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. 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型肌纤维介导：该肌纤维在肢体骨骼肌中表达目前已知最快的肌球蛋白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则表现出IIb型肌纤维与MYH4表达的显著减少或完全缺失，更倾向于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目前仍不甚明晰。此前我们已在小鼠中鉴定出大Maf转录因子家族（large Maf transcription factor family，包含Mafa、Mafb、Maf）是IIb型肌纤维定型的核心调控因子。本研究首次证实，大MAFs可在包括人类与牛在内的大型哺乳动物的骨骼肌中特异性诱导MYH4表达。此外，大MAFs介导的MYH4诱导表达可显著增强人类肌管的糖酵解能力，通量分析仪数据与RNA测序（RNA-seq）结果均显示，大量与葡萄糖代谢相关的基因表达上调。最后，对人类肌肉活检样本的RNA-seq分析显示，MAFA、MAF与MYH4的表达呈正相关。本研究结果表明，大MAFs可促进大型哺乳动物骨骼肌中最快收缩的IIb型肌纤维的形成。这一发现不仅阐明了大型哺乳动物IIb型肌纤维缺失的潜在机制，还为提升运动表现以及拮抗衰老相关的快缩肌纤维流失提供了潜在应用价值。本研究对分化第6天的对照细胞与过表达大MAFs的细胞开展了RNA-seq转录组分析。