Growth on stiffer substrates impacts animal health and longevity in C. elegans

Abstract

Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo model organism setting to study the impact of altering substrate stiffness on aging by changing the stiffness of solid agar medium used for growth of C. elegans. We found that greater substrate stiffness had limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our study reveals that altering substrate stiffness of growth medium for C. elegans has only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.

Fig 1. Growth on stiff substrate results in mild lifespan extension but a decrease in organismal health.

(A) N2 wild-type animals grown on empty vector (EV) RNAi bacteria on either control (2%, blue) or stiff (4%, purple) agar plates from L1. Lifespans were scored every 2 days. Data is representative of 4 biological replicates, sample size is represented in the legend in parentheses, and statistical analysis is available in S2 Table. ** p = 0.0031. (B) N2 wild-type animals grown on EV RNAi bacteria on either control (2%, blue) or stiff (4%, purple) agar plates from L1. Animals are grown on UV-treated bacteria with no FUDR. Lifespans were scored every 2 days. Data is representative of 2 biological replicates, sample size is represented in legend in parentheses, and statistical analysis is available in S2 Table. *** p < 0.0001. (C) N2 wild-type animals were grown on EV RNAi bacteria on either 2% or 4% NGM plates from L1. At day 1, 5, and 9 of adulthood, animals were collected in M9 solution and video recordings were taken on an M205 stereoscope for 10 seconds. Body bends per 10 seconds were counted by eye for each individual worm. (D) N2 wild-type animals were grown on EV RNAi bacteria on either 2% or 4% agar plates from L1. At the L4 stage, animals were singled out and moved daily. Number of live progenies were counted for each animal with n > 50 per sample. Data is pooled from 3 independent biological replicates with n = 10 per replicate. * = p < 0.05, ** = p < 0.01 through non-parametric Mann-Whitney testing. Lines are median and interquartile range, and each dot represents a single animal where blue dots are animals grown on 2% control plates and purple dots are animals grown on 4% stiff plates.

Fig 2. Growth on stiff substrates does not affect induction of stress responses.

(A) Representative fluorescent images of day 1 adult animals expressing hsp-6p::GFP grown on EV or cco-1 RNAi bacteria from L1. Data is representative of 3 independent replicates. Quantification is presented as arbitrary fluorescent units, which are integrated fluorescent intensity measurements using ImageJ Fiji, dots are independent replicates, and lines represent standard deviation. Data is representative of 3 independent replicates. ns = not significant, p > 0.05. (B) Representative fluorescent images of day 1 adult animals expressing hsp-4p::GFP grown on EV or tag-335 RNAi bacteria from L1. Data is representative of 3 independent replicates. Quantification is presented as arbitrary fluorescent units, which are integrated fluorescent intensity measurements using ImageJ Fiji, dots are independent replicates, and lines represent standard deviation. Data is representative of 3 independent replicates. ns = not significant, p > 0.05. (C) Representative fluorescent images of day 1 adult animals expressing hsp-16.2p::GFP grown on EV RNAi bacteria from L1. Animals were heat-shocked at 34°C for 2 hours followed by a 2-hour recovery at 20°C. Data is representative of 3 independent replicates. Quantification of is presented as arbitrary fluorescent units, which are integrated fluorescent intensity measurements using ImageJ Fiji, dots are independent replicates, and lines represent standard deviation. Data is representative of 3 independent replicates. ns = not significant, p > 0.05. (D) Representative fluorescent images of day 1 adult animals expressing gst-4p::GFP grown on EV RNAi bacteria from L1. Animals were treated with 1 mM tert-butyl hydroperoxide (TBHP) rotating in an M9-TBHP solution for 2 hours at 20°C. TBHP was then washed with M9 solution and worms were recovered on OP50 bacteria for 16 hours prior to imaging. Data is representative of 3 independent replicates. Quantification is presented as arbitrary fluorescent units, which are integrated fluorescent intensity measurements using ImageJ Fiji, dots are independent replicates, and lines represent standard deviation. Data is representative of 3 independent replicates. ns = not significant, p > 0.05.

Fig 3. Transcriptome analysis reveals minor changes in gene expression in animals grown on stiffer substrates.

(A) Volcano plot of differentially expressed genes of animals grown on stiff (4%) agar compared to standard (2%) agar. Every dot is a single gene, where red dots indicate genes with p-value < 0.05 and black dots indicate genes with p-value > 0.05. (B) Heat map of all differentially expressed genes in worms grown on 4% agar where warmer colors indicate higher expression and cooler colors indicate lower expression. A list of all genes is available in S2 Table. (C) Gene ontology enrichments for differentially expressed genes (p-value < 0.05) in worms grown on 4%`agar.

Fig 4. Growth on stiff surfaces results in increased actin stability, which can drive lifespan extension.

(A) Representative fluorescent images of body wall muscle actin (myo-3p::LifeAct::mRuby) are shown. Animals were grown on empty vector (EV) RNAi bacteria from L1. Single-slice images were captured on a Leica THUNDER Imager. Scale bar is 10 μm. (B) Representative max-projection fluorescent images of hypodermal actin (col-19p::LifeAct::mRuby) are shown. Animals were grown on EV RNAi bacteria from L1. Z-stack images were captured on a Leica THUNDER Imager using system-optimized z-slices. Scale bar is 10 μm. (C) Representative max-projection fluorescent images of intestinal actin (gly-19p::LifeAct::mRuby) are shown. Animals were grown on EV RNAi bacteria from L1. Z-stack images were captured on a Leica Stellaris using system-optimized z-slices. Scale bar is 10 μm. (D) N2 wild-type animals grown on EV or act-1 RNAi bacteria diluted 10/90 act-1/EV on either control (2%) or stiff (4%) agar plates from L1. Lifespans were scored every 2 days. Data is representative of 3 biological replicates, sample size is represented in the legend in parentheses, and statistical analysis is available in S1 Table. * p = 0.0012, *** p < 0.0001.

Fig 5. Variable sources of agar formulations have significant differences in stiffness.

Standard 2% agar-based solid NGM plates were made as described in Materials and Methods using different sources of agar. Stiffness of each agar plate was measured using an oscillatory rheometer. Each dot represents a single technical replicate performed on an individual plate and lines represent median plus interquartile range. X-axis indicates brand and catalog number of each agar source.