Large Maf Transcription Factors Reawaken Evolutionarily Dormant Fast-Glycolytic Type IIb Myofibers in Human Skeletal Muscle

Small mammals, such as mice, rely on the rapid contraction of skeletal muscles to achieve swift movements. These capabilities are enabled by type IIb myofibers, which express the fastest myosin IIb in limb muscles, encoded by MYH4. In contrast, larger mammals, including humans, exhibit a marked reduction or complete absence of type IIb myofibers and MYH4 expression, favoring slower-contracting myofiber types. The evolutionary shift away from MYH4 expression in larger mammals and the underlying regulatory mechanisms remain poorly understood. Previously, we identified the large Maf transcription factor family (Mafa, Mafb, Maf) as principal regulators of type IIb myofiber determination in mice. Here, we provide the first evidence that the large MAFs specifically induces MYH4 expression in the skeletal muscles of large mammals, including humans and bovine. Furthermore, induction of MYH4 by large MAFs significantly enhances the glycolytic capacity of human myotubes, as evidenced by flux analyzer data and RNA-seq showing the upregulation of numerous genes associated with glucose metabolism. Lastly, RNA-seq analysis of human muscle biopsies reveals a positive correlation between MAFA, MAF, and MYH4 expression. Our findings propose that the large MAFs promotes the formation of the fastest type IIb myofibers in the skeletal muscles of large mammals. This discovery not only elucidates the mechanisms underlying the loss of type IIb myofibers in larger mammals but also holds potential implications for enhancing athletic performance and counteracting the loss of fast-twitch myofibers associated with aging. Overall design: RNA-seq profiling of control cells and large MAFs overexpressing cells at Day6 of differentiation.

小型哺乳动物（如小鼠）依靠骨骼肌的快速收缩实现迅捷运动。这类运动能力由IIb型肌纤维（type IIb myofibers）赋予，该类肌纤维在肢体肌肉中表达速度最快的肌球蛋白IIb（myosin IIb），其编码基因为MYH4。与之相反，包括人类在内的大型哺乳动物，其IIb型肌纤维与MYH4的表达量显著降低甚至完全缺失，转而偏好收缩速度较慢的肌纤维类型。大型哺乳动物中MYH4表达的进化转变及其潜在调控机制仍有待深入阐释。此前，本团队已鉴定出大Maf转录因子家族（large Maf transcription factor family，包含Mafa、Mafb、Maf三个成员）是小鼠IIb型肌纤维分化的核心调控因子。本研究首次证实，大MAF家族可特异性诱导包括人类、牛在内的大型哺乳动物骨骼肌细胞中MYH4的表达。此外，通过通量分析仪（flux analyzer）数据与RNA测序（RNA-seq）结果均可证明，大MAF家族诱导MYH4表达可显著增强人类肌管的糖酵解能力，上调大量与葡萄糖代谢相关的基因。最后，对人类肌肉活检样本的RNA测序（RNA-seq）分析显示，MAFA、MAF与MYH4的表达水平呈显著正相关。本研究结果表明，大MAF家族可促进大型哺乳动物骨骼肌中最快收缩类型的IIb型肌纤维的形成。该发现不仅阐明了大型哺乳动物丢失IIb型肌纤维的分子机制，同时为提升运动表现、对抗衰老相关的快缩肌纤维（fast-twitch myofibers）流失提供了潜在应用方向。总体实验设计：对细胞分化第6天时的对照细胞与过表达大MAF家族的细胞进行RNA测序（RNA-seq）转录组分析。