An "active enzyme" muscle model

A new model of skeletal muscle contraction is presented from a unified view of muscle physiology, chemical energetics and newly obtained experimental data concerning actomyosin ATPase in vitro.In this model an interaction between actin and myosin, involving two distinct active sites, is considered to be the essential elementary mechanism for muscle contractions. These two sites are located on myosin. One site, forming a myosin-ADP-P, complex, has stored energy derived from ATP splitting before the beginning of a contraction. Another site, forming a myosin-ATP complex, upon interacting with actin, catalyzes ATP hydrolysis, using a fraction of the stored energy. The hydrolysis at the latter site is responsible for tension development, while the stored energy is released to drive the contractile reaction between actin and myosin unidirectionally. (Thus, the two sites act co-operatively and they can be viewed as forming an active enzyme.)There has been a difficulty in explaining the shortening heat production with apparent lack of corresponding chemical change at the early stage of contraction. The active enzyme model accounts for the shortening heat as the irreversible release of the stored energy. The heat production appears to precede its corresponding ATP splitting for “refueling” which occurs after complete exhaustion of the stored energy, while the actomyosin ATP hydrolysis takes place proportionally to the work. At the macroscopic level, the model is compatible with Hill's tension-velocity and heat relation.     

EMG activity in "compensatory" muscle hypertrophy

The functional elimination of synergistic muscles leads to dramatic muscle hypertrophy. However, neither resting EMG activity recorded by an implanted electrode array, nor activity during locomotion have substantiated the assumption that the hypertrophy in the rat soleus muscle is caused by hyperactivity.     

Quantitation of "autophagic flux" in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure "autophagic flux" in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3II protein may render possible misinterpretations since LC3II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3II, a technique aptly named the "autophagometer". In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a "colchicine block." Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an "in vivo autophagometer" study using colchicine in skeletal muscle.     

"Donor" muscle structure and function after end-to-side neurorrhaphy

End-to-end nerve coaptation is the preferred surgical technique for peripheral nerve reconstruction after injury or tumor extirpation. However, if the proximal nerve stump is not available for primary repair, then end-to-side neurorrhaphy may be a reasonable alternative. Numerous studies have demonstrated the effectiveness of this technique for muscle reinnervation. However, very little information is available regarding the potential adverse sequelae of end-to-side neurorrhaphy on the innervation and function of muscles innervated by the "donor" nerve. End-to-side neurorrhaphy is hypothesized to (1) acutely produce partial donor muscle denervation and (2) chronically produce no structural or functional deficits in muscles innervated by the donor nerve. Adult Lewis rats were allocated to one of two studies to determine the acute (2 weeks) and chronic (6 months) effects of end-to-side neurorrhaphy on donor muscle structure and function. In the acute study, animals underwent either sham exposure of the peroneal nerve (n = 13) or end-to-side neurorrhaphy between the end of the tibial nerve and the side of the peroneal nerve (n = 7). After a 2-week recovery period, isometric force (F(0) was measured, and specific force (sF(0) was calculated for the extensor digitorum longus muscle ("donor" muscle) for each animal. Immunohistochemical staining for neural cell adhesion molecule (NCAM) was performed to identify populations of denervated muscle fibers. In the chronic study, animals underwent either end-to-side neurorrhaphy between the end of the peroneal nerve and the side of the tibial nerve (n = 6) or sham exposure of the tibial nerve with performance of a peroneal nerve end-to-end nerve coaptation approximately 6), to match the period of anterior compartment muscle denervation in the end-to-side neurorrhaphy group. After a 6-month recovery period, contractile properties of the medial gastrocnemius muscle ("donor" muscle) were measured. Acutely, a fivefold increase in the percentage of denervated muscle fibers (1 +/0 0.7 percent to 5.4 +/-2.7 percent) was identified in the donor muscles of the animals with end-to-side neurorrhaphy (p < 0.001). However, no skeletal muscle force deficits were identified in these donor muscles. Chronically, the contractile properties of the medial gastrocnemius muscles were identical in the sham and end-to-side neurorrhaphy groups. These data support our two hypotheses that end-to-side neurorrhaphy causes acute donor muscle denervation, suggesting that there is physical disruption of axons at the time of nerve coaptation. However, end-to-side neurorrhaphy does not affect the long-term structure or function of muscles innervated by the donor nerve.     

Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle

Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pKa of 7.3 in 30 mM imidazole. PFK binding increased at lower pH values; at 150 mM KCl the apparent pKa value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium--with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F.D. Shaw, and D.J. Morton (1980) Biochem. J. 186, 105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.     

The "all-or-none" law in skeletal muscle and nerve fibres

In 1905 the Cambridge physiologist Keith Lucas extended the "all-or-none" principle (introduced by H. P. Bowditch for the cardiac tissue) to skeletal muscle and nerve fibres. Nevertheless, in a short time it was clear that nerve fibres obey this law, but also that frequency of discharge is another relevant factor in the nervous conduction.     

The "levator septi nasi muscle" and its clinical significance

It is strange that all textbooks of anatomy describe the depressor septi nasi muscle singly, without an antagonist. Incidentally, in 1986, a small rod of soft tissue was found between the medial crura of the two alar cartilages during a rhinoplastic operation with the external approach technique of Anderson and Ries. From 1990 through 1995, anatomic dissections of the nasolabial region under 3.5x loupe magnification were performed on 14 Chinese formalin-preserved cadavers, one fresh Chinese cadaver, and one fresh American white female cadaver. The small soft-tissue rod was found in every one of the dissected cadavers, and it was seen to be a pair of muscles. Each one of these paired small muscles arose from the aponeurosis on the dorsum of the nose and inserted into the muscular substance of the upper lip at the base of the columella and to the anterior spine of the maxilla. Histologic examinations of these muscles stained with hematoxylin and eosin and Masson trichrome showed that they were striated muscles. According to its origin and insertion, this newly found muscle was called the "levator septi nasi." Its clinical significance in cleft lip deformity and its relations to the orbicularis oris muscle, the dermocartilaginous ligament of Pitanguy, and the nasal superficial musculoaponeurotic system of Letourneau and Daniel are all discussed.     

"Red" fibres of pigeon pectoralis major muscle are "type II red".

On the basis of the histochemical activity of succinic dehydrogenase, only two fibre-types are distinguished in pigeon pectoralis major muscle. These are narrow "Red" and broad "White". The histochemical activity of myofibrillar ATPase was studied in these two distinct fibre-types. Both fibre-types showed high activity for the ATPase. "Red" fibres of pigeon pectoralis were not alkali-labile, at incubation pH 9.4, as were the "Type I" fibres of both avian and mammalian muscles. Again unlike "Type I" fibres, the "Red" fibres of pigeon pectoralis lacked the characteristic activation of acid-preincubated ATPase reaction. Pigeon pectoralis "Red" fibres are known to possess some characteristics of fast-twitch fibres (e.g. high fat, considerable phosphorylase, fibrillenstruktur myofibrillar arrangement, focal "en plaque" pattern of nerve endings). It is emphasized, therefore, that the pigeon pectoralis "Red" fibres are not equivalent to "Type I or slow-twitch", muscle fibres, but they are possibly "fast-twitch fatigue resistent or Type II Red" muscle fibres.     

Mitochondrial regular arrangement in muscle cells: a "crystal-like" pattern

The aim of this work was to characterize quantitatively the arrangement of mitochondria in heart and skeletal muscles. We studied confocal images of mitochondria in nonfixed cardiomyocytes and fibers from soleus and white gastrocnemius muscles of adult rats. The arrangement of intermyofibrillar mitochondria was analyzed by estimating the densities of distribution of mitochondrial centers relative to each other (probability density function). In cardiomyocytes (1,820 mitochondrial centers marked), neighboring mitochondria are aligned along a rectangle, with distance between the centers equal to 1.97 ± 0.43 and 1.43 ± 0.43 µm in the longitudinal and transverse directions, respectively. In soleus (1,659 mitochondrial centers marked) and white gastrocnemius (621 pairs of mitochondria marked), mitochondria are mainly organized in pairs at the I-band level. Because of this organization, there are two distances characterizing mitochondrial distribution in the longitudinal direction in these muscles. The distance between mitochondrial centers in the longitudinal direction within the same I band is 0.91 ± 0.11 and 0.61 ± 0.07 µm in soleus and white gastrocnemius, respectively. The distance between mitochondrial centers in different I bands is 3.7 and 3.3 µm in soleus and gastrocnemius, respectively. In the transverse direction, the mitochondria are packed considerably closer to each other in soleus than in white gastrocnemius, with the distance equal to 0.75 ± 0.22 µm in soleus and 1.09 ± 0.41 µm in gastrocnemius. Our results show that intermyofibrillar mitochondria are arranged in a highly ordered crystal-like pattern in a muscle-specific manner with relatively small deviation in the distances between neighboring mitochondria. This is consistent with the concept of the unitary nature of the organization of the muscle energy metabolism. confocal microscopy; quantitative analysis; cardiac and skeletal muscles; probability density function; unitary structure of cells     

The "roll and lock" mechanism of force generation in muscle

Muscle force results from the interaction of the globular heads of myosin-II with actin filaments. We studied the structure-function relationship in the myosin motor in contracting muscle fibers by using temperature jumps or length steps combined with time-resolved, low-angle X-ray diffraction. Both perturbations induced simultaneous changes in the active muscle force and in the extent of labeling of the actin helix by stereo-specifically bound myosin heads at a constant total number of attached heads. The generally accepted hypothesis assumes that muscle force is generated solely by tilting of the lever arm, or the light chain domain of the myosin head, about its catalytic domain firmly bound to actin. Data obtained suggest an additional force-generating step: the "roll and lock" transition of catalytic domains of non-stereo-specifically attached heads to a stereo-specifically bound state. A model based on this scheme is described to quantitatively explain the data.