The number of Z-repeats and super-repeats in nebulin greatly varies across vertebrates and scales with animal size

Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of ∼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations.

Download Full-text

Abstract 958: Cardiac MyBP-C Modulates Actomyosin Interactions: Evidence From Cardiac MyBP-c−/ − Mice

Circulation ◽

10.1161/circ.116.suppl_16.ii_189-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Yves Lecarpentier ◽

Nicolas Vignier ◽

Patricia Oliviero ◽

Miguel Cortes-Morichetti ◽

Lucie Carrier ◽

...

Keyword(s):

Thin Filament ◽

Isometric Force ◽

Binding Protein ◽

Critical Role ◽

Cardiac Contractility ◽

Left Ventricular ◽

Actin Binding ◽

Power Stroke ◽

Shortening Velocity

The precise role of cardiac myosin binding protein C (cMyBP-C) on actomyosin interaction (AMI) remains unknown. We hypothesized that the lack of cMyBP-C impaired cardiac AMI. Experiments were performed on 16 weeks old cMyBP-C −/− (KO) and age-matched wild-type (WT) mice (n=20/group). In vitro mechanical and energetics properties were performed on left ventricular (LV) papillary muscles and Huxley’s equations were used to characterize AMI. In vitro motility assays were performed using myosin purified from LV. Myosin-based sliding velocities of actin filaments were analyzed at baseline, after pretreatment of the myosin solution with 10 umol of the catalytic subunit of PKA and/or in the presence of increasing amount of α-actinin, an actin-binding protein that acts as an internal load thereby providing an index of relative isometric force. Western-blot analysis was used to quantify cMyBP-C and phosphorylated cMyBP-C in myosin solutions. Compared to WT, both total tension and maximum shortening velocity were lower in KO (p<0.001). The probability for myosin to be weakly bound to actin was higher in KO than in WT (8.6±0.3 vs. 5.4±0.2%, p<0.05), whereas the number of strongly bound, high-force generated state cross-bridges was lower in KO (6.4±0.9 vs. 11.6±1.0 10 9 /mm 2 , p<0.001). The unitary force per AMI was lower in KO than in WT (p<0.01). At baseline, myosin-based velocities of actin were slower in KO than in WT (1.65±0.01 vs. 1.98±0.01 um/s, p<0.01). The minimum amount of α-actinin needed to completely arrest the thin filament motility was significantly higher in WT than in KO (73.3±1.1 vs 29.1±0.1 ug/l, p<0.001). As expected, cMyBP-C was present in WT myosin solution whereas cMyBP-C was not detected in KO. In WT, PKA induced a 1.6-fold increased in cMyBP-C phosphorylation (p<0.01) associated with a 53±1% increase in the amount of α-actinin required to arrest thin filament motility (p<0.001). PKA did not modify sliding velocity in WT. In KO, PKA had no effect on myosin sliding. We conclude that cMyBP-C regulates AMI by limiting inefficient cross-bridge formation and by enhancing the power stroke step. Phosphorylation status of cMyBP-C appears to play a critical role on cardiac contractility through a direct effect on the myosin molecular motor.

Download Full-text

The nebulin repeat protein Lasp regulates I-band architecture and filament spacing in myofibrils

The Journal of Cell Biology ◽

10.1083/jcb.201401094 ◽

2014 ◽

Vol 206 (4) ◽

pp. 559-572 ◽

Cited By ~ 27

Author(s):

Isabelle Fernandes ◽

Frieder Schöck

Keyword(s):

Thin Filament ◽

Muscle Protein ◽

Nemaline Myopathy ◽

Single Amino Acid ◽

Actin Binding ◽

Filament Length ◽

Thin Filaments ◽

Single Amino Acid Change ◽

Filament Spacing

Mutations in nebulin, a giant muscle protein with 185 actin-binding nebulin repeats, are the major cause of nemaline myopathy in humans. Nebulin sets actin thin filament length in sarcomeres, potentially by stabilizing thin filaments in the I-band, where nebulin and thin filaments coalign. However, the precise role of nebulin in setting thin filament length and its other functions in regulating power output are unknown. Here, we show that Lasp, the only member of the nebulin family in Drosophila melanogaster, acts at two distinct sites in the sarcomere and controls thin filament length with just two nebulin repeats. We found that Lasp localizes to the Z-disc edges to control I-band architecture and also localizes at the A-band, where it interacts with both actin and myosin to set proper filament spacing. Furthermore, introducing a single amino acid change into the two nebulin repeats of Lasp demonstrated different roles for each domain and established Lasp as a suitable system for studying nebulin repeat function.

Download Full-text

Causes of fatigue in slow-twitch rat skeletal muscle during dynamic activity

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.91043.2008 ◽

2009 ◽

Vol 297 (3) ◽

pp. R900-R910 ◽

Cited By ~ 13

Author(s):

Morten Munkvik ◽

Per Kristian Lunde ◽

Ole M. Sejersted

Keyword(s):

Skeletal Muscle ◽

Isometric Force ◽

Force Production ◽

Maximum Force ◽

Stimulation Protocol ◽

Shortening Velocity ◽

Dynamic Activity ◽

Initial Values ◽

Isotonic Shortening

Skeletal muscle fatigue is most often studied in vitro at room temperature and is classically defined as a decline in maximum force production or power output, exclusively linked to repeated isometric contractions. However, most muscles shorten during normal use, and we propose that both the functional correlate of fatigue, as well as the fatigue mechanism, will be different during dynamic contractions compared with static contractions. Under isoflurane anesthesia, fatigue was induced in rat soleus muscles in situ by isotonic shortening contractions at 37°C. Muscles were stimulated repeatedly for 1 s at 30 Hz every 2 s for a total of 15 min. The muscles were allowed to shorten isotonically against a load corresponding to one-third of maximal isometric force. Maximal unloaded shortening velocity (V0), maximum force production (Fmax), and isometric relaxation rate (−dF/d t) was reduced after 100 s but returned to almost initial values at the end of the stimulation protocol. Likewise, ATP and creatine phosphate (CrP) were reduced after 100 s, but the level of CrP was partially restored to initial values after 15 min. The rate of isometric force development, the velocity of shortening, and isotonic shortening were also reduced at 100 s, but in striking contrast, did not recover during the remainder of the stimulation protocol. The regulatory myosin light chain (MLC2s) was dephosphorylated after 100 s and did not recover. Although metabolic changes may account for the changes of Fmax, −dF/d t, and V0, dephosphorylation of MLC2s may be involved in the fatigue seen as sustained slower contraction velocities and decreased muscle shortening.

Download Full-text

Work-dependent deactivation of a crustacean muscle

Journal of Experimental Biology ◽

10.1242/jeb.202.18.2551 ◽

1999 ◽

Vol 202 (18) ◽

pp. 2551-2565 ◽

Cited By ~ 6

Author(s):

R.K. Josephson ◽

D.R. Stokes

Keyword(s):

Muscle Activation ◽

Isometric Force ◽

Carcinus Maenas ◽

Stimulus Frequency ◽

Force Production ◽

Muscle Length ◽

Background Level ◽

Shortening Velocity ◽

Shortening Deactivation ◽

Work Done

Active shortening of respiratory muscle L2B from the crab Carcinus maenas results in contractile deactivation, seen as (1) a decline of force during the course of isovelocity shortening, (2) a reduction in the rate of force redevelopment following shortening, (3) a depression of the level of isometric force reached following shortening, and (4) an accelerated relaxation at the end of stimulation. The degree of deactivation increases with increasing distance of shortening, decreases with increasing shortening velocity, and is approximately linearly related to the work done during shortening. Deactivation lasts many seconds if stimulation is maintained, but is largely although not completely removed if the stimulation is temporarily interrupted so that the force drops towards the resting level. Deactivation for a given distance and velocity of shortening increases with increasing muscle length above the optimum length for force production. Stimulating muscle L2B at suboptimal frequencies gives tetanic contractions that are fully fused but of less than maximal amplitude. The depression of force following shortening, relative to the force during an isometric contraction, is independent of the stimulus frequency used to activate the muscle, indicating that deactivation is not a function of the background level of stimulus-controlled muscle activation upon which it occurs. Deactivation reduces the work required to restretch a muscle after it has shortened, but it also lowers the force and therefore the work done during shortening. The net effect of deactivation on work output over a full shortening/lengthening cycle is unknown.

Download Full-text

Disruption of cardiac thin filament assembly arising from a mutation in LMOD2: A novel mechanism of neonatal dilated cardiomyopathy

Science Advances ◽

10.1126/sciadv.aax2066 ◽

2019 ◽

Vol 5 (9) ◽

pp. eaax2066 ◽

Cited By ~ 4

Author(s):

Rebecca C. Ahrens-Nicklas ◽

Christopher T. Pappas ◽

Gerrie P. Farman ◽

Rachel M. Mayfield ◽

Tania M. Larrinaga ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Cardiac Transplantation ◽

Thin Filament ◽

Force Production ◽

Actin Binding ◽

Isolated Cardiomyocytes ◽

Thin Filaments ◽

Familial Dilated Cardiomyopathy ◽

Filament Assembly ◽

Explanted Heart

Neonatal heart failure is a rare, poorly-understood presentation of familial dilated cardiomyopathy (DCM). Exome sequencing in a neonate with severe DCM revealed a homozygous nonsense variant in leiomodin 2 (LMOD2, p.Trp398*). Leiomodins (Lmods) are actin-binding proteins that regulate actin filament assembly. While disease-causing mutations in smooth (LMOD1) and skeletal (LMOD3) muscle isoforms have been described, the cardiac (LMOD2) isoform has not been previously associated with human disease. Like our patient, Lmod2-null mice have severe early-onset DCM and die before weaning. The infant’s explanted heart showed extraordinarily short thin filaments with isolated cardiomyocytes displaying a large reduction in maximum calcium-activated force production. The lack of extracardiac symptoms in Lmod2-null mice, and remarkable morphological and functional similarities between the patient and mouse model informed the decision to pursue cardiac transplantation in the patient. To our knowledge, this is the first report of aberrant cardiac thin filament assembly associated with human cardiomyopathy.

Download Full-text

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.

The Journal of Cell Biology ◽

10.1083/jcb.116.6.1369 ◽

1992 ◽

Vol 116 (6) ◽

pp. 1369-1380 ◽

Cited By ~ 120

Author(s):

L Hemmings ◽

P A Kuhlman ◽

D R Critchley

Keyword(s):

Smooth Muscle ◽

Binding Site ◽

Muscle Protein ◽

Actin Binding ◽

In Vitro Assays ◽

E Coli ◽

Cos Cells ◽

Terminal Domain ◽

Actin Binding Site

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.

Download Full-text