Supplementary Material for: Changes in serial sarcomere number of five hindlimb muscles across adult aging in rats

Introduction: The age-associated loss of muscle mass is partly accounted for by a reduction in muscle fascicle length (FL). Studies on rodents have confirmed this reduced FL is driven by a loss of sarcomeres aligned in series (serial sarcomere number; SSN) along a muscle. However, studies on rodents have focused primarily on rat plantar flexor SSN at two aging timepoints, leaving an incomplete view of age-related changes in SSN. Hence, this study investigated SSN as a contributor to the age-related loss of muscle mass in five hindlimb muscles across four aging timepoints in rats. Methods: The soleus, medial gastrocnemius (MG), plantaris, tibialis anterior (TA), and vastus lateralis (VL) were obtained from 5 young (8 months), 5 middle-aged (20 months), 5 old (32 months), and 5 very old (36 months) male F344BN rats. After fixation of muscles in formalin and digestion in nitric acid, fascicles were teased out end-to-end to measure FL. SSN was determined by dividing FL by sarcomere length measured via laser diffraction. Muscle wet weight, anatomical cross-sectional area (ACSA), and physiological cross-sectional area (PCSA) were also determined for insight on age-related losses of whole-muscle mass and in-parallel muscle morphology. Results: Age-related SSN loss was apparent after middle age for all muscles, with the plantaris showing the smallest (8%) and the VL the greatest (21%) differences between age groups. The MG and VL appeared to plateau in their SSN loss by 32 months, while the soleus and TA demonstrated continued decline from 32 to 36 months. In all muscles, an age-related lower SSN evidently contributed in part to the smaller muscle mass, alongside less contractile tissue in parallel (indicated by ACSA and PCSA). Conclusion: As SSN is closely tied to biomechanical function, these findings present SSN as a distinct target for improving muscle performance in older adults.