Factors affecting the reactivation of the oligomycin-sensitive adenosine 5'-triphosphatase and the release of ATPase inhibitor protein during the re-energization of intact mitochondria from ischemic cardiac muscle

In the present study we examined factors affecting the reversal of the ischemia-induced protonic inhibition of the mitochondrial ATPase described earlier (Rouslin, W. (1983) J. Biol. Chem. 258, 9657-9661). It was found that ATPase reactivation and accompanying inhibitor protein release during the re-energization of intact mitochondria isolated from 20-min ischemic canine heart muscle could be blocked completely by either carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin but was unaffected by valinomycin at 35 mM K+. At higher K+ concentrations, valinomycin also blocked ATPase reactivation but not quite as completely as did nigericin. These observations suggest that ATPase reactivation and inhibitor protein release are particularly dependent upon either the trans-inner membrane pH gradient (delta pH) or possibly upon matrix pH per se and slightly less dependent upon membrane potential (delta psi) in intact cardiac muscle mitochondria. The addition of FCCP at the end of the re-energization incubations limited partially the extent of both ATPase reactivation and inhibitor protein release. This latter effect appears to have been mediated by a partial reassociation of the inhibitor protein with the enzyme, and it was accentuated (when FCCP was added at the end of the incubations) or mimicked (when FCCP was absent) by lowering the pH of the re-energization medium. A close examination of the first 10 min of the time course of enzyme activation and of inhibitor protein release revealed that while the former process was essentially finished in 1 min or less, the latter required approximately 10 min for completion. This observation led to the proposal of a two-site model of enzyme-inhibitor interaction which is discussed.     

Characterization of the giant myofiber in bovine skeletal muscle

Semitendinosus (ST) muscle samples were excised pre- and postrigor from 32 experimental animals (Bos taurus). Four intact and 4 castrate males were exsanguinated when they reached age endpoints of 8, 12, 16, and 20 months. The samples were sectioned, histochemically fiber-typed for SDH and ATPase activity, and examined for giant myofibers. Of the 32 muscles analyzed, only one (8-month intact male) postrigor ST contained a giant myofiber that was hematoxylin and eosin positive, SDH negative, ATPase positive, and 2 X the size of the surrounding fibers. Subsequent studies demonstrated these giant type II white myofibers are an anomaly of muscle contraction and not a distinct fiber type as has been previously reported. This is a first report of giant white or type II myofibers in postrigor bovine skeletal muscle.     

Isolation of two ATPase inhibitor proteins from mitochondria of rat skeletal muscle

An ATPase inhibitor protein was isolated from mitochondria of rat skeletal muscle by alkaline extraction and then was purified, It differed in definitive ways from the ATPase inhibitor protein isolated previously by Ca²⁺-stripping of submitochondrial particles of rat skeletal muscle. The two ATPase inhibitor proteins were shown to be present together in intact mitochondria.     

Regulation of the mitochondrial adenosine 5'-triphosphatase in situ during ischemia and in vitro in intact and sonicated mitochondria from slow and fast heart-rate hearts

In the present study we examined the regulation of the cardiac muscle mitochondrial ATPase both in situ and in vitro in intact and sonicated mitochondria from rabbit, pigeon, and rat. We chose to study these three species because each is representative of one of the three classes into which all species thus far studied may be placed with respect to the in situ activity of their cardiac muscle mitochondrial ATPase inhibitor and with respect to the amount of ATPase inhibitor present in their cardiac muscle mitochondria (1). Class A species (rabbit) contain a full complement of ATPase inhibitor and show a marked ATPase inhibition during ischemia. Class B species (pigeon) also contain a full complement of inhibitor but exhibit only a low level of ATPase inhibition in situ. Class C species (rat) contain only low levels of inhibitor and, like class B species, don't appear to utilize the inhibitor they possess during ischemia in situ. We found that, while hearts from all three species developed a marked cytosolic acidosis during ischemia, only rabbit exhibited a marked ATPase inhibition in situ. In in vitro experiments in which matrix pH values close to 6.2 and delta psi values close to zero were measured in intact mitochondria from all three species, matrix pH appeared to be an important factor regulating ATPase inhibition in rabbit, but it had little effect upon ATPase--inhibitor interaction in pigeon and rat despite the lack of membrane potential. However, a pH-dependent further release of ATPase inhibitor was observed in sonicated pigeon heart mitochondria only. This latter observation suggests that, while slow heart-rate heart mitochondria appear to be designed for ATPase down regulation during ischemia by inhibitor binding to the ATPase, fast heart-rate heart mitochondria appear to be designed primarily for ATPase up regulation by a further release of inhibitor from the enzyme.     

A new histochemical method for magnesium actomyosin adenosine triphosphatase at physiological pH

A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics.     

Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction

The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type IIA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

Summary This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type HA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

Rapid anisotropic motion of the Ca2+-transport ATPase of the rabbit skeletal muscle sarcoplasmic reticulum.

1H nuclear magnetic resonance techniques were used to study the binding of uridine 5'-triphosphate to the Ca2+-transport ATPase (EC 3.6.1.3) of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. The nuclear spin relaxation times determined for the bound nucleotide are used to characterize the rotational motion of the ATPase to which the nucleotide is bound. The results, assuming an anisotropic model for the motion of the ATPase in the membrane, place a low upper limit on the rotational correlation time of the ATPase. This indicates that the motion of the ATPase in the membrane is quite rapid when compared, for example, with the motion found for other membrane-bound proteins such as rhodopsin.     

Cross-bridge model of muscle contraction. Quantitative analysis

We recently presented, in a qualitative manner, a cross-bridge model of muscle contraction which was based on a biochemical kinetic cycle for the actomyosin ATPase activity. This cross-bridge model consisted of two cross-bridge states detached from actin and two cross-bridge states attached to actin. In the present paper, we attempt to fit this model quantitatively to both biochemical and physiological data. We find that the resulting complete cross-bridge model is able to account reasonably well for both the isometric transient data observed when a muscle is subjected to a sudden change in length and for the relationship between the velocity of muscle contraction in vivo and the actomyosin ATPase activity in vitro. This model also illustrates the interrelationship between biochemical and physiological data necessary for the development of a complete cross-bridge model of muscle contraction.     

A constitutive description of contracting papillary muscle and its implications to the dynamics of the intact heart

A simple mathematical model capable of simulating the major biomechanical attributes of contracting cardiac muscle is presented. This model is based on the phenomenological observations on heart muscle. The form of the equation can be readily extended to describe the pressure-volume-time-velocity of the intact heart as well, thus allowing a direct bridge between the dynamics of papillary muscle and the dynamics of intact heart. Parameters that are sensitive to inotropic state of the muscle can be obtained directly from the isometric tension-time record of the muscle or the isovolumic pressure-time record of the ventricle. These parameters have the potential to serve as quantitative measures of cardiac health.     

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca²⁺ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca²⁺ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca²⁺ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca²⁺ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca²⁺ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca²⁺ and the role of the Ca²⁺ ATPase in intact myocytes as well as in genetically altered heart cells.     

Characterization of a caldesmon fragment that competes with myosin-ATP binding to actin.

The protein caldesmon inhibits actin-activated ATP hydrolysis of myosin and inhibits the binding of myosin.ATP to actin. A fragment isolated from a chymotryptic digest of caldesmon contains features of the intact molecule that make it useful as a selective inhibitor of the binding of myosin.ATP complexes to actin without having the complexity of binding to myosin. The COOH-terminal 20 kDa region of caldesmon binds to actin with one-sixth the affinity of caldesmon with a stoichiometry of binding of one fragment per two actin monomers. This contrasts with the 1:6-9 stoichiometry of intact caldesmon. The binding of the 20 kDa fragments to actin is totally reversed by Ca(2+)-calmodulin and, like intact caldesmon, the 20 kDa fragments are competitive with the binding of myosin subfragments to actin. This competition with myosin binding is largely responsible for the inhibition of ATP hydrolysis, although both the fragments and intact caldesmon also reverse the potentiation of ATPase activity caused by tropomyosin. These polypeptides are useful both in defining the function of caldesmon and in studying the role of weakly bound cross-bridges in muscle.     

Calcium binding and the activation of fibrillar insect flight muscle

A new method has been used to measure calcium binding in intact glycerol extracted muscle fibres; results with rabbit psoas muscle are in agreement with previous work. Lethocerus cordofanus flight muscle bound up to 140 μM calcium at high affinity in the presence of ATP; removal of the ATP increased the maximum amount bound to 210 μM and the affinity approx. 3-fold. Calcium binding in the presence of ATP correlated with calcium activation of the ATPase but no changes in calcium binding occurred when the muscle was further activated by stretching.     

Mitochondrial coupling in vivo in mouse skeletal muscle

The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with ³¹P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 ± 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 ± 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought. P/O; oxidative phosphorylation; proton leak; optical spectroscopy     

The ATPase core of a clathrin uncoating protein

Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.     

A proteolytic NH2-terminal truncation of cardiac troponin I that is up-regulated in simulated microgravity

In a tail suspension rat model, we investigated changes in myofilament protein during cardiac adaptation in simulated microgravity. Contractile force and velocity of cardiac muscle were decreased in the tail suspension rats as compared with the control. Ca(2+)-dependent actomyosin ATPase activity was also decreased; however, sensitivity of cardiac muscle to Ca(2+) activation was unchanged. There was no change in expression of myosin heavy chain, tropomyosin, troponin T, or troponin I isoforms in hearts of tail suspension rats. A novel finding is a fragment of cardiac troponin I (cTnI) that had increased amounts in the heart of tail suspension rats. Binding of this cTnI fragment by a monoclonal antibody that specifically recognizes the COOH terminus indicates an intact COOH terminus. NH(2)-terminal sequence analysis of the cTnI fragment revealed truncations primarily of amino acids 1-26 and 1-27 and smaller amounts of 1-30, including Ser(23) and Ser(24), which are substrates of protein kinase A phosphorylation. This cTnI fragment is present in normal cardiac muscle and incorporated into myofibrils, indicating a role in regulating contractility. This proteolytic modification of cTnI up-regulated during simulated microgravity suggests a potential role of the NH(2)-terminal segment of cTnI in functional adaptations of cardiac muscle.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

Characterization of swimming muscle in the Atlantic mackerel, Scomber scombrus L.

Both red and white muscle were removed from juvenile and adult Atlantic mackerel, Scomber scombrus L., for histochemical characterization of the muscle fibre types. Staining of white muscle for myosin ATPase, SDH, NADH diaphorase, GPDH and LDH revealed that these fibres are homogeneous. Red muscle was shown to be heterogeneous, of at least two fibre types recognizable on the basis of myosin ATPase staining with preincubation at a pH of 9·8. These two red types are dispersed throughout the red muscle and are present in both juveniles and adults. Red muscle is located both deep within the myotomes and as a superficial layer of muscle fibres. A third group of muscle fibres, intermediate in nature, was distinguished at the apex of the red muscle 'triangle,' between the epaxial and hypaxial muscle, using NADH diaphorase and myosin ATPase stains. This paper discusses the possibility that functionally different muscle fibres occur in the red swimming muscle of the Atlantic mackerel.