Background
Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene ...mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe.
Methods
Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro.
Results
MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway.
Conclusions
Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability ...of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.
Myosin binding protein-C (MyBP-C), a major thick filament associated sarcomeric protein, plays an important functional and structural role in regulating sarcomere assembly and crossbridge formation. ...Missing or aberrant MyBP-C proteins (both cardiac and skeletal) have been shown to cause both cardiac and skeletal myopathies, thereby emphasising its importance for the normal functioning of the sarcomere. Mutations in cardiac MyBP-C are a major cause of hypertrophic cardiomyopathy (HCM), while mutations in skeletal MyBP-C have been implicated in a disease of skeletal muscle—distal arthrogryposis type 1 (DA-1). Here we report the first detailed electron microscopy studies on human cardiac and skeletal tissues carrying MyBP-C gene mutations, using samples obtained from HCM and DA-1 patients. We have used established image averaging methods to identify and study the axial distribution of MyBP-C on the thick filament by averaging profile plots of the A-band of the sarcomere from electron micrographs of human cardiac and skeletal myopathy specimens. Due to the difficulty of obtaining normal human tissue, we compared the distribution to the A-band structure in normal frog skeletal, rat cardiac muscle and in cardiac muscle of MyBP-C-deficient mice. Very similar overall profile averages were obtained from the C-zones in cardiac HCM samples and skeletal DA-1 samples with MyBP-C gene mutations, suggesting that mutations in MyBP-C do not alter its mean axial distribution along the thick filament.
Abstract
Nanoparticles (NPs) may come into contact with circulating blood elements including platelets following inhalation and translocation from the airways to the bloodstream or during proposed ...medical applications. Studies with model polystyrene latex nanoparticles (PLNPs) have shown that NPs are able to induce platelet aggregation in vitro suggesting a poorly defined potential mechanism of increased cardiovascular risk upon NP exposure. We aimed to provide insight into the mechanisms by which NPs may increase cardiovascular risk by determining the impact of a range of concentrations of PLNPs on platelet activation in vitro and in vivo and identifying the signaling events driving NP-induced aggregation. Model PLNPs of varying nano-size (50 and 100 nm) and surface chemistry unmodified (uPLNP), amine-modified (aPLNP) and carboxyl-modified (cPLNP) were therefore examined using in vitro platelet aggregometry and an established mouse model of platelet thromboembolism. Most PLNPs tested induced GPIIb/IIIa-mediated platelet aggregation with potencies that varied with both surface chemistry and nano-size. Aggregation was associated with signaling events, such as granule secretion and release of secondary agonists, indicative of conventional agonist-mediated aggregation. Platelet aggregation was associated with the physical interaction of PLNPs with the platelet membrane or internalization. 50 nm aPLNPs acted through a distinct mechanism involving the physical bridging of adjacent non-activated platelets leading to enhanced agonist-induced aggregation in vitro and in vivo. Our study suggests that should they translocate the pulmonary epithelium, or be introduced into the blood, NPs may increase the risk of platelet-driven events by inducing or enhancing platelet aggregation via mechanisms that are determined by their distinct combination of nano-size and surface chemistry.
Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal ...arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis.
Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are ...regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.
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•The structure of CsfB is unique among anti-sigma factors•CsfB assembles into a tight homodimer of treble-clef zinc finger domains•CsfB dimerization is essential for inhibition of two alternative sigma factors
Martínez-Lumbreras, Alfano et al. have solved the structure of the anti-sigma factor CsfB and explored its role in inhibiting two alternative sigma factors during Bacillus subtilis spore formation. The results provide insight into the molecular mechanism underlying a gene expression switch in bacteria.
Nanoparticles (NPs) may come into contact with circulating blood elements including platelets following inhalation and translocation from the airways to the bloodstream or during proposed medical ...applications. Studies with model polystyrene latex nanoparticles (PLNPs) have shown that NPs are able to induce platelet aggregation in vitro suggesting a poorly defined potential mechanism of increased cardiovascular risk upon NP exposure. We aimed to provide insight into the mechanisms by which NPs may increase cardiovascular risk by determining the impact of a range of concentrations of PLNPs on platelet activation in vitro and in vivo and identifying the signaling events driving NP-Induced aggregation. Model PLNPs of varying nano-size (50 and 100 nm) and surface chemistry unmodified (uPLNP), a mine-modified (aPLNP) and carboxyl-modified (cPLNP) were therefore examined using in vitro platelet aggregometry and an established mouse model of platelet thromboembolism. Most PLNPs tested induced GPIIb/llla-medlated platelet aggregation with potencies that varied with both surface chemistry and nano-size. Aggregation was associated with signaling events, such as granule secretion and release of secondary agonists, indicative of conventional agonist-mediated aggregation. Platelet aggregation was associated with the physical interaction of PLNPs with the platelet membrane or internalization. 50 nm aPLNPs acted through a distinct mechanism involving the physical bridging of adjacent non-activated platelets leading to enhanced agonist-induced aggregation in vitro and in vivo. Our study suggests that should they translocate the pulmonary epithelium, or be introduced into the blood, NPs may increase the risk of platelet-driven events by inducing or enhancing platelet aggregation via mechanisms that are determined by their distinct combination of nano-size and surface chemistry.
The 70kDa members of the heat shock protein family (eg. Hsp70) function as molecular chaperones by binding to exposed hydrophobic patches on nascent polypeptides forming non-covalent interactions, ...thereby preventing their aggregation and facilitating their proper folding. The folding reaction comprises of cyclic binding and release of the unfolded substrate powered by ATP hydrolysis. Hsp70 requires the assistance of a co-chaperone, generally provided by the Hsp40 group of proteins, for the cycle of protein folding. Biochemical analyses have mapped the possible sites of interaction between the Hsp70 and Hsp40 proteins and predicted a bipartite mode of interaction between Hsp70 and Hsp40. However, structural investigations into the mechanistic features of the folding cycle have been hampered by the transient nature of interaction. The underlying theme for this work was to therefore structurally understand the assembly of Hsp70 proteins with the Hsp40 co-chaperones as a complex during the Hsp70-assisted folding cycle. This study was carried out using human Hsc70 and HSJ1b as representatives of the Hsp70 and Hsp40 families respectively. The first step to understand this co-operation was to develop a strategy to isolate a complex of Hsc70 and HSJ1b suitable for structural studies. Previous studies have reconstituted the Hsp70/Hsp40 complex in vitro by combining the two proteins in molar ratios in the presence of ATP. In this work a co-expression system was developed and a recombinant form of the human Hsc70/HSJ1b complex was successfully purified using a bacterial expression system. Biochemical characterisation revealed that this chaperone complex can protect ~85% of substrate protein from thermal aggregation. Gel filtration analysis revealed that the complex was composed of a heterogenous mix of ~220 kDa and hetero-oligomeric co-polymer species. Analytical ultracentrifugation confirmed that these hetero-oligomeric co-polymer species were not aggregates, and molecular weight for this species was estimated to be 1.1 MDa. These two species represent potentially two different states of association between Hsc70 and HSJ1b. ATP and heat treatment at 42oC with luciferase were identified as factors which promote the conversion of the oligomeric Hsc70/HSJ1b species to the ~220 kDa Hsc70/HSJ1b species. Domain variants of Hsc70 were then generated and their ability to complex with HSJ1b was investigated. Using these Hsc70 domain variants, the region on Hsc70 paramount for polymerisation was identified. The C-terminal 10 kDa lid region was found to be essential for the chaperone/co-chaperone interaction, since the removal of this zone alters binding, function and conformational properties of the Hsc70 and HSJ1b interaction. X-ray crystallography studies on the full length and the domain complexes were carried out, leading to the structure of the apo form of the nucleotide binding domain of Hsc70. Preliminary electron microscopy (EM) analysis was undertaken of the recombinant Hsc70/HSJ1b complex. The preliminary results from negative staining revealed mostly circular particles and were extremely encouraging. Currently work is being carried out to improve sample homogeneity, which will facilitate further EM studies. Thus the recombinant complex generated in this study is an attractive tool to further our understanding of the functional and structural features of the interactions of Hsp70 with Hsp40.
The 70kDa members of the heat shock protein family (eg. Hsp70) function as molecular chaperones by binding to exposed hydrophobic patches on nascent polypeptides forming non-covalent interactions, ...thereby preventing their aggregation and facilitating their proper folding. The folding reaction comprises of cyclic binding and release of the unfolded substrate powered by ATP hydrolysis. Hsp70 requires the assistance of a co-chaperone, generally provided by the Hsp40 group of proteins, for the cycle of protein folding. Biochemical analyses have mapped the possible sites of interaction between the Hsp70 and Hsp40 proteins and predicted a bipartite mode of interaction between Hsp70 and Hsp40. However, structural investigations into the mechanistic features of the folding cycle have been hampered by the transient nature of interaction. The underlying theme for this work was to therefore structurally understand the assembly of Hsp70 proteins with the Hsp40 co-chaperones as a complex during the Hsp70-assisted folding cycle. This study was carried out using human Hsc70 and HSJ1b as representatives of the Hsp70 and Hsp40 families respectively. The first step to understand this co-operation was to develop a strategy to isolate a complex of Hsc70 and HSJ1b suitable for structural studies. Previous studies have reconstituted the Hsp70/Hsp40 complex in vitro by combining the two proteins in molar ratios in the presence of ATP. In this work a co-expression system was developed and a recombinant form of the human Hsc70/HSJ1b complex was successfully purified using a bacterial expression system. Biochemical characterisation revealed that this chaperone complex can protect ~85% of substrate protein from thermal aggregation. Gel filtration analysis revealed that the complex was composed of a heterogenous mix of ~220 kDa and hetero-oligomeric co-polymer species. Analytical ultracentrifugation confirmed that these hetero-oligomeric co-polymer species were not aggregates, and molecular weight for this species was estimated to be 1.1 MDa. These two species represent potentially two different states of association between Hsc70 and HSJ1b. ATP and heat treatment at 42oC with luciferase were identified as factors which promote the conversion of the oligomeric Hsc70/HSJ1b species to the ~220 kDa Hsc70/HSJ1b species. Domain variants of Hsc70 were then generated and their ability to complex with HSJ1b was investigated. Using these Hsc70 domain variants, the region on Hsc70 paramount for polymerisation was identified. The C-terminal 10 kDa lid region was found to be essential for the chaperone/co-chaperone interaction, since the removal of this zone alters binding, function and conformational properties of the Hsc70 and HSJ1b interaction. X-ray crystallography studies on the full length and the domain complexes were carried out, leading to the structure of the apo form of the nucleotide binding domain of Hsc70. Preliminary electron microscopy (EM) analysis was undertaken of the recombinant Hsc70/HSJ1b complex. The preliminary results from negative staining revealed mostly circular particles and were extremely encouraging. Currently work is being carried out to improve sample homogeneity, which will facilitate further EM studies. Thus the recombinant complex generated in this study is an attractive tool to further our understanding of the functional and structural features of the interactions of Hsp70 with Hsp40.
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