Unraveling the pathophysiology of Bethlem Myopathy using a unique zebrafish model for the disease – FishandCol6

- The silicone clamp technique and single channel patch clamp recording will be used to explore intramembrane charge movements, action potentials characteristics and biophysical properties of voltage-activated, stretch-regulated and resting channels in larval and 1-year old fish isolated trunk muscle fibers.
- Resting intracellular Ca2+ concentrations under voltage control and depolarization-induced Ca2+ changes will be measured in voltage-clamped fibers using a Ca2+ dye dialyzed in the patch pipette to explore sarcoplasmic reticulum (SR) Ca2+ release and SR Ca2+ pump efficiencies.
- The influx of Ca2+ coming from outside will be measured using the high-resolution technique of Mn2+ quenching of fluorescence in voltage-clamped fibers to monitor the rate of Mn2+ entry at resting potential and in response to voltage pulses inducing SR Ca2+ depletion.
- Fish swimming performance and O2 consumption during natural motor activity will be assessed by placing fish in a swimming tunnel and flow speed to cause fatigue. Specific twitch and maximal tetanus muscle force developed by electrically-evoked trunk contractions will be measured at larvae and adult stage in fish using a home-made isometric force transducer.
- Immunolocalization of ion channels and proteins involved in Ca2+ homeostasis will be analyzed with fluorescent confocal microscopy.
- Spatiotemporal expression pattern of ColVI and its binding partners in the myomatrix will be investigated using immunostaining and biochemistry and by creating a transgenic reporter line expressing a fluorescent protein under the control of col6a1 promoter.
- The supramolecular assembly of trimeric protein containing a truncated a1 chain at different stages of zebrafish life span will be assessed using agarose-acrylamide composite native gels designed for high-molecular-weight proteins.
- Transcriptomic analysis using RNAseq will be carried out to identify dysregulated genes in col6a1?ex14-/- larvae and adult muscles, specifically genes encoding matrisome proteins and proteins involved in Ca2+ signaling. A selection of dysregulated genes will be validated at the protein level.

Using current- and voltage-clamp combined with intracellular Ca2+ measurements on isolated fibers from 1-year old col6a1?ex14-/- fish, which were found to be more severely affected than young fish, we have demonstrated that (i) muscle action potentials are unchanged in mutant fish as compared to wild-type, (ii) the voltage-dependence of charge movements produced by depolarization-induced activation of dihydropyridine receptors, that control sarcoplasmic reticulum Ca2+ release, is shifted toward negative potentials in mutant fish, (iii) concomitantly, the voltage-dependence of depolarization-evoked intracellular Ca2+ transients is shifted toward negative voltages over the whole membrane voltage range, promoting in this way a pathogenic SR Ca2+ leak at resting membrane potentials that could likely explain muscle wasting observed in BM. These data thus valid our initial working hypothesis on the pathogenic mechanisms involved in BM. In parallel, we performed a comprehensive characterization of the different steps of excitation-contraction coupling process in wild-type zebrafish, which revealed unexpected superfast kinetics of activation of zebrafish muscle fibers. We also identified and cloned a two-pore domain K+ channel in adult zebrafish muscle fiber never described in vertebrates’ skeletal muscle that confers sensitivity to stretch and heat to zebrafish muscle fiber excitation. The zebrafish genome contains 2 paralogs of the human gene, knck4a and b. Using qPCR, we showed that the second paralog knck4b starts to be expressed and is fully functional in skeletal muscle after metamorphosis.
Thanks to the newly generated rabbit antibodies against the zebrafish ColVIa1 chain, we showed that, contrary to wild type, ColVI immunoreactivity was abnormally present as irregular patches in the trunk muscle of col6a1?ex14-/- larvae and almost disappeared over time. This can explain the worsening of the skeletal muscle phenotype as fish aged. No immunoreactivity of ColVIa1 chain was observed neither in the trunk of col6a1-null larvae and adults (a mutant line kindly provided by P. Bonaldo, Italy). In the meantime, we have generated guinea pig antibodies to zebrafish ColVIa1 chain and confirmed these data. These new antibodies allow to perform double staining with muscle and extracellular matrix markers and thus to explore in more details the col6a1?ex14-/- mutant phenotype. These antibodies work well with western blot and allowed us to confirm our immunostaining data. Finally, a first set of swimming performance experiments with a limited number of fish was conducted and showed that the critical swimming speed (Ucrit, expressed as body length/s) was lower in mutant fish as compared to controls. A second set of experiments with col6a1?ex14-/- fish is ongoing.

At this stage, the following data may be highlighted: (i) demonstration of superfast properties of excitation-contraction coupling in adult zebrafish fast skeletal muscle fibers, (ii) Identification and functional characterization of a mechano-gated K+ channel in adult zebrafish skeletal muscle, (iii) Alteration of the control of the efflux of Ca2+ from the sarcoplasmic reticulum in muscle fibers from a zebrafish model of BM, (iv) the truncated ColVI protein is initially secreted but does not assemble correctly in the myomatrix and then disappeared in adults, (v) col6a1?ex14 heterozygous fish show reduced muscle performance compared to wild type fish.
The next step will be now to analyze muscle performance in response to electrically-evoked contractions. These force measurement data should correlate with measurement of swimming performance. Additionally, RNA-seq of isolated skeletal muscles of WT vs mutant trunks will be carried out to understand how alteration of an ECM protein can disturb intracellular muscle cell function as planned in our proposal, except that scRNA-seq might be preferred to bulk RNA-seq.
The next period will be also devoted to the study of resting trans-sarcolemmal Ca2+ influx and store-operated Ca2+ entry (SOCE) in col6a1?ex14 zebrafish muscle fibers. Our finding that a SR Ca2+ leak is exacerbated at resting membrane potentials in mutant fish indeed may imply that a chronic SR Ca2+ depletion over-activates SOCE as observed in other dystrophic conditions. Secondly, we plan to determine if a direct or an indirect interaction exists between ColVI and the main subunit of DHPR using histochemical, Proximity Ligation Assays (PLA) and co-immunoprecipitation methodologies using the newly generated guinea pig antibodies to zebrafish ColVIa1 chain.

- List of multi-partner publications
Peer-reviewed articles:
Idoux R, Bretaud S, Berthier C, Jacquemond V, Ruggiero F, Allard B. Étude physiopathologique de la myopathie de Bethlem à l’aide d’un modèle de poisson zèbre – 16èmes Journées de la Société Française de Myologie (JSFM) - Prix Master 2018 R. Idoux. Med. Sci. (Paris) 35 Hors-série n° 2:39-42 (2019).

Communications (conferences):
1. Idoux R, Bretaud S, Berthier C, Jacquemond V, Ruggiero F, Allard B. Investigation of excitability and Ca2+ handling defects in skeletal muscle fibers from a zebrafish model of Bethlem myopathy. Communication orale 17èmes JSFM - Brest (Nov 2020).
2. Idoux R, Bretaud S, Berthier C, Jacquemond V, Ruggiero F, Allard B. Investigation of excitability and Ca2+ handling defects in skeletal muscle fibers from a zebrafish model of Bethlem myopathy. Oral presentation (visio-conference) Muscle Science Talks (Janv 2021).

Conferences (lay):
Idoux R, Bretaud S, Berthier C, Jacquemond V, Ruggiero F, Allard B. Présentation vulgarisée du projet FishandCol6 dans le cadre de l’opération « 1000 chercheurs dans les écoles » Saint- Etienne (Nov 2020).

- List of mono-partner publications
Communications (conferences)
Idoux R, Berthier C, Jacquemond V, Allard B. Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling: Calcium Signaling and Excitation–Contraction in Cardiac, Skeletal and Smooth Muscle. Oral communication (visio-conference) - EC coupling meeting 2021. J Gen Physiol 154:e2021ecc14 (2022).
Idoux R, Berthier C, Jacquemond V, Allard B. Comprehensive characterization of excitation-contraction coupling in isolated adult zebrafish fast skeletal muscle fibers reveals superfast excitation-contraction coupling properties. Poster 18èmes JSFM Saint-Etienne (Nov 2021).

Bethlem myopathy (BM) is a misunderstood disorder characterized by abnormal contractures and muscle weakness. BM is caused by mutations in one of the genes encoding the 3 major a-chains of collagen VI (ColVI), a component of the myomatrix. In skeletal muscle, ColVI is produced by interstitial fibroblasts and the signaling pathway that transduces ColVI extracellular signals within muscle fibers remains to be identified. In an attempt to address this issue, a ColVI-null mouse model (Col6a1-/-) was generated. Absence of ColVI resulted in reduced muscle strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR), suggesting abnormal SR Ca2+ handling, together with defects in mitochondria structure and function. Morpholino-based knockdown of different ColVI a-chains in developing zebrafish resulted in comparable defects in skeletal muscle. Although mouse and zebrafish models have significantly contributed to a better understanding of ColVI-related diseases, none of these models consistently reproduced mutations occurring in BM patients. Moreover morpholinos injection induced transient protein knockdown while BM is a progressive disease. In order to circumvent these limitations, Partner 2 has established a zebrafish line exhibiting a mutation that is the most frequently found in BM patients and consisting in an in-frame skipping of exon 14 in col6a1 mRNA (col6a1?ex14 a.k.a. col6a1ama605003). Ultrastructural analysis of mutant fish revealed abnormal myofibers with disorganized myofibrils, enlarged SR, altered mitochondria and misaligned sarcomeres that are all hallmarks of BM. The general objective of the present project is to use this BM zebrafish model to unravel the pathophysiological mechanisms involved in this pathology. Besides the uniqueness of this BM model, the zebrafish has emerged as an excellent model organism for gaining insight into the pathophysiology of human diseases and developmental and genetic analysis. Our working hypothesis is that lack of ColVI compromises muscle excitability and/or intracellular Ca2+ handling. This assumption is based on the observations that (i) in the different col6a-deficient animal models used so far, the structure of the SR which plays a pivotal role in Ca2+ homeostasis was found to be altered, (ii) matrix components have been shown to regulate ion channels, (iii) intracellular Ca2+ is under the tight control of ion channels and plays a pivotal role in muscle contraction, development and disease, (iv) preliminary data obtained by the two partners revealed in col6a1?ex14 fish reduced SR Ca2+ release channel immunostaining and reduced density of charge movements that reflect activity of the protein complex controlling Ca2+ release.
This research program will be conducted by two research groups with complementary expertise in muscle cell electrophysiology and intracellular Ca2+ measurements in health and disease (Partner 1), and in collagen biology and pathology and zebrafish model (Partner 2). The proposal is organized in 5 tasks which, in comparing larval and adult col6a1?ex14 and wildtype siblings, aim to (i) perform a comprehensive analysis of skeletal muscle phenotype from transcriptome profiling, to protein synthesis and assembly (ii) explore excitable properties, plasmalemmal ion channels and effect of membrane pressure-induced stress on ion conductances, (iii) measure resting intracellular Ca2+ and analyze excitation-contraction coupling properties, (iv) explore trans-sarcolemmal resting Ca2+ influx and store-operated Ca2+ entry, (v) measure swimming performance and quantify trunk muscle force.
In a fundamental point of view, our proposal should importantly contribute to better understand how the myomatrix can transduce signals within skeletal muscle. In identifying the disrupted muscle functions in BM, the successful execution of the project should also allow in the long term to determine potential therapeutic targets for this currently incurable pathology.