Teresa Jagla

The ryanodine receptor (RYR) genes encode evolutionarily conserved calcium release channels involved in a wide range of calcium-dependent biological processes. Here, we show that the sole Drosophila RYR gene ( dRyR ) functions in differentiated somatic and cardiac muscle as well as in developing embryonic myotubes. In the larval body wall muscles, dRyR protein localizes at the SR membranes, and dRyR knockdown adversely affects muscle contractility, suggesting its conserved role in calcium-triggered E-C coupling. After dRyR attenuation, sarcomere, and mitochondrial patterns are severely impaired, showing dRyR involvement in structural muscle properties. However, dRyR is also prominently expressed and functionally required in growing embryonic muscles. dRyR loss of function leads to myotube growth defects and thin myofiber phenotypes, while its overexpression induces myofiber splitting. Given the structural and functional conservation of dRyR , we used Drosophila to test the impact of one human RYR1 variant of unknown significance (VUS). Larvae carrying p.Met4881Ile RYR1 VUS showed impaired mobility and altered structural muscle properties reminiscent of those seen in dRyR knockdown, thus indicating it is likely pathogenic. Overall, we show that Drosophila dRyR plays a conserved role in setting muscle contractility and structural muscle features. Our findings underline the still under-investigated role of dRyR as a promyogenic factor and provide a first example of the impact assessment of a human RYR1 VUS in Drosophila .

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA‐binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down‐regulation of muscle‐ and heart‐specific miRNA, miR‐1 , have been reported in DM1 patients, but the impact of reduced miR‐1 on DM1 pathogenesis has been unknown. Here, we use Drosophila DM1 models to explore the role of miR‐1 in cardiac dysfunction in DM1. We show that miR‐1 down‐regulation in the heart leads to dilated cardiomyopathy (DCM), a DM1‐associated phenotype. We combined in silico screening for miR‐1 targets with transcriptional profiling of DM1 cardiac cells to identify miR‐1 target genes with potential roles in DCM. We identify Multiplexin (Mp) as a new cardiac miR‐1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila . Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM‐developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR‐1 and consecutive up‐regulation of its target Mp/Col15A1 might be critical in DM1‐associated DCM.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in theDrosophilaheart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila . We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin ( Gel ) and dCryAB , both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.

Heat shock proteins (Hsps) form a large family of evolutionarily conserved molecular chaperones that help balance protein folding and protect cells from various stress conditions. However, there is growing evidence that Hsps may also play an active role in developmental processes. Here, we take the example of developmental expression and function of one class of Hsps characterized by low molecular weight, the small Hsps (sHsps). We discuss recent reports and genome-wide datasets that support vital sHsps functions in the developing nervous system, reproductive system, and muscles. This tissue- and time-specific sHsp expression is developmentally regulated, so that the enhancer sequence of an sHsp gene expressed in developing muscle, in addition to stress-inducible elements, also carries binding sites for myogenic regulatory factors. One possible reason for sHsp genes to switch on during development and in non-stress conditions is to protect vital developing organs from environmental insults.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a complex toxic CTG repeat disorder involving misbalance between two RNA- binding factors, MBNL1 and CELF1. How this pathogenic DM1 condition translates into cardiac conduction disorders remains poorly understood. Here, we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and identified associated gene deregulations using TU-tagging based transcriptional profiling of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels and among them increased expression of straightjacket/α2δ3 that encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heart beating, a hallmark of affected conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans but significantly elevated in ventricular muscles from DM1 patients with conduction defects. Taken together, this suggests that reducing the straightjacket/α2δ3 transcript levels in ventricular cardiomyocytes could represent a strategy to prevent conduction defects and in particular intraventricular conduction delay associated with DM1 pathology.

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. Here, we report the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, we observed a different impact on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryABR120G, which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.

This review focuses on the question how sHSPs, still relatively poorly understood proteins, contribute to the development and function of three types of muscle tissue: skeletal, cardiac and smooth.

Significance: We report that glycolytic genes are synchronously activated in midembryogenesis in developing Drosophila muscles and that muscle-targeted attenuation of their expression leads to reduced muscle size and the presence of unfused myoblasts. Importantly, a “thin muscle” phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, strongly suggesting a conserved role of glycolysis in promoting myoblast fusion-based muscle growth. We also show that insulin positively regulates glycolysis and that blocking the insulin pathway phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. These findings led us to propose that setting metabolism to glycolysis-based high-rate biomass production is part of a core myogenic program that promotes formation of syncytial muscles.