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Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

ABSTRACT: Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.

SUBMITTER: Rudolph F

PROVIDER: S-EPMC7305127 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

Rudolph Franziska F Fink Claudia C Hüttemeister Judith J Kirchner Marieluise M Radke Michael H MH Lopez Carballo Jacobo J Wagner Eva E Kohl Tobias T Lehnart Stephan E SE Mertins Philipp P Gotthardt Michael M

Nature communications 20200619 1

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signa ...[more]

PMID: 32561764

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Project description:The "Woody" or "Wooden" breast disease is a severe myopathy of pectoralis major muscle recently identified within rapidly growing broiler lines all around the world with a prevalence rate around 20%, or even higher. Although of significant ethical and economic impact, little is known regarding the structural and functional aspects of the contractile apparatus in the woody breast muscle. The aim of the present study was to determine physiological properties of the contractile system in the morphologically intact muscle fibers of focally damaged woody breast in comparison with normal muscle fibers to gain insight into the muscle function of the animal and possibly mechanisms involved in the disease development. Muscle samples were taken from woody breast (non-lesioned areas) and normal breast muscles from broilers. Length-tension curves, maximal active stress, maximal shortening velocity, calcium sensitivity, rate of tension development, lattice spacing and muscle biochemical composition were investigated on single skinned fibers. Sarcomeres of woody breast fibers were more compliant, which is very likely related to the wider spacing (18% wider compared to controls) between thick and thin filament. No differences were found in optimal sarcomere length (2.68 ± 0.04 vs. 2.65 ± 0.05 ?m) nor in maximal active stress (116 ± 17 vs. 125 ± 19 mN mm-2). However, woody breast fibers had less steep descending arm as shown in length-tension curve. Woody breast muscle fibers had 40% bigger sarcomeric volume compared to controls. Content of contractile proteins (myosin and actin), and maximal shortening velocity were unchanged indicating that the growth in woody breast muscle fiber was associated with synthesis of new contractile units with unaltered kinetics. Calcium sensitivity was decreased in woody breast muscle fibers significantly. In conclusion, the results show that the rapid growth of muscle in woody breast disease is associated with significant structural and functional changes in the pectoralis major musculature, associated with alterations in the mechanical anchoring of contractile filaments.

Project description:The E143K (Glu???Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling.The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts.As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.

Dataset Information

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

Publications

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

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