Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan

Abstract

In muscles, sarcopenia, the loss of muscle mass, is the major cause of aging-related functional decline and frailty. Several factors are correlated with sarcopenia during aging, including contraction-related cellular injury, oxidative stress, endocrine changes and reduced regenerative potential. However the involvement of these factors has not been experimentally investigated. Here, we report that contraction-related injury may significantly promote the progression of sarcopenia in the pharynx of the nematode, Caenorhabditis elegans, a model of aging in non-regenerative tissues. Both functional and structural declines in the pharynx during aging were significantly delayed in mutants with reduced muscle contraction rates. We also examined the role of bacteria in pharynx muscle decline during aging, as previous studies reported that antimicrobial treatments could extend C. elegans lifespan. Although microbial infection may have enhanced functional decline in the pharynx during aging, it was not the sole cause of decreased pumping rates in old animals. This study identifies contraction-related injury as a factor affecting the initiation and progression of sarcopenia during aging. Further, characterization of the specific types of damage induced by muscle contraction will be helpful for understanding the underlying causes of sarcopenia.

Fig. 1

Aging is associated with structural and functional declines in the C. elegans pharynx. (A–E) Structural decline in the C. elegans pharynx over lifespan, between adult days 2 (A) and day 10 (E) in fem-1(hc17) adults. In these populations, 50% survival was approximately 15 days. (A) The anatomical regions of the pharynx are the corpus (C), isthmus (I), and terminal bulb (TB). After adult day 6, structural declines were evident in the terminal bulb (D, arrow), or isthmus (E, arrow). Vacuole-like pits appeared in the terminal bulb region (*). The isthmus also became plugged with bacteria (D, arrowheads). Scale bars, 20 μm.

Fig. 2

Bacteria were a minor contributor to pharynx functional decline during aging in fem-1(hc17) animals. (A) Functional decline in pharynx of animals cultured on growing bacteria ( ), or ampicillin-treated non-growing bacteria ( ), or transferred to ampicillin-treated bacteria on adult day 6 ( ). *P=0.006, live versus ampicillin-treated bacteria; #P=0.019, live bacteria versus transferred on day 6; (n), 20 animals, except n=10 animals for days 8, 10 on live bacteria. (B) Average pumping rate in aged (days 7–10) adults with or without bacterial plugging. Error bars, SEM; plugged, n=10 animals; unplugged, n=49 animals; P=0.2, t-test. (C) Fraction of day 7–10 adult animals that pumped fewer than 50 times/min; plugged, n=10 animals; unplugged pharynx, n=49 animals.

Fig. 3

Pharynx functional decline during aging in the presence of serotonin and in pumping-impaired mutants. (A) Serotonin stimulated pumping at all ages ( untreated; 5 mg/mL serotonin creatine sulfate) and the change between untreated and serotonin-treatment was not different at different ages ( change in pump rate; fold change by serotonin); **P<0.001, t-test versus day 2, untreated; n=32–44, except n=12 for day 12. (B) Pumping rate during aging in fem-1(hc17) (◆), tph-1(mg280) (△), eat-2(ad465) (□) and eat-18(ad1110) (○) animals. (C) Relative decline of pumping rate, expressed as a fraction of pumping rate on adult day 2, in strains as presented in part (B); n> 14 animals; *P<0.05, t-test versus same-age wildtype.

Fig. 4

Slow pumping delayed structural aging of the pharynx in eat-2(ad465) animals. (A–C) Pharynx structure in representative adult day 8 fem-1(hc17) (A), eat-2(ad465) (B) or tph-1(mg280) (C) animals. Scale bars, 20 μrn. (D, E) Classification of pharynx structure in fem-1(hc17) versus eat-2(ad465) (D) or fem-1(hc17) versus tph-1(mg280) (E) animals at adult days 2 and 8. Each diamond designates the average score for structural integrity in the terminal bulb of each pharynx examined. Smaller scores indicate better-organized structure, i.e. more like younger animals. Mean score for all images is shown as an orange diamond. Significance, West for average of all images, (D) day 8 eat-2(ad465) versus fem-1(hc17),P =0.007 (**), day 2 P = 0.127; day 2 versus 8 fem-1(hc17), P <0.0001; day 2 versus 8 eat-2(ad465), P=0.0002; (E) day 8 tph-1(mg280) versus fem-1(hc17), P=0.51, day 2 P=0.34; day 2 versus 8 fem-1(hc17), P=0.0004; day 2 versus 8 tph-1(mg280), P=0.0001.

Fig. 5

Body muscle aging was not delayed in eat-2(ad465). (A) Body movement in fem-1(hc17) (■) and eat-2(ad465) (□) adults declined between days 2 and 8 of adulthood (25 °C); **P=0.00005 for day 2 fem-1(hc17) versus day 2 eat-2(ad465), t-test. (B, C) Structural damage occurred similarly in both fem-1(hc17) and eat-2(ad465) animals between days 2 and 8. (B) The fraction of animals with damage (wrinkled or faded) to sarcomeric actin, visualized by phalloidin;fem-1(hc17), day 2, n = 27 animals, day 8, n = 29; eat-2(ad465), day 2, n = 17; day 8, n= 16. (C) Representative images of phalloidin-stained sarcomeric actin in body wall muscles; scale bars, 20 μm.

Fig. 6

Slow pumping only in adulthood could protect against functional decline during aging in daf-2(e1370) animals. Functional decline of the pharynx in (A) daf-2(e1370) animals that reduce pumping at 25 °C and (B) daf-2(e1368) animals that do not. Pumping rates across lifespan are shown for animals maintained throughout adulthood at 20 °C ( ) or transferred to 20 °C after 2 days ( ) or 15 days ( ) at 25 °C. Significance between e1370 and e1368 pumping rates after d15 transfer, P = 3 × 10⁻⁹, t-test, measured on d1 6; n ≥ 20 animals for populations maintained at 20 °C throughout adulthood; for transfers, n=30 (e1370, d2 transfer), 34 (e1370, d15 transfer) and n=29 (e1368 d2 transfer), 33 (d15 transfer).

Fig. 7

Structural aging was delayed in daf-2(e1370) animals that reduce pumping rate. (A) Structural decline correlated with functional decline. Pharynxs of day 20 daf-2(e1370) (upper) and daf-2(e1368) (lower) animals showed different extents of structural deterioration. Terminal bulb deterioration was reduced in daf-2(e1370) animals maintained at 25 °C as adults, than in daf-2(e1368) animals (brackets), although the isthmus of daf-2(e1370) animals appeared thinner and weaker (arrows). Scale bars, 20 μm. (B) Classification of terminal bulb structure from daf-2(e1368) and daf-2(e1370) after 15 or 20 adult days at 25 °C, evaluated by human scorers as Fig. 4, statistical analysis: d15, P=0.0002; d20, P=3 × 10⁻⁸, e1370 versus e1368, average of all images, t-test.