) from Auerbach's plexus of guinea-pig ileum has been measured in eserinized Krebs solution using longitudinal muscle strip preparations. 2. Removal of the external K ions enhanced both the resting and stimulated release of ACh from the plexus. This effect was not affected by tetrodotoxin. 3. On readmission of K+ to tissues which had been suspended in K-free Krebs solution the release of ACh was promptly reduced in both stimulated and unstimulated tissues. The extent of the reduction of ACh release depended on the exposure time to K-free solution, the recovery being delayed by longer exposure. 4. The ACh releasing effect of (1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) was completely inhibited by the readmission of K ions to tissue which had been kept in K-free Krebs solution. 5. Rb+ substitution for K+ produced no change in ACh release and addition of 5-9 mM-Rb after K removal reduced the release of ACh as K did readmission. When the K ions were substituted by Cs+, both the resting and stimulated release were enhanced. The amount of ACh released by a stimulus was enhanced both at low and high frequency of sustained stimulation. 6. Removal of the external K ions increased the release of tritiated noradrenaline (NA), from isolated rat iris; however, when K+ (5-9 mM) was readmitted the release was reduced even below the control value. 7. It is concluded that the stimulation of (Na+-K+)-activated ATP-ase in the membrane inhibits the release of transmitter, and under physiological condition Ca-fluxes and the subsequent inhibition of membrane ATP-ase may be involved in triggering the release of transmitter.     

The generation of the cardiac action potential: After the first millisecond

After an initial, transient voltage-and time-dependent burst of sodium current (equivalent to that occurring in nerve), the membrane current of cardiac muscle reverses in sign to a maximum value that is orders of magnitude smaller than that seen in nerve. The membrane of cardiac muscle, rather than exchanging an increased permeability to sodium ions (Na⁺) for one to potassium ions (K⁺), appears to become relatively impermeable to a variety of ions. It is argued that in a tissue such as cardiac muscle where the time when the cell is active is comparable to that when it is quiescent, the current generated by the active electrogenic transport/exchange of Na⁺, K⁺, and Ca²⁺ must be comparable to the corresponding currents generated by the passive transport of these ions. Consequently, the complex voltage and time dependency of the membrane current on the time scale of repolarization and beyond is generated, at least in part, by the complex time and voltage dependency of these transport/exchange processes. Measurement of the electrochemical properties of such transport/exchange mechanisms must ultimately be made on the individual mechanisms in isolation, e.g., in artificial membrane systems, before their contribution to the generation of the cardiac action potential can be unequivocably determined.     

Histological diagnosis of myocardial injury. Comparison of hematoxylin-basic fuchsin-picric acid (HBFP)-stained sections obtained during autopsy with isolated viable rat cardiac myocytes exposed to anoxia.

The light microscopic appearances in hematoxylin-basic fuchsin-picric acid (HBFP)-stained histological sections from cardiac and skeletal muscle tissue were put in relation to the reactions of isolated viable rat cardiac myocytes exposed to anoxia in suspension and their morphology in paraffin-embedded sections. Special attention was paid to prenecrotic phases of myocytic injury which were followed, in viable rat cardiac myocytes, by light microscopy, and confirmed with biochemical assays indicating increased plasma membrane permeability. In cases of sudden death, and traumatic injury to the heart and skeletal muscle, there was good agreement between alterations demonstrated with the HBFP technique and alterations of viable rat cardiac myocytes exposed to anoxia. In isolated myocytes, these alterations were associated with irregular contractility which, when occurring in situ, might have influenced the cardiac function prior to death. Moreover, the changes develop at a much faster rate than the inflammatory reaction following tissue injury and may therefore be regarded as an early vital phenomenon of significance in clinico-pathological and medico-legal considerations.