Skeletal muscles play a central role in converting biological fuel to functional force via the contraction of myofibrils. Until recently, myofibrils were characterized as individual cylindrical structures that run parallel to each other along the length of the muscle cell. However, using nanoscale 3D imaging, our group demonstrated that the contractile apparatus of vertebrate muscle cells is comprised of a unified myofibrillar matrix in which myofibrils frequently connect to each other through branching and merging events. While this work established myofibrillar connectivity as a fundamental component of all striated muscle cell types in vertebrate models, including mice and humans, it is unknown whether myofibrillar connectivity is evolutionarily conserved across invertebrate models commonly used to investigate muscle cell biology. Here, we use 3-dimensional focused-ion beam electron microscopy (FIB-SEM) to evaluate myofibrillar ultrastructure in different striated muscle cell types from Drosophila melanogaster. All procedures were approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee and performed in accordance with the guidelines described in the Animal Care and Welfare Act (7 USC 2142 § 13). 3D reconstruction of myofibrillar structures revealed that, similar to vertebrates, the myofibrils within direct flight (DF), jump, and leg muscles of Drosophila link together to form highly connected myofibrillar networks. Alternatively, we found that myofibrils in indirect flight (IF) muscles do not connect to each other as each successive sarcomere was connected in series to form isolated myofibrils. Quantitative assessment of DF, jump, and leg muscles revealed that the frequency of sarcomere branching differs by muscle type with leg muscles exhibiting the highest branching compared to no branching in IF muscles (see Supporting Figures). The percentage of myofibrils within the field of view of our datasets with at least one branching sarcomere is as follows: 94 ± 4.2% (mean ± SE, 3 cells, 150 myofibrils, 690 sarcomeres) in leg muscles, 77 ± 5.0% (4 cells, 200 myofibrils, 1461 sarcomeres) in DF muscles, 50 ± 3.7% (3 cells, 150 myofibrils, 1713 sarcomeres) in jump muscles, and 0 ± 0 % (3 cells, 114 myofibrils, 982 sarcomeres) in IF muscles. Classifying the analyzed muscles by specialized biological functions (slow oxidative: leg muscle; and fast oxidative/glycolytic: jump, DF, and IF muscles) suggests that branching events are more frequent in slow-twitch muscles compared to fast-twitch muscles. Since this frequency pattern arises in both vertebrates and invertebrates, we posit that sarcomere branching is an evolutionarily conserved adaptation. Therefore, the long-standing belief that sarcomeres are only arranged in a linear fashion (i.e., end to end in series) must be revisited to improve our understanding of how muscle architecture impacts biological processes and musculoskeletal pathologies.
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