Improved health by combining dietary restriction and promoting muscle growth in DNA repair-deficient progeroid mice. Improved health by combining dietary restriction and promoting muscle growth in DNA repair-deficient progeroid mice

Ageing is a complex multifactorial process, impacting all organs and tissues, with DNA damage accumulation serving as a common underlying cause. To decelerate ageing, various strategies have been applied to model organisms and evaluated for health and lifespan benefits. Dietary restriction (DR, also known as caloric restriction) is a well-established long-term intervention recognised for its universal anti-ageing effects. DR temporarily suppresses growth, and when applied to progeroid DNA repair-deficient mice doubles lifespan with systemic health benefits. Counterintuitively, attenuation of myostatin/activin signalling by soluble activin receptor (sActRIIB), boosts the growth of muscle, and in these animals prevents muscle wasting, improves kidney functioning, and compresses morbidity. Here we investigated a combined approach, applying an anabolic regime (sActRIIB) at the same time as DR to Ercc1d/- progeroid mice. Following both single treatments and combined, we monitored global effects on body weight, lifespan and behaviour, and local effects on muscle and tissue weight, muscle morphology and function, and ultrastructural and transcriptomic changes in muscle and kidney. Lifespan was mostly influenced by DR (extended from approx. 20 to 40-weeks; p<0.001), with sActRIIB clearly increasing muscle mass (35-65%) and tetanic force (p<0.001). The combined regime yielded a stable uniform body weight, but increased compared to DR alone, synergistically improved motor coordination and further delayed the onset and development of balance problems. sActRIIB significantly increased muscle fibre size (p<0.05) in mice subjected to DR and lowered all signs of muscle damage. In the neuromuscular junction of Ercc1d/- mice the junctional folding of the basement membrane at the postsynaptic side was either rudimentary or completely missing. Single interventions by sActRIIB treatment or DR only partially rescued this phenotype, while in the double intervention group the regularly shaped junctional foldings were maintained. In kidney of Ercc1d/- mice we observed a mild but significant foot process effacement, which was restored by either intervention. Transcriptome analysis also pointed towards reduced levels of DNA damage in muscle and kidney by DR, but not sActRIIB, while these levels retained lower in the double intervention. In muscle we found synergistic effects of combining sActRIIB with DR, but not in kidney, with an overall better health in the double intervention group. Crucially, the benefits of each single intervention are not lost when administered in combination, but rather strengthened, even when sActRIIB was applied late in life, opening opportunities for translation to human. Overall design: RNA sequencing was performed on mouse quadriceps muscle and kidney specimens of 16-week-old male Ercc1d/- progeroid mice subjected to sActRIIB, dietary restriction, or double intervention, with mock ad libitum Ercc1d/- and Ercc1+/+ wildtype controls.

衰老是一种复杂的多因素生理过程，可累及全身所有器官与组织，而DNA损伤积累是其共同的潜在核心机制。为延缓衰老进程，研究者已针对多种模式生物开发了诸多干预策略，并评估其对机体健康与寿命的增益效果。饮食限制（Dietary Restriction, DR），又称热量限制（Caloric Restriction），是一种被广泛证实的长期干预手段，因其广谱的抗衰老效应得到学界公认。DR可暂时性抑制机体生长，当应用于早衰型DNA修复缺陷小鼠时，可使其寿命翻倍并带来系统性健康获益。

与之相悖的是，通过可溶性激活素受体IIB（soluble Activin Receptor IIB, sActRIIB）减弱肌抑素/激活素信号通路，可促进肌肉生长；在该类小鼠中，该干预可预防肌肉萎缩、改善肾功能并延缓发病进程。本研究探索了联合干预策略：在对Ercc1d/-早衰小鼠实施饮食限制的同时，给予合成代谢方案sActRIIB。我们分别监测了单干预与联合干预下，小鼠体重、寿命与行为学的全局变化，以及肌肉与组织重量、肌肉形态与功能、肌肉与肾脏的超微结构及转录组学改变等局部效应。

寿命主要受DR调控（从约20周延长至40周；p<0.001），而sActRIIB可显著提升肌肉质量（35%~65%）并增强强直收缩力（p<0.001）。联合干预可使小鼠体重维持稳定均匀，且较单纯DR组更高，同时协同改善运动协调能力，进一步延迟平衡障碍的发作与进展。sActRIIB可显著增大DR处理小鼠的肌纤维横截面积（p<0.05），并缓解所有肌肉损伤征象。在Ercc1d/-小鼠的神经肌肉接头（neuromuscular junction）处，突触后侧（postsynaptic side）的基底膜连接褶皱要么发育不全，要么完全缺失。单应用sActRIIB或DR仅能部分挽救该表型，而联合干预组则可维持形态规则的连接褶皱。在Ercc1d/-小鼠的肾脏中，我们观察到轻度但显著的足突脱落（foot process effacement）现象，两种单干预手段均可使其恢复至正常水平。转录组分析显示，DR可降低肌肉与肾脏中的DNA损伤水平，而sActRIIB无此效果；但联合干预组的DNA损伤水平仍维持在较低水平。在肌肉中，我们发现sActRIIB与DR联合存在协同效应，但肾脏中未观察到此现象，联合干预组的整体健康状况更佳。至关重要的是，联合给药并未抵消单一干预的获益，反而进一步增强了效果，即便sActRIIB在小鼠晚年才开始给药，这为向人类临床转化提供了可行契机。

整体实验设计：对16周龄雄性Ercc1d/-早衰小鼠分别给予sActRIIB、饮食限制或联合干预，以自由进食的Ercc1d/-小鼠与Ercc1+/+野生型（wildtype）小鼠作为对照组，对其股四头肌（quadriceps muscle）与肾脏标本进行RNA测序（RNA sequencing）。