Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice.

Myoglobin is a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers and facilitates oxygen delivery during periods of high metabolic demand. Myoglobin content in skeletal muscle increases in response to hypoxic conditions. However, we previously reported that myoglobin-null mice are viable and fertile. In the present study, we define important functional, cellular, and molecular compensatory adaptations in the absence of myoglobin. Mice without myoglobin manifest adaptations in skeletal muscle that include a fiber type transition (type I to type II in the soleus muscle), increased expression of the hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1alpha and HIF-2 (endothelial PAS domain protein), stress proteins such as heat shock protein 27, and the angiogenic growth factor vascular endothelial growth factor (soleus muscle), as well as increased nitric oxide metabolism (extensor digitorum longus). The resulting changes in angiogenesis, nitric oxide metabolism, and vasomotor regulation are likely to account for preserved exercise capacity of animals lacking myoglobin. These results demonstrate that mammalian organisms are capable of a broad spectrum of adaptive responses that can compensate for a potentially serious defect in cellular oxygen transport.

Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction.

Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.

Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle.

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

Ca2+-independent activity of nitric oxide synthase.

Ca2+-independent forms of nitric-oxide synthase have significant activity when the endogenous calmodulin subunit is Ca2+ free. Further activation is seen when Ca2+ is added. We have examined the activation of a Ca2+-independent nitric-oxide synthase variant and its two point mutants that are more dependent on Ca2+ for activation using mutant calmodulins containing non-functional Ca2+-binding sites. These studies provide evidence that the Ca2+-independent activity of these enzymes can be exerted through specific adapted interactions between the enzyme and the Ca2+-binding site 2 of calmodulin. Further, the results suggest that EGTA-sensitive metals other than Ca2+ complexed to calmodulin may be involved in maximal activation of these nitric-oxide synthase variants.

Nitric oxide contributes to vascular smooth muscle relaxation in contracting fast-twitch muscles.

During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers or from endothelial cells (eNOS) may relax vascular smooth muscle contributing to functional hyperemia. To examine the relative importance of these pathways, smooth muscle myosin regulatory light chain (smRLC) phosphorylation was assessed as an index of vascular tone in isolated extensor digitorum longus (EDL) muscles from C57, nNOS(-/-), and eNOS(-/-) mice. The smRLC phosphorylation (in mol phosphate per mol smRLC) in C57 resting muscles (0.12 +/- 0.04) was increased 3.7-fold (0.44 +/- 0.03) by phenylephrine (PE). Reversal of this increase with electrical stimulation (to 0.19 +/- 0.03; P < 0.05) was partially blocked by N(omega)-nitro-l-arginine (NLA). In nNOS(-/-) EDL, the PE-induced increase in smRLC phosphorylation (0.10 +/- 0.02 to 0.49 +/- 0.04) was partially decreased by stimulation (0.25 +/- 0.04). In eNOS(-/-) EDL, the control value for smRLC was increased (0.24 +/- 0.04), and PE-induced smRLC phosphorylation (0.36 +/- 0.06) was decreased by stimulation even in the presence of NLA (to 0.20 +/- 0.02; P < 0.05). These results suggest that in addition to NO-independent mechanisms, NO derived from both nNOS and eNOS plays a role in the integrative vascular response of contracting skeletal muscle.

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Activation of myosin light chain kinase requires translocation of bound calmodulin.

A novel translocation step is inferred from structural studies of the interactions between the intracellular calcium receptor protein calmodulin (CaM) and one of its regulatory targets. A mutant of CaM missing residues 2-8 (DeltaNCaM) binds skeletal muscle myosin light chain kinase with high affinity but fails to activate catalysis. Small angle x-ray scattering data reveal that DeltaNCaM occupies a position near the catalytic cleft in its complex with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Thus, CaM residues 2-8 appear to facilitate movement of bound CaM away from the vicinity of the catalytic cleft.

Localization and activity of myosin light chain kinase isoforms during the cell cycle.

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.

Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin-glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alpha-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD.

Myosin light chain kinase binding to actin filaments.

Smooth muscle myosin light chain kinase (MLCK) plays important roles in contractile-motile processes of a variety of cells. Three DFRxxL motifs at the kinase N-terminus (residues 2-63) are critical for high-affinity binding to actin-containing filaments [Smith et al. (1999) J. Biol. Chem. 274, 29433-29438]. A GST fusion protein containing residues 1-75 of MLCK (GST75-MLCK) bound maximally to both smooth muscle myofilaments and F-actin at 0.28 and 0.31 mol GST75-MLCK/mol actin with respective K(D) values of 0.1 microM and 0.8 microM. High-affinity binding of MLCK to actin-containing filaments may be due to each DFRxxL motif binding to one actin monomer in filaments.

Neuronal nitric oxide synthase localizes through multiple structural motifs to the sarcolemma in mouse myotubes.

In skeletal muscle, neuronal nitric oxide synthase is localized at the sarcolemma in association with the dystrophin glycoprotein complex (DGC). The nNOS N-terminal 231 amino acids comprise a PDZ domain (residues 1-100) and a beta-hairpin finger loop (residues 101-130) which binds alpha-syntrophin located in the DGC. Endogenous nNOS and GFP-tagged nNOS localize to the sarcolemma in mouse C2C12 myotubes. Expression of GFP-tagged nNOS domains in C2C12 myotubes reveals that the PDZ domain and the beta-hairpin finger loop of nNOS are independently capable of localizing to the sarcolemma of C2C12 myotubes. Binding studies indicate that alpha-syntrophin binds only to the beta-hairpin finger loop and not the PDZ domain of nNOS. nNOS may bind to proteins in addition to alpha-syntrophin at muscle sarcolemma.

nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle.

Nitric oxide (NO) from Ca(2+)-dependent neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers may modulate vascular tone by a cGMP-dependent pathway similar to NO derived from NOS in endothelial cells (eNOS). In isolated fast-twitch extensor digitorum longus (EDL) muscles from control mice, cGMP formation increased approximately 166% with electrical stimulation (30 Hz, 15 s). cGMP levels were not altered in slow-twitch soleus muscles. The NOS inhibitor N(omega)-nitro-l-arginine abolished the contraction-induced increase in cGMP content in EDL muscles, and the NO donor sodium nitroprusside (SNP) increased cGMP content approximately 167% in noncontracting EDL muscles. SNP treatment but not electrical stimulation increased cGMP formation in muscles from nNOS(-/-) mice. cGMP formation in control and stimulated EDL muscles from eNOS(-/-) mice was less than that obtained with similarly treated muscles from control mice. Arteriolar relaxation in contracting fast-twitch mouse cremaster muscle was attenuated in muscles from mice lacking either nNOS or eNOS. These findings suggest that increases in cGMP and NO-dependent vascular relaxation in contracting fast-twitch skeletal muscle may require both nNOS and eNOS.

Mutations at lysine 525 of inducible nitric-oxide synthase affect its Ca2+-independent activity.

Calmodulin binding to inducible nitric-oxide synthase may play an important role in its Ca(2+)-independent activity. Studies of inducible nitric-oxide synthase chimeras containing the calmodulin binding sequence of neuronal or endothelial nitric-oxide synthases show that the calmodulin binding sequence of inducible nitric-oxide synthase is necessary but not sufficient for the Ca(2+)-independent activity. The mutations at lysine 525 located at the C terminus of the calmodulin binding sequence of inducible nitric-oxide synthase were examined for the effects on the Ca(2+)-independent activity with chimeras containing the oxygenase or reductase domains of inducible or neuronal nitric-oxide synthases. Results show that the Ca(2+)-independent binding of calmodulin is not solely responsible for maximal Ca(2+)-independent activity of inducible nitric-oxide synthase. Lysine 525 of inducible nitric-oxide synthase may also play an important role in coordinating the maximal Ca(2+)-independent activity.

Functional assembly of fragments from bisected smooth muscle myosin light chain kinase.

The C-terminal regulatory segment of smooth muscle myosin light chain kinase folds back on its catalytic core to inhibit kinase activity. This regulatory segment consists of autoinhibitory residues linking the catalytic core to the calmodulin-binding sequence and perhaps additional C-terminal residues including an immunoglobulin-like motif. However, mutational and biochemical analyses showed no specific involvement of residues C-terminal to the calmodulin-binding sequence. To obtain additional insights on the proposed mechanisms for autoinhibition and Ca(2+)/calmodulin activation of the kinase, the polypeptide backbone chain of myosin light chain kinase was cleaved by genetic means to produce N- and C-terminal protein fragments. The N-terminal fragment containing the catalytic core was catalytically inactive when expressed alone. Co-expression of the N-terminal fragment with the C-terminal fragment containing the regulatory segment restored kinase activity. Deletion of the autoinhibitory linker residues without or with the calmodulin-binding sequence prevented restoration of kinase activity. In the presence or absence of Ca(2+)/calmodulin, regulatory segment binding occurred through the linker region connecting the catalytic core to the calmodulin-binding sequence. Collectively, these results indicate that residues C-terminal to the calmodulin-binding sequence (including the immunoglobulin-like motif) are not functional components of the regulatory segment. Furthermore, the principal autoinhibitory motif is contained in the sequence linking the catalytic core of myosin light chain kinase to the calmodulin-binding sequence.

Conformational requirements for Ca(2+)/calmodulin binding and activation of myosin light chain kinase.

Myosin light chain kinase contains a regulatory segment consisting of an autoinhibitory region and a calmodulin-binding sequence that folds back on its catalytic core to inhibit kinase activity. It has been proposed that alpha-helix formation may be involved in displacement of the regulatory segment and activation of the kinase by Ca(2+)/calmodulin. Proline mutations were introduced at putative non-interacting residues in the regulatory segment to disrupt helix formation. Substitution of proline residues immediately N-terminal of the Trp in the calmodulin-binding sequence had most significant effects on Ca(2+)/calmodulin binding and activation. Formation of an alpha-helix in this region upon Ca(2+)/calmodulin binding may be necessary for displacement of the regulatory segment allowing phosphorylation of myosin regulatory light chain.

Role for alpha-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies.

A dystrophin-containing glycoprotein complex (DGC) links the basal lamina surrounding each muscle fibre to the fibre's cytoskeleton, providing both structural support and a scaffold for signalling molecules. Mutations in genes encoding several DGC components disrupt the complex and lead to muscular dystrophy. Here we show that mice deficient in alpha-dystrobrevin, a cytoplasmic protein of the DGC, exhibit skeletal and cardiac myopathies. Analysis of double and triple mutants indicates that alpha-dystrobrevin acts largely through the DGC. Structural components of the DGC are retained in the absence of alpha-dystrobrevin, but a DGC-associated signalling protein, nitric oxide synthase, is displaced from the membrane and nitric-oxide-mediated signalling is impaired. These results indicate that both signalling and structural functions of the DGC are required for muscle stability, and implicate alpha-dystrobrevin in the former.