Fluvastatin-induced myofibrillar damage is associated with elevated ROS, and impaired fatty acid oxidation, and is preceded by mitochondrial morphological changes.

Previously, we showed that fluvastatin treatment induces myofibrillar damage and mitochondrial phenotypes in the skeletal muscles of Drosophila. However, the sequential occurrence of mitochondrial phenotypes and myofibril damage remains elusive. To address this, we treated flies with fluvastatin for two and five days and examined their thorax flight muscles using confocal microscopy. In the two-day fluvastatin group, compared to the control, thorax flight muscles exhibited mitochondrial morphological changes, including fragmentation, rounding up and reduced content, while myofibrils remained organized in parallel. In the five-day fluvastatin treatment, not only did mitochondrial morphological changes become more pronounced, but myofibrils became severely disorganized with significantly increased thickness and spacing, along with myofilament abnormalities, suggesting myofibril damage. These findings suggest that fluvastatin-induced mitochondrial changes precede myofibril damage. Moreover, in the five-day fluvastatin group, the mitochondria demonstrated elevated H2O2 and impaired fatty acid oxidation compared to the control group, indicating potential mitochondrial dysfunction. Surprisingly, knocking down Hmgcr (Drosophila homolog of HMGCR) showed normal mitochondrial respiration in all parameters compared to controls or five-day fluvastatin treatment, which suggests that fluvastatin-induced mitochondrial dysfunction might be independent of Hmgcr inhibition. These results provide insights into the sequential occurrence of mitochondria and myofibril damage in statin-induced myopathy for future studies.

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity.

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila.

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing single-molecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells.

Insights and perspectives on the enigmatic alary muscles of arthropods.

Three types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles.

Statins Induce Locomotion and Muscular Phenotypes in Drosophila melanogaster That Are Reminiscent of Human Myopathy: Evidence for the Role of the Chloride Channel Inhibition in the Muscular Phenotypes.

The underlying mechanisms for statin-induced myopathy (SIM) are still equivocal. In this study, we employ Drosophila melanogaster to dissect possible underlying mechanisms for SIM. We observe that chronic fluvastatin treatment causes reduced general locomotion activity and climbing ability. In addition, transmission microscopy of dissected skeletal muscles of fluvastatin-treated flies reveals strong myofibrillar damage, including increased sarcomere lengths and Z-line streaming, which are reminiscent of myopathy, along with fragmented mitochondria of larger sizes, most of which are round-like shapes. Furthermore, chronic fluvastatin treatment is associated with impaired lipid metabolism and insulin signalling. Mechanistically, knockdown of the statin-target Hmgcr in the skeletal muscles recapitulates fluvastatin-induced mitochondrial phenotypes and lowered general locomotion activity; however, it was not sufficient to alter sarcomere length or elicit myofibrillar damage compared to controls or fluvastatin treatment. Moreover, we found that fluvastatin treatment was associated with reduced expression of the skeletal muscle chloride channel, ClC-a (Drosophila homolog of CLCN1), while selective knockdown of skeletal muscle ClC-a also recapitulated fluvastatin-induced myofibril damage and increased sarcomere lengths. Surprisingly, exercising fluvastatin-treated flies restored ClC-a expression and normalized sarcomere lengths, suggesting that fluvastatin-induced myofibrillar phenotypes could be linked to lowered ClC-a expression. Taken together, these results may indicate the potential role of ClC-a inhibition in statin-associated muscular phenotypes. This study underlines the importance of Drosophila melanogaster as a powerful model system for elucidating the locomotion and muscular phenotypes, promoting a better understanding of the molecular mechanisms underlying SIM.

Drosophila, an Integrative Model to Study the Features of Muscle Stem Cells in Development and Regeneration.

Muscle stem cells (MuSCs) are essential for muscle growth, maintenance and repair. Over the past decade, experiments in Drosophila have been instrumental in understanding the molecular and cellular mechanisms regulating MuSCs (also known as adult muscle precursors, AMPs) during development. A large number of genetic tools available in fruit flies provides an ideal framework to address new questions which could not be addressed with other model organisms. This review reports the main findings revealed by the study of Drosophila AMPs, with a specific focus on how AMPs are specified and properly positioned, how they acquire their identity and which are the environmental cues controlling their behavior and fate. The review also describes the recent identification of the Drosophila adult MuSCs that have similar characteristics to vertebrates MuSCs. Integration of the different levels of MuSCs analysis in flies is likely to provide new fundamental knowledge in muscle stem cell biology largely applicable to other systems.

Notch Mediates Inter-tissue Communication to Promote Tumorigenesis.

Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, we show that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, we provide evidence that signaling can be mediated by cytonemes from Delta-expressing cells. We, thus, propose that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating.

Characterization of Drosophila ATPsynC mutants as a new model of mitochondrial ATP synthase disorders.

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.

A population of adult satellite-like cells in Drosophila is maintained through a switch in RNA-isoforms.

Adult stem cells are important for tissue maintenance and repair. One key question is how such cells are specified and then protected from differentiation for a prolonged period. Investigating the maintenance of Drosophila muscle progenitors (MPs) we demonstrate that it involves a switch in zfh1/ZEB1 RNA-isoforms. Differentiation into functional muscles is accompanied by expression of miR-8/miR-200, which targets the major zfh1-long RNA isoform and decreases Zfh1 protein. Through activity of the Notch pathway, a subset of MPs produce an alternate zfh1-short isoform, which lacks the miR-8 seed site. Zfh1 protein is thus maintained in these cells, enabling them to escape differentiation and persist as MPs in the adult. There, like mammalian satellite cells, they contribute to muscle homeostasis. Such preferential regulation of a specific RNA isoform, with differential sensitivity to miRs, is a powerful mechanism for maintaining a population of poised progenitors and may be of widespread significance.

Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles.

BACKGROUND: A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs) expressed by its FC. Here, we addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? RESULTS: The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. CONCLUSIONS: Transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.

Notch directly regulates the cell morphogenesis genes Reck, talin and trio in adult muscle progenitors.

There is growing evidence that activation of the Notch pathway can result in consequences on cell morphogenesis and behaviour, both during embryonic development and cancer progression. In general, Notch is proposed to coordinate these processes by regulating expression of key transcription factors. However, many Notch-regulated genes identified in genome-wide studies are involved in fundamental aspects of cell behaviour, suggesting a more direct influence on cellular properties. By testing the functions of 25 such genes we confirmed that 12 are required in developing adult muscles, consistent with roles downstream of Notch. Focusing on three, Reck, rhea/talin and trio, we verify their expression in adult muscle progenitors and identify Notch-regulated enhancers in each. Full activity of these enhancers requires functional binding sites for Su(H), the DNA-binding transcription factor in the Notch pathway, validating their direct regulation. Thus, besides its well-known roles in regulating the expression of cell-fate-determining transcription factors, Notch signalling also has the potential to directly affect cell morphology and behaviour by modulating expression of genes such as Reck, rhea/talin and trio. This sheds new light on the functional outputs of Notch activation in morphogenetic processes.

An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila.

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates.

Tup/Islet1 integrates time and position to specify muscle identity in Drosophila.

The LIM-homeodomain transcription factor Tailup/Islet1 (Tup) is a key component of cardiogenesis in Drosophila and vertebrates. We report here an additional major role for Drosophila Tup in specifying dorsal muscles. Tup is expressed in the four dorsal muscle progenitors (PCs) and tup-null embryos display a severely disorganized dorsal musculature, including a transformation of the dorsal DA2 into dorsolateral DA3 muscle. This transformation is reciprocal to the DA3 to DA2 transformation observed in collier (col) mutants. The DA2 PC, which gives rise to the DA2 muscle and to an adult muscle precursor, is selected from a cluster of myoblasts transiently expressing both Tinman (Tin) and Col. The activation of tup by Tin in the DA2 PC is required to repress col transcription and establish DA2 identity. The transient, partial overlap between Tin and Col expression provides a window of opportunity to distinguish between DA2 and DA3 muscle identities. The function of Tup in the DA2 PC illustrates how single cell precision can be reached in cell specification when temporal dynamics are combined with positional information. The contributions of Tin, Tup and Col to patterning Drosophila dorsal muscles bring novel parallels with chordate pharyngeal muscle development.

Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo.

Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of 'identity' transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, we show here that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. We conclude that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity.