Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multi-nucleated mammalian muscle formation.
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided
- H Lee Sweeney
- H Lee Sweeney
- H Lee Sweeney
- David W Hammers
- Richard E Cheney
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the University of Florida. Protocol #201910602.
- Pekka Lappalainen, University of Helsinki, Finland
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here, we report that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays we showed that PI4KIIIα, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIα to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. Our results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.
Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.
To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.
Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.
We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.
Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH ; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant  to DM and OB.