NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction

[Purpose]

The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis.

[Methods]

Primary skeletal muscle cells were isolated from the gastrocnemius in C57BL/6 mice. The muscle cells were stimulated by electrical current at 1Hz for 3 minutes in conditions of normal or NAD metabolism related inhibitor treatment. NAD/NADH level, Sirt1 and mitochondria biogenesis related signal factor’s changes were examined in normal or NAD metabolism related inhibitor treated cells.

[Results]

Electrical stimulation (ES) induced muscle contractions significantly increased NAD/NADH levels, NAMPT inhibitor FK-866 inhibited ES-induced NAD formation, which caused SIRT1 expression and PGC-1α deacetylation to decrease. Moreover, NAMPT inhibition decreased mitochondrial biogenesis related mRNA, COX-1 and Tfam levels. Along with AMPK inhibitor, compound C decreases SIRT1 expression, PGC-1α deacetylation and muscle contraction induced mitochondrial biogenesis related mRNA increment. These results indicated that the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis. Inhibition of the AMPK upregulator, Camkkβ, STO-609 decreased AMPK phosphorylation and SIRT1 expression but did not decrease PGC-1α deacetylation. However, CAMKII inhibition via AIP decreased PGC-1α deacetylation.

[Conclusion]

In conclusion, the results indicate that NAMPT plays an important role in NAD metabolism and mitochondrial biogenesis. However, mitochondrial biogenesis is also controlled by different calcium binding protein signals including Camkkβ and CAMKII. [Keyword] Muscle contraction, NAD metabolism, SIRT1, PGC-1 α, mitochondria biogenesis.     

Smooth muscle contraction and relaxation

This brief review serves as a refresher on smooth muscle physiology for those educators who teach in medical and graduate courses of physiology. Additionally, those professionals who are in need of an update on smooth muscle physiology may find this review to be useful. Smooth muscle lacks the striations characteristic of cardiac and skeletal muscle. Layers of smooth muscle cells line the walls of various organs and tubes in the body, and the contractile function of smooth muscle is not under voluntary control. Contractile activity in smooth muscle is initiated by a Ca(2+)-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. Ca(2+) sensitization of the contractile proteins is signaled by the RhoA/Rho kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase, thereby maintaining force generation. Removal of Ca(2+) from the cytosol and stimulation of myosin phosphatase initiate the process of smooth muscle relaxation.     

Conformation of the troponin core complex in the thin filaments of skeletal muscle during relaxation and active contraction

Contraction of skeletal and cardiac muscles is regulated by Ca(2+) binding to troponin in the actin-containing thin filaments, leading to an azimuthal movement of tropomyosin around the filament that uncovers the myosin binding sites on actin. Here, we use polarized fluorescence to determine the orientation of the C-terminal lobe of troponin C (TnC) in skeletal muscle cells as a step toward elucidating the molecular mechanism of troponin-mediated regulation. Assuming, as shown by X-ray crystallography, that this lobe of TnC is part of a well-defined troponin domain called the IT arm, we show that the coiled coil formed by troponin components I and T makes an angle of about 55° with the thin filament axis in relaxed muscle, in contrast with previous models based on electron microscopy in which this angle is close to 0°. The E helix of TnC makes an angle of about 45° with the thin filament axis. Both the IT coiled coil and the TnC E helix tilt by about 10° on muscle activation. By combining in situ measurements of the orientation of the IT arm and regulatory domain of troponin, which together form the troponin core complex, with published intermolecular distances between thin filament components, we derive models of thin filament structure in which the IT arm of troponin holds its regulatory domain close to the actin surface. Although the structure and function of troponin regions outside the core complex remain to be characterized, the present results provide useful constraints for molecular models of the mechanism of muscle regulation.     

Calcium transients and calcium binding to troponin at the contraction threshold in skeletal muscle.

Antipyrylazo III calcium transients from voltage-clamped, cut skeletal muscle fibers of the frog were recorded, and the calcium binding to the regulatory sites of troponin C was calculated. The strength-duration curve for the contraction threshold was determined. It was found that the increase in myoplasmic calcium concentration necessary to produce the same level of contractile activation, i.e., the just visible movement, was approximately 60% higher at more positive membrane potentials resulting from short depolarizing pulses than at rheobase. However, using biochemical data for the kON and kOFF rate coefficients of the binding sites, the calculated maximums of the calcium binding curves were about the same at different voltages, and the time to maximum saturation was roughly equal to the latency of the contractions. To characterize the calcium binding in intact fibers more accurately, those values of the kON and kOFF rate coefficients that gave equal peak saturations during threshold movement at different membrane potentials were determined.     

Heat production and metabolism during the contraction of mammalian skeletal muscle

Methods are described whereby initial processes of muscular contraction may be investigated in a mammalian preparation, the soleus muscle of the rat. Conditions are chosen so that recovery is avoided. An isometric tetanus is investigated and an energy balance sheet is drawn up. It is found that there is more heat evolved than can be accounted for in terms of measured chemical reaction. This discrepancy is discussed with reference to the similar results that have been obtained using frog muscle.     

Two elementary models for the regulation of skeletal muscle contraction by calcium

It is shown by use of an extremely simple explicit two-state model that two basic ideas may be sufficient to understand at least qualitatively the sensitive activation of isometric muscle contraction by Ca2+. (a) Ca2+ binds much more strongly on troponin if myosin is already attached to actin. The steady state analogue of this is that the single rate constant (in the two-state model) for myosin attachment plus Pi release is much larger if Ca2+ is bound to troponin. (b) End-to-end tropomyosin interactions are responsible for positive cooperativity. Although these ideas seem to be sufficient, this of course does not mean that they are necessary. These same ingredients were used in two previous, more elaborate models for the cooperative equilibrium binding of myosin subfragment-1 on actin-tropomyosin-troponin, with and without Ca2+, and for a study of the steady state ATPase activity of the same system. Essentially as an appendix, the above-mentioned simple treatment is extended to a somewhat more realistic and complicated model of isometric contraction.     

Intramuscular fluid pressure during isometric contraction of human skeletal muscle

Intramuscular fluid pressures were recorded in the vastus medialis of seven healthy male volunteers. Pressures were measured simultaneously at three different sites in the muscle by a catheter-tip transducer with extremely low volume-displacement characteristics and by two extracorporeal transducers connected to slit catheters. All three recording systems gave qualitatively similar results provided the catheters had inner diameters exceeding 0.53 mm and allowed measurement of pressures lasting as short as 1 s. Wick catheters yielded slower responses than slit catheters. At any position intramuscular fluid pressure increased linearly with force up to maximal voluntary contraction (MVC). However, slopes of these curves varied greatly mainly because the pressure was also a linear function of the distance from the fascia. The highest recorded pressure was 570 Torr. At prolonged submaximal contractions intramuscular fluid pressure oscillated independent of contraction force. The linearity of both the pressure-force relationship and the pressure-depth relationship is compatible with a simple model based on the law of Laplace because the muscle fibers are curved during contraction in this muscle. It is hypothesized that blood flow is first compromised deep in the muscle where pressure is highest and in general at lower stress or tension in short bulging muscles with great curvature of the fibers compared with long slender ones.     

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.     

Spatial interaction between tissue pressure and skeletal muscle perfusion during contraction

The vascular waterfall theory attributes decreased muscle perfusion during contraction to increased intramuscular pressure (P(IM)) and concomitant increase in venous resistance. Although P(IM) is distributed during contractions, this theory does not account for heterogeneity. This study hypothesises that pressure heterogeneity could affect the interaction between P(IM) rise and perfusion. Regional tissue perfusion during submaximum (100kPa) tetanic contraction is studied, using a finite element model of perfused contracting skeletal muscle. Capillary flow in muscles with one proximal artery and vein (SIM(1)) and with an additional distal artery and vein (SIM(2)) is compared. Blood flow and pressures at rest and P(IM) during contraction ( approximately 25kPa maximally) are similar between simulations, but capillary flow and venous pressure differ. In SIM(2), venous pressure and capillary flow correspond to P(IM) distribution, whereas capillary flow in SIM(1) is less than 10% of flow in SIM(2), in the muscle half without draining vein. This difference is caused by a high central P(IM), followed by central venous pressure rise, in agreement with the waterfall theory. The high central pressure (SIM(1)), obstructs outflow from the distal veins. Distal venous pressure rises until central blood pressure is reached, although local P(IM) is low. Adding a distal vein (SIM(2)) restores the perfusion. It is concluded that regional effects contribute to the interaction between P(IM) and perfusion during contraction. Unlike stated by the vascular waterfall theory, venous pressure may locally exceed P(IM). Although this can be explained by the principles of this theory, the theory does not include this phenomenon as such.     

The mechanics of mouse skeletal muscle when shortening during relaxation

The dynamic properties of relaxing skeletal muscle have not been well characterised but are important for understanding muscle function during terrestrial locomotion, during which a considerable fraction of muscle work output can be produced during relaxation. The purpose of this study was to characterise the force-velocity properties of mouse skeletal muscle during relaxation. Experiments were performed in vitro (21 degrees C) using bundles of fibres from mouse soleus and EDL muscles. Isovelocity shortening was applied to muscles during relaxation following short tetanic contractions. Using data from different contractions with different shortening velocities, curves relating force output to shortening velocity were constructed at intervals during relaxation. The velocity component included contributions from shortening of both series elastic component (SEC) and contractile component (CC) because force output was not constant. Early in relaxation force-velocity relationships were linear but became progressively more curved as relaxation progressed. Force-velocity curves late in relaxation had the same curvature as those for the CC in fully activated muscles but V(max) was reduced to approximately 50% of the value in fully activated muscles. These results were the same for slow- and fast-twitch muscles and for relaxation following maximal tetani and brief, sub-maximal tetani. The measured series elastic compliance was used to partition shortening velocity between SEC and CC. The curvature of the CC force-velocity relationship was constant during relaxation. The SEC accounted for most of the shortening and work output during relaxation and its power output during relaxation exceeded the maximum CC power output. It is proposed that unloading the CC, without any change in its overall length, accelerated cross-bridge detachment when shortening was applied during relaxation.     

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.     

Changes in glucose 1,6-bisphosphate content in rat skeletal muscle during contraction.

Glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glycogen, lactate and other glycolytic metabolites were measured in rat gastrocnemius muscle, which was electrically stimulated in situ via the sciatic nerve. Both the frequency and the duration of stimulation were varied to obtain different rates of glycolysis. There was no apparent relationship between fructose 2,6-bisphosphate content and lactate accumulation in contracting muscle. In contrast, glucose 1,6-bisphosphate content increased with lactate concentration during contraction. It is suggested that the increase in glucose 1,6-bisphosphate could play a role in phosphofructokinase stimulation and in the activation of the glycolytic flux during muscle contraction.     

Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C

The influences of [Ca(2+)] and Ca(2+) dissociation rate from troponin C (TnC) on the kinetics of contraction (k(Ca)) activated by photolysis of a caged Ca(2+) compound in skinned fast-twitch psoas and slow-twitch soleus fibers from rabbits were investigated at 15 degrees C. Increasing the amount of Ca(2+) released increased the amount of force in psoas and soleus fibers and increased k(Ca) in a curvilinear manner in psoas fibers approximately 5-fold but did not alter k(Ca) in soleus fibers. Reconstituting psoas fibers with mutants of TnC that in solution exhibited increased Ca(2+) affinity and approximately 2- to 5-fold decreased Ca(2+) dissociation rate (M82Q TnC) or decreased Ca(2+) affinity and approximately 2-fold increased Ca(2+) dissociation rate (NHdel TnC) did not affect maximal k(Ca). Thus the influence of [Ca(2+)] on k(Ca) is fiber type dependent and the maximum k(Ca) in psoas fibers is dominated by kinetics of cross-bridge cycling over kinetics of Ca(2+) exchange with TnC.     

Ryanodine receptor purified from crayfish skeletal muscle

The ryanodine receptor was isolated from the sarcoplasmic reticulum of crayfish skeletal muscle. Ryanodine binding to the native fraction was measured by Scatchard analysis and values of 60 nmol/l and 9 pmol/mg were obtained for KD and Bmax respectively. The identity of purified receptor was confirmed by electron microscopy, electrophoresis and incorporation into planar lipid bilayers. At least two conductance states (100 pS and 50 pS) were observed in 100 mmol/l NaCl both for native and purified receptor.     

Regulation of Limulus skeletal muscle contraction

Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin-dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono- and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon C2+ or Ca2+/calmodulin activation, the fibers developed force (357+/-78.7 mN/mm2; 338+/-69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.     

Intracellular calcium and smooth muscle contraction

Excitation-contraction coupling in smooth muscle involves many processes, some of which are outlined in this article. The total amount of Ca2+ released on excitation is considerably in excess of the free Ca2+ concentration and this implies a high capacity, high affinity Ca2+ buffer system. The two major Ca2+-binding proteins are calmodulin and myosin. Only calmodulin has the appropriate binding affinity to act as a component of the Ca2+-buffer system. The Ca2+-calmodulin complex activates myosin light chain kinase and thus is involved in the regulation of contractile activity. Phosphorylation of myosin stabilizes an active conformation and promotes cross bridge cycling and is essential for the initiation of contraction. During the initial contractile response phosphorylation correlates to tension development and velocity of shortening. However, as contraction continues the extent of myosin phosphorylation and velocity often decreases but tension is maintained. In general, the Ca2+ transient is reflected by the extent of phosphorylation that in turn correlates with shortening velocity. Maintenance of tension at low phosphorylation levels is not accounted for within our understanding of the phosphorylation theory and thus alternative regulatory mechanisms have been implicated. Some of the possibilities are discussed.