published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Pharmacology / Master / Master of Research in Medicine

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Experiments have been designed to investigate the physiological factors influencing the interrelationship between excitation and force generation that may counteractt he processesle ading to a decline in force (fatigue) during stimulatedi sometric contractions of the human adductor pollicis in vivo. Indices of isometric force, relaxation and contraction rates and evoked compound muscle action potentials (CMAP) were measured during defined patterns of stimulated activity (via the motor nerve). A computerized stimulator controller for precise generation of trains of electrical impulses was developed for this purpose. Forces generated at different frequencies were reproducible on separate occasions. Using an ascending frequency stimulation protocol (1-100Hz) the relationship between force decline and excitation (measured as the amplitude of the surface evoked CMAP) appeared to be dependent on stimulation frequency during ischaemic and nonoccluded activity. At high frequencies (50-100Hz), a `safety factor' was apparent, allowing preservation of force despite a marked fall in excitation, whereas at low frequencies (1-10Hz) force initially potentiated and then declined in excess of excitation. Maximum relaxation rate was reduced at all stimulation frequencies and was independent of stimulation frequency. Contractile activity performed was shown to be linearly related to maximum relaxation rate over a frequency range of 20-100Hz for up to 30max. seconds. Contractile activity performed was therefore used as a measure of the metabolic cost of a contraction. Force failure appeared to depend upon the numbers of stimuli delivered, independent of frequency, rather than on contractile activity performed, suggesting that electrophysiological factors are of importance in contributing to fatigue. Further studieso n CMAP characteristicsd emonstrateda broadeningo f the action potential, reflecting a slowing of conduction velocity, which is thought to lead to `runin' of action potentials, and hencet he reduction of CMAP amplitude associatedw ith the high-frequency `safety factor'. The broadening of the action potential recovered immediately during ischaemic conditions at 100Hz following 2400 stimuli but did not recover following prolonged activity at 20Hz until circulation was restored, whereas CMAP amplitude recovered immediately at both frequencies, suggesting that slowing of conduction velocity may be dependent on metabolic factors at low stimulation frequencies which in turn may depend on the contractile history of the muscle. Patients with myophosphorylase deficiency (and thus unable to utilize glycogen), were studied to investigate the importance of energy supply. A failure of ischaemic recovery of the CMAP amplitude and no broadening of the CMAP after stimulated activity at 20Hz was observed, suggesting a failure of excitation of individual muscle cells occurs resulting in force failure in these individuals. Reversing the pattern of stimulation resulted in an initial enhancement of low frequency (10Hz) force and a prolonged maintenance of this force throughout the period of contraction studied. This was independent of slowing of relaxation or excitation. The initial force enhancement may result from the increased slowing of relaxation, and in addition, a form of post-tetanic twitch potentiation operates to counteract the decline in force despite a loss in excitation. In conclusion, during stimulated contractile activity of the adductor pollicis, mechanisms act to maintain or increase force generated per action potential distal to the sarcolemmal membrane, at both high and low frequencies of stimulation, thereby counteracting mechanisms that lead to fatigue. It is postulated that the alterations in intramuscular processes may allow voluntary isometrically contracting muscle to optimize force production at the onset of a contraction where high motor unit discharge rates are initially developed, delaying or eliminating the influence of excitation failure which would lead to contractile failure once maximal force is achieved, and subsequently to optimize contractile activation in the light of possible excitation failure as motor unit discharge rates decline. These findings may have important functional implications and may form the basis of physiological strategies for optimizing force production in the development of stimulation regimes for `functional electrical stimulation' or to any area of skeletal muscle research in which fatigue resistance is of importance.

Nous avons généré des souris déficientes pour les gènes codant pour les échangeurs Na/Ca de type I (NCX1) et de type III (NCX3) afin d'étudier, in vivo, le rôle de ces deux protéines. L‘analyse phénotypique des souris adultes totalement déficientes pour le gène Ncx1 (Ncx1-/-) n'a pu être menée étant donné que ces souris décèdent au cours du développement embryonnaire. Les souris déficientes pour le gène Ncx3 (Ncx3-/-) sont viables et fertiles. Nous avons analysé l'effet de l'inactivation du gène Ncx3 dans le muscle squelettique et plus particulièrement au niveau de la jonction neuromusculaire. L'analyse histologique des muscles squelettiques de souris Ncx3-/- a révélé des altérations des fibres musculaires caractérisées par la présence de foyers de fibres nécrotiques et d'infiltrats de cellules mononuclées. L'analyse électromyographique classique a montré un électromyogramme anormal du muscle gastrocnémien de souris Ncx3-/-, révélant une affection neuromusculaire pré- et post-synaptique caractérisée par (i) la petitesse de l'amplitude de la réponse M au repos, (ii) le décrément après stimulation répétitive à basse fréquence, (iii) l'incrément après stimulation répétitive à haute fréquence et (iv) la facilitation post-exercice. L'électromyographie à fibre unique a révélé une MCD élevée et des blocages anormaux de la transmission neuromusculaire, reflétant une atteinte post-synaptique de la jonction neuromusculaire chez les souris Ncx3-/-. L'ensemble de ces anomalies électromyographiques sont les caractéristiques du syndrome myasthénique de Lambert-Eaton. Finalement, pour déterminer les conséquences de l'inactivation du gène Ncx3 sur l'activité physique des souris Ncx3-/-, nous avons réalisé des tests comportementaux sur ces souris. Ces tests ont permis de détecter un épuisement et une faiblesse musculaire accrus à l'effort chez ces souris. En conclusion, nos observations montrent que les souris Ncx3-/- présentent des anomalies électromyographiques similaires à celles du syndrome myasthénique de Lambert-Eaton. Ces résultats suggèrent que l'échangeur NCX3 est peut-être impliqué dans la pathogenèse de certaines formes de cette maladie. Des études supplémentaires afin de confirmer notre hypothèse devront donc être réalisées. / We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein which mediates cellular Ca2+ efflux (forward mode) or Ca2+ influx (reverse mode) and thus controls intracellular Ca2+ concentration. NCX3-deficient mice (Ncx3-/-) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities including low compound muscle action potential amplitude, a decremental response at low frequency nerve stimulation, an incremental response and a prominent post-exercise facilitation at high frequency nerve stimulation as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3-/- neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high frequency nerve stimulation. It is suggested that Ca2+ entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca2+ and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca2+ concentrations in the skeletal muscle fibers and at the neuromuscular junction.

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