MicroRNAs (miRNAs) are small noncoding endogenous RNAs, typically 21-23 nucleotides long, that regulate gene expression, usually post-transcriptionally, by binding to the 3'-UTR of target mRNA, thus blocking translation. The expression of several miRNAs is significantly altered during cardiac hypertrophy, myocardial ischemia, fibrosis, heart failure, and other cardiac myopathies. Recent studies have implicated miRNA-9 (miR-9) in myocardial hypertrophy. However, a detailed mechanism remains obscure. In this study, we have addressed the roles of miR-9 in muscle development and function using a genetically tractable model system, the indirect flight muscles (IFMs) of Drosophila melanogaster Bioinformatics analysis identified 135 potential miR-9a targets, of which 27 genes were associated with Drosophila muscle development. Troponin-T (TnT) was identified as major structural gene target of miR-9a. We show that flies overexpressing miR-9a in the IFMs have abnormal wing position and are flightless. These flies also exhibit a loss of muscle integrity and sarcomeric organization causing an abnormal muscle condition known as "hypercontraction." Additionally, miR-9a overexpression resulted in the reduction of TnT protein levels while transcript levels were unaffected. Furthermore, muscle abnormalities associated with miR-9a overexpression were completely rescued by overexpression of TnT transgenes which lacked the miR-9a binding site. These findings indicate that miR-9a interacts with the 3'-UTR of the TnT mRNA and downregulates the TnT protein levels by translational repression. The reduction in TnT levels leads to a cooperative downregulation of other thin filament structural proteins. Our findings have implications for understanding the cellular pathophysiology of cardiomyopathies associated with miR-9 overexpression.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

Dynamic changes in mitochondrial shape and size are vital for mitochondrial health and for tissue development and function. Adult Drosophila indirect flight muscles contain densely packed mitochondria. We show here that mitochondrial fusion is critical during early muscle development (in pupa) and that silencing of the outer mitochondrial membrane fusion gene, Marf, in muscles results in smaller mitochondria that are functionally defective. This leads to abnormal muscle development resulting in muscle dysfunction in adult flies. However, post-developmental silencing of Marf has no obvious effects on mitochondrial and muscle phenotype in adult flies, indicating the importance of mitochondrial fusion during early muscle development.

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.

The mechanisms of cell cycle exit by myoblasts during skeletal muscle development are poorly understood. Cell cycle arrest is known to be a prerequisite for myoblast fusion and subsequent differentiation. Despite tremendous knowledge on myoblast fusion and differentiation, tissue-specific factors that spatio-temporally regulate the cell cycle exit are not well known. In this paper, we show that the transcriptional factor/co-activator "Erect wing" (Ewg) synchronises myoblast cell cycle exit with that of the fusion process. Ewg-null myoblasts show delayed temporal development of dorsal longitudinal muscles (DLMs), a group of indirect flight muscles (IFMs), which culminates to abnormal and asymmetric muscle pattern. A role for Ewg in cell cycle exit at G1/S stage is also shown. Reducing Cyclin E dose in Ewg-null mutant rescues the lack of IFMs and flight ability. Thus, we show that Ewg repression of Cyclin E expression is required for the arrest of myoblast proliferation and initiate myoblast fusion and terminal differentiation.

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging.

Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut, salm, and H15.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block-face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase-quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age-related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age-related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue-dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.