Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.     

Subunit composition of rodent isomyosins and their distribution in hindlimb skeletal muscles

Three adult skeletal muscle sarcomeric myosin heavy chain (MHC) genes have been identified in the rat, suggesting that the expressed native myosin isoforms can be differentiated, in part, on the basis of their MHC composition. This study was undertaken to ascertain whether the five major native isomyosins [3 fast (Fm1, Fm2, Fm3), 1 slow (Sm), and 1 intermediate (Im)], typically expressed in the spectrum of adult rat skeletal muscles comprising the hindlimb, could be further differentiated on the basis of their MHC profiles in addition to their light chain composition. Results show that in muscles comprised exclusively of fast-twitch glycolytic (FG) fibers and consisting of Fm1, Fm2, and Fm3, such as the tensor fasciae latae, only one MHC, designated as fast type IIb, could be resolved. In soleus muscle, comprised of both slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers and expressing Sm and Im, two MHC bands were resolved and designated as slow/cardiac beta-MHC and fast type IIa MHC. In muscles expressing a mixture of all three fiber types and a full complement of isomyosins, as seen in the plantaris, the MHC could be resolved into three bands. Light chain profiles were characterized for each muscle type, as well as for the purified isomyosins. These data suggest that Im (IIa) consists of a mixture of fast and slow light chains, whereas Fm (IIb) and Sm (beta) isoforms consist solely of fast- and slow-type light chains, respectively. Polypeptide mapping of denatured myosin extracted from muscles expressing contrasting isoform phenotypes suggests differences in the MHC primary structure between slow, intermediate, and fast myosin isotypes. These findings demonstrate that 1) Fm, Im, and Sm isoforms are differentiated on the bases of both their heavy and light chain components and 2) each isomyosin is distributed in a characteristic fashion among rat hindlimb skeletal muscles. Furthermore, these data suggest that the ratio of isomyosins in a given muscle or muscle region is of physiological importance to the function of that muscle during muscular activity.     

Parkinson's disease-like neuromuscular defects occur in prenyl diphosphate synthase subunit 2 (Pdss2) mutant mice

The Pdss2 gene product is needed for the isoprenylation of benzoquinone to generate coenzyme Q (CoQ). A fatal kidney disease occurs in mice that are homozygous for a missense mutation in Pdss2, which can be recapitulated in conditional Pdss2 knockouts targeted to glomerular podocytes. We now report that homozygous missense mutants also demonstrate significant neuromuscular deficits, as validated by behavioral and coordination assays, and these deficits are recapitulated in conditional Pdss2 knockouts targeted to dopaminergic neurons. Both conditional knockout and missense mutant mice demonstrate deficiencies in tyrosine hydroxylase-positive neurons in the substantia nigra, implicating a pathology similar to sporadic Parkinson's disease (PD).     

Phosphorylation of 4E-BP1 in the Mammalian Brain Is Not Altered by LRRK2 Expression or Pathogenic Mutations

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson''s disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.     

NMR study of non-structural proteins–part III: <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and side-chain resonance assignment of macro domain from <Emphasis Type="Italic">Chikungunya</Emphasis> virus (CHIKV)

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130–190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.     

Oxidative modifications, mitochondrial dysfunction, and impaired protein degradation in Parkinson's disease: how neurons are lost in the Bermuda triangle

In AtT-20 cells ACTH secretion is regulated by both Ca²⁺ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca²⁺ binding protein which regulates Alzheimer's β-amyloid precursor protein (APP) biogenesis, binds both Ca²⁺ as well as Gα subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with Gαi1/2 and Gαi3 at the PM and on RSG. Cytosolic calnuc(ΔSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with Gαi1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of Gαi1/2 on the PM whereas the distribution of Gβ subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(ΔSS)-CFP enhances ACTH secretion two-fold triggered by mastoparan or GTPγS but does not significantly affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-Gαi1/2, Gαi3, Gβ or calnuc IgG into permeabilized cells but not when Gα12 or preimmune IgG is introduced. The results suggest that calnuc binds to Gα subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of Gαi subunits to the PM and RSG, indicating that calnuc plays a role in dynamic distribution of only Gα but not Gβ subunits. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion.     

Effect of anabolic steroids on skeletal muscle mass during hindlimb suspension

The efficacy of anabolic steroid treatment [0.3 or 0.9 mg nandrolone decanoate (Deca-Durabolin) per day] was examined in the context of sparing rodent fast-twitch plantaris and slow-twitch soleus muscle weight, sparing subcellular protein, and altering isomyosin expression in response to hindlimb suspension. Female rats were assigned to four groups (7 rats/group for 6 wk): 1) normal control (NC), 2) normal steroid (NS), 3) normal suspension (N-SUS), and 4) suspension steroid (SUS-S). Compared with control values for the plantaris and soleus muscles, suspension induced 1) smaller body and muscle weight (P less than 0.05), 2) losses in myofibril content (mg/muscle, P less than 0.05), and 3) shifts in the relative expression (expressed as %of total isomyosin) of isomyosins which favored lesser slow myosin and greater fast myosin isotypes (P less than 0.05). Steroid treatment of suspended animals (SUS-S vs. N-SUS) partially spared body and muscle weight (P less than 0.05) and spared plantaris but not soleus myofibril content (mg/muscle, P less than 0.05). However, steroid treatment did not modify the isomyosin pattern induced by suspension. In normal rats (NS vs. NC), steroid treatment enhanced body and plantaris muscle weight but not soleus weight (P less than 0.05) and did not alter isomyosin expression in either muscle type. Collectively these data suggest that in young female rats anabolic steroids 1) enhance the body weight and the weight of a fast-twitch ankle extensor in normal rats, 2) ameliorate the loss in body weight, fast-twitch muscle weight and protein content and slow-twitch muscle weight associated with hindlimb suspension.(ABSTRACT TRUNCATED AT 250 WORDS)