Third party funded individual grant
Start date : 01.01.2020
End date : 31.12.2021
Skeletal muscle defines our motoric quality of life. Although most muscle diseases, either inherited or acquired, present with reduced muscle performance and clinical weakness, some other genetic mutations can come along with gain-of-function or modified fine-tuning towards a specific performance regime. The actn3 gene encodes for alpha-actinin-3, an isoform of a-actinin inherent to fast-twitch skeletal muscle. The protein ACTN3 belongs to the cytoskeleton protein family of actinins that mechanically stabilize the actin filaments at the z-disks. ACTN3 is exclusively expressed in fast-twitch type-II, i.e. glycolytic, skeletal muscle fibres while ACTN2 is expressed in all fibre types. About 20% of the world population presents with a common nonsense polymorphism in the actn3 gene that completely blunts ACTN3 expression in fast fibres. However, this mutation is not associtaed with a disease phenotype, but rather with a genetic predisposition towards enudrance performance and cold pre-acclimatization of fast muscle (Head et al. 2015, PLoS Genet). This phenotype is not associated with molecular switches in the expression of slow myosin isoforms in fast muscle but more with metabolic changes and changes in Ca2+ homeostasis that resemble the slow muscle, i.e. oxidative, phenotype. However, since ACTN3 is also a structural cytoskeletal protein, the question arises whether altered structural cytoarchitecture in 3D might be associated with the aforementioned changes that would render myofibrillar architecture more disordered and thus, limit maximum force in actn3 null fast muscle to that seen in more ordered slow muscle. Also, since actn2 is also upregulated in the null fibres, there is currently no information available of whether an intrinsic genetically enforced upregulation of actn2 may also be responsible for cytoarchitectural changes. Multiphoton Second Harmonic Generation (SHG) microscopy in conjunction with quantitative morphometry of muscle cytoarchitecture in single fibres and whole muscle using optical clearing techniques (all having been established in the labs of the German partner) is suggested to be a suitable and feasible set of tools to address this question in a focussed international collaborative research project.
The specific scientific goals during this collaboration initiative are:
1) To assess the 3D cytoarchitecture of the myofibrillar lattice of single EDL muscle fibres from adult and old ACTN3 KO mice using Second Harmonic Generation microscopy. The ultrastructural parameters cosine angle sum (CAS) and Vernier Density (VD) will be assessed at those two ages to quantify cellular remodeling related to the null mutation and age. Single soleus (SOL) muscle fibres will serve as internal controls expressing predominantly ACTN2 as well as from wt EDL muscle fibres expressing predominantly ACTN3.
2) To assess the 3D cytoarchitecture and intercellular collagen fibril distribution of the extracellular matrix in whole EDL muscles following optical clearing with thiodiethanol (TDE) protocols (established at the German partner’s labs). SOL muscles will serve as control, as well as wt EDL muscles.
3) To correlate structural cytoarchitecture data with direct isometric force recordings in single fibres from ACTN3 KO fibres. Control wt results have been already obtained in a current study of the German partner using a novel engineered MechaMorph system to combine biomechatronics with SHG recordings.