The influence of denervation on DNA content in skeletal muscle fibers

In this paper we describe a significant reduction of nuclear DNA content in skeletal muscle fibers after denervation. Some properties of an endogenous DNAase activity in normal and denervated muscle are also reported.     

Effect of denervation on ATP consumption rate of diaphragm muscle fibers.

Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC(2X) isoform but not in fibers expressing MHC(slow) and MHC(2A). Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP(iso)) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC(2X). In single-type-identified, permeabilized DIAm fibers, ATP(iso) was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V(o)) and cross-bridge cycling rate [estimated from the rate constant for force redevelopment (k(TR)) following quick release and restretch] was also examined. Two weeks after DNV, ATP(iso) was significantly reduced in fibers expressing MHC(2X), but unaffected in fibers expressing MHC(slow) and MHC(2A). This effect of DNV on fibers expressing MHC(2X) persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V(o) and k(TR) were slowed in fibers expressing MHC(2X), consistent with the effect on ATP(iso). The difference between V(max) ATPase and ATP(iso) reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC(2X). DNV-induced reductions in ATP(iso) and V(max) ATPase of fibers expressing MHC(2X) reflect the underlying decrease in MHC content, while reduction in ATP(iso) also reflects a slowing of cross-bridge cycling rate.     

Electrophysiological properties of biventer cervicis muscle fibers of normal and roller pigeons

Cable parameters, excitability characteristics, and contractile response to acetylcholine were measured in biventer cervicis muscles from Helmet pigeons, Racing Homer pigeons and Parlor (nonflying) Roller pigeons. Cable parameters for the three strains, were respectively: calculated diameter, 30.1, 42.5, and 37.3 m̈m; membrane resistance, 450, 556, and 386 ω · cm²; membrane capacitance, 4.2, 3.9, and 4.5 m̈F/cm², and myoplasmic resistivity, 79, 185, and 116 ω · cm. Significant differences between excitability characteristics of Homer pigeon and Roller pigeon fibers were a 17% shorter maximal latency for spike initiation (P < 0.025) and 24% lower rheobasic current (P < 0.05) in Roller fibers. Doseresponse curves of isolated biventer cervicis to acetylcholine revealed slight, but significant, differences between Helmets and Rollers. These are the first electrophysiological data from pigeon skeletal muscle and the first from any avian biventer cervicis. The biventer muscles of chickens contain mainly “slow” fibers, but our results show that pigeon biventer fibers have properties similar to the “fast” PLD fibers of the chicken. Furthermore, the existence of different myoplasmic resistivities for each strain of pigeons used in this study suggests the need for more careful determination of this parameter in electrophysiological investigations. Although our results show that Roller pigeon fibers differ from those of nonrolling pigeons in the respects described above, these differences are minor in comparison to the severe behavioral abnormalities of Roller pigeons. Some yet untested component of neuromuscular transmission may be directly involved in the rolling phenomenon, but the differences we report may simply be due to strain differences, muscle hypertrophy, or a more severe defect elsewhere in the nervous system.     

大鼠单一肌梭的电生理特征

本研究旨在观察大鼠单一肌梭的电生理特征.从大鼠比目鱼肌中分离单一肌梭,用空气隔绝法观察大鼠单一肌梭感觉末梢在不同液体环境中的放电活动.结果显示:在基础生理盐水溶液中,大鼠单一肌梭的自发放电频率很低,平均(51.78±25.63) impulse/1000s(n=13);在加有适量氨基酸的生理盐水溶液中,其自发放电频率明...     

Electrophysiological properties of biventer cervicis muscle fibers of normal and roller pigeons.

Cable parameters, excitability characteristics, and contractile response to acetylcholine were measured in biventer cervicis muscles from Helmet pigeons, Racing Homer pigeons and Parlor (nonflying) Roller pigeons. Cable parameters for the three strains, were respectively: calculated diameter, 30.1, 42.5, and 37.3 mum; membrane resistance, 450, 556, and 386 omega-cm2; membrane capacitance, 4.2, 3.9, and 4.5 muF/cm2, and myoplasmic resistivity, 79, 185, and 116 omega-cm. Significant differences between excitability characteristics of Homer pigeon and Roller pigeon fibers were a 17% shorter maximal latency for spike initiation (P less than 0.025) and 24% lower rheobasic current (P less than 0.05) in Roller fibers. Dose-response curves of isolated biventer cervicis to acetylcholine revealed slight, but significant, differences between Helmets and Rollers. These are the first electrophysiological data from pigeon skeletal muscle and the first from any avian biventer cervicis. The biventer muscles of chickens contain mainly "slow" fibers, but our results show that pigeon biventer fibers have properties similar to the "fast" PLD fibers of the chicken. Furthermore, the existence of different myoplasmic resistivities for each strain of pigeons used in this study suggests the need for more careful determination of this parameter in electrophysiological investigations. Although our results show that Roller pigeon fibers differ from those of nonrolling pigeons in the respects described above, these differences are minor in comparison to the severe behavioral abnormalities of Roller pigeons. Some yet untested component of neuromuscular transmission may be directly involved in the rolling phenomenon, but the differences we report may simply be due to strain differences, muscle hypertrophy, or a more severe defect elsewhere in the nervous system.     

Degeneration of the afferent fibers simulating the effects of motor denervation on skeletal muscle

Degeneration of afferent nerve fibres was induced in rats in order to observe its effects on the properties of the extra-junctional membrane of soleus muscle fibres. In one approach, removal of dorsal root ganglia L4 and L5 was accomplished in preparations with intact or impulse-blocked (with tetrodotoxin containing cuffs around the sciatic nerve) efferent innervation. Spike resistance to tetrodotoxin developed in the inactive deafferented preparations earlier and to a greater extent than in control, that is only impulse-blocked, preparations. In another series of experiments, efferent denervation alone proved to be less effective than the association of efferent and afferent denervation. On the other hand, section of the afferent fibres central to the dorsal root ganglia was without effect. These results are consistent with the interpretation that products of nerve degeneration contribute together with inactivity to the development of the extrajunctional membrane changes observed in skeletal muscle after denervation.     

Effect of unilateral denervation on maximum specific force in rat diaphragm muscle fibers.

We hypothesize that 1) the effect of denervation (DNV) is more pronounced in fibers expressing fast myosin heavy chain (MHC) isoforms and 2) the effect of DNV on maximum specific force reflects a reduction in MHC content per half sarcomere or the number of cross bridges in parallel. Studies were performed on single Triton X-100-permeabilized fibers activated at a pCa (-log Ca2+ concentration) of 4.0. MHC content per half sarcomere was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. After 2 of wk DNV, the maximum specific force of fibers expressing MHC2X was reduced by approximately 40% (MHC(2B) expression was absent), whereas the maximum specific force of fibers expressing MHC2A and MHC(slow) decreased by only approximately 20%. DNV also reduced the MHC content in fibers expressing MHC2X, with no effect on fibers expressing MHC2A and MHC(slow). When normalized for MHC content per half sarcomere, force generated by DNV fibers expressing MHC2X and MHC2A was decreased compared with control fibers. These results suggest the force per cross bridge is also affected by DNV.     

Effects of denervation and colchicine treatment on the chloride conductance of rat skeletal muscle fibers

Membrane potentials, cable parameters, and component resting conductances were measured in extensor digitorum longus (EDL) muscle fibers from adult rats in vitro at 24°C, after 15 to 18 days of denervation by nerve section, and at seven to ten days following epineural injection of 100 to 450 μg of colchicine in the peroneal nerve. The denervated muscles were paralyzed throughout the experimental period, whereas the colchicine-treated preparations showed no clinical paralysis except for the first day or two. The EDL from the untreated side served as a control. Both the denervated and colchicine-treated fibers were depolarized, showed signs of fibrillation, had tetrodotoxin-resistant action potentials, and membrane resistance was increased two- to sevenfold. In the denervated fibers, mean chloride conductance G_Cl dropped from a control value of 3196 to 596 μmhos/cm² while mean potassium conductance G_K showed a tendency to rise from 260 to 332 μmhos/cm². Colchicine-treated fibers while showing a similar fall in mean G_Cl from 2993 to 1066 μmhos/cm², also showed a significant fall in mean G_K from 213 to 116 μmhos/cm². It was concluded that factors transported by the microtubular system are important for the maintenance of the high resting G_Cl of mammalian skeletal muscle fibers.     

Early changes of type 2B fibers after denervation of rat EDL skeletal muscle.

Skeletal muscle type 2B fibers normally receive a moderate level of motoneuron discharge. As a consequence, we hypothesize that type 2B fiber properties should be less sensitive to the absence of the nerve. Therefore, we have investigated the response of sarcoplasmic reticulum and myofibrillar proteins of type 2B fibers isolated from rat extensor digitorum longus muscle after denervation (2 and 7 days). Single fibers were identified by SDS-PAGE of myosin heavy chain isoforms. Electrophysiological and isometric contractile properties of the whole muscle were also analyzed. The pCa-tension relationship of type 2B single fibers was shifted to the left at 2 days and to right at 7 days after denervation, with significant differences in the Hill coefficients and pCa threshold values in 2- vs. 7-day-denervated fibers. The sarcoplasmic reticulum Ca2+ uptake capacity and rate significantly decreased after 2 days of denervation, whereas both increased at 7 days. Caffeine sensitivity of sarcoplasmic reticulum Ca2+ release was transitory and markedly increased in 2-day-denervated fibers. Our results indicate that type 2B fiber functional properties are highly sensitive to the interruption of nerve supply. Moreover, most of 2-day-denervated changes were reverted at 7 days.     

Effect of olivomycin on denervation changes in the membrane of frog tonic muscle fibers]

Membrane properties of frog denervated tonic muscle fibers were investigated after the action of olivomycin, which is an inhibitor of protein synthesis. Olivomycin injected as a single dose (7.5 mg) during 1--2 days after the denervation of m. pyriformis decreases the regenerative action potentials. This action is decreased three weeks after denervation. After denervation, olivomycin failed to prevent the appearance of extrajunctional cholinoergic receptors, but blocked the increase of junctional ones. The data support the view that some neuronal factor may influence the synthesis of functional membrane proteins.     

Effects of denervation and colchicine treatment on the chloride conductance of rat skeletal muscle fibers.

Membrane potentials, cable parameters, and component resting conductances were measured in extensor digitorum longus (EDL) muscle fibers from adult rats in vitro at 24 degrees C, after 15 to 18 days of denervation by nerve section, and at seven to ten days following epineural injection of 100 to 450 mug of colchicine in the peroneal nerve. The denervated muscles were paralyzed throughout the experimental period, whereas the colchicine-treated preparations showed no clinical paralysis except for the first day or two. The EDL from the untreated side served as a control. Both the denervated and colchicine-treated fibers were depolarized, showed signs of fibrillation, had tetrodotoxin-resistant potentials, and membrane resistance was increased two- to sevenfold. In the denervated fibers, mean chloride conductance GC1 dropped from a control value of 3196 to 596 mumhos/cm2 while mean potassium conductance GK showed a tendency to rise from 260 to 332 muhos/cm2. Colchicine-treated fibers while showing a similar fall in mean GC1 from 2993 to 1066 mumhos/cm2, also showed a significant fall in mean GK from 213 to 116 mumhos/cm2. It was concluded that factors transported by the microtubular system are important for the maintenance of the high resting GC1 of mammalian skeletal muscle fibers.     

Intersarcomere dynamics of single muscle fibers during fixed-end tetani

The contraction dynamics of end and center regions of single fibers have been measured during fixed-end tetani. Experimental control and data acquisition are provided by a digital system that can acquire diffraction data as fast as every 260 microseconds for 300-700 ms. Tension records are simultaneously displayed on a storage oscilloscope. Resting sarcomere length variation between the end and center regions was analogous to that of Gordon et al. (1966). During the rapid rise in force (less than 45 ms), the end regions contract almost twice as fast as the center regions. During the slow rise in force, the velocity of contraction of the end regions was 3.8 times the velocity of stretch of the center regions. In addition, factors that affected the rate and extent of the slow rise in tension also affected the rate and extent of end shortening. In 58% of the cases studied, the amount of shortening observed in the end region was enough to explain the extent of the slow rise in tension. These data support the explanation of creep first proposed by A. V. Hill (1953) and used by Gordon et al. (1966) to justify their use of the back-extrapolation technique in measuring the isometric force-generating capability of a single fiber. These data also indicate that the laser diffraction technique may provide an effective, noninvasive method for studying sarcomere dynamics during creep and related phenomena.