Sex Differences in Mitochondrial Function in Mouse Skeletal Muscle Aging

Sex differences in lifespan and age-associated phenotypes are pervasive across species, yet the mechanisms remain poorly understood. Mitochondrial dysfunction is a major hallmark of aging, but whether skeletal muscle mitochondria age along sex specific trajectories remains incompletely defined. Here, we profiled mitochondrial bioenergetics and DNA integrity in flexor digitorum brevis (FDB) muscle from young (3–4 months) and aged (20–24 months) male and female C57BL/6 mice.

We quantified cellular respiration in intact myofibers, measured mitochondrial DNA (mtDNA) copy number, and assessed expression of genes involved in mitochondrial dynamics, electron transport chain (ETC) function, and mtDNA maintenance. Cellular respiration differed by sex at baseline and changed with age in a sex dependent manner. Aged females exhibited a lower basal and ATP-linked respiration than aged males. In contrast, spare respiratory capacity increased in aged females relative to aged males, consistent with age- and sex-specific remodeling of the bioenergetic reserve. mtDNA copy number increased with age in both sexes, with a greater increase in mtDNA content in aged males. Gene expression analyses revealed age- and/or sex-dependent changes, including lower Pink1 expression in females compared to males, an age-related increase in the mtDNA maintenance gene Polg2 only in males, though most genes were not significantly different. As an exploratory systemic readout, we additionally assessed DNA damage responsiveness in whole-blood leukocytes using the alkaline comet assay following oxidative challenge; young females exhibited greater induced DNA damage than young males.

Together, these data define sex- and age-associated mitochondrial remodeling in FDB and provide an initial assessment of sex-dependent inducible DNA damage responses in blood, underscoring the importance of sex as a biological variable in studies of aging.

Included Data:
qPCR file for Mitochondrial Genomic DNA: Graphpad file of the average qPCR results for the genomic mitochondrial DNA genes, excel files with the raw CT values and calculations
OCR file: Graphpad Prism file of the average oxygen consumption rate, Graphpad Prism file for the OCR trace graphs
Gene expression qPCR file: File with Graphpad Prism file of the average qPCR results for the gene expression, excel files with the raw CT values and calculations
Comet assay data file: Comet images, excel file of the number of comets counted per mouse per condition, graphpad file of the average comet assay data

不同物种中普遍存在寿命与衰老相关表型的性别差异，但其背后的调控机制仍未被充分阐明。线粒体功能障碍是衰老的核心特征之一，但骨骼肌线粒体的衰老是否遵循性别特异性轨迹，目前尚未完全明确。本研究对年轻（3~4月龄）与老龄（20~24月龄）C57BL/6雄性及雌性小鼠的趾短屈肌（flexor digitorum brevis, FDB）进行了线粒体生物能学与DNA完整性分析。

我们对完整肌纤维的细胞呼吸进行了定量检测，测定了线粒体DNA（mitochondrial DNA, mtDNA）拷贝数，并分析了参与线粒体动态调节、电子传递链（electron transport chain, ETC）功能及mtDNA维持的基因的表达水平。细胞呼吸基线水平存在性别差异，且随年龄的变化也呈现性别依赖性。老龄雌性小鼠的基础呼吸及ATP关联呼吸水平均低于老龄雄性小鼠；与之相反，老龄雌性小鼠的储备呼吸能力较老龄雄性小鼠有所提升，这与生物能储备的年龄及性别特异性重塑相一致。两性的mtDNA拷贝数均随年龄增长而升高，但老龄雄性小鼠的mtDNA含量增幅更为显著。基因表达分析显示存在年龄及/或性别依赖性的表达变化，例如雌性小鼠的Pink1表达水平低于雄性，仅在雄性小鼠中观察到mtDNA维持基因Polg2的表达随年龄升高，不过多数基因的表达差异未达到统计学显著性。作为探索性的系统生物学检测指标，我们还经氧化应激刺激后采用碱性彗星试验分析了全血白细胞的DNA损伤应答能力；结果显示年轻雌性小鼠的诱导性DNA损伤水平高于年轻雄性小鼠。

综上，本研究明确了FDB肌肉中线粒体的年龄及性别特异性重塑模式，并首次评估了血液中性别依赖性的诱导性DNA损伤应答，凸显了性别作为衰老研究中生物学变量的重要性。

数据集包含：
1. 线粒体基因组DNA qPCR文件：线粒体基因组基因qPCR平均结果的GraphPad Prism文件，以及包含原始CT值与计算数据的Excel文件
2. 氧消耗率（OCR）文件：平均氧消耗率的GraphPad Prism文件，以及OCR轨迹图的GraphPad Prism文件
3. 基因表达qPCR文件：基因表达平均qPCR结果的GraphPad Prism文件，以及包含原始CT值与计算数据的Excel文件
4. 彗星试验数据文件：彗星图像、每个小鼠各实验条件下的彗星计数Excel文件，以及平均彗星试验数据的GraphPad Prism文件