Elevated CO2 levels affect development, motility, and fertility and extend life span in Caenorhabditis elegans

Abstract

Hypercapnia (high CO(2) levels) occurs in a number of lung diseases and it is associated with worse outcomes in patients with chronic obstructive lung disease (COPD). However, it is largely unknown how hypercapnia is sensed and responds in nonneuronal cells. Here, we used C. elegans to study the response to nonanesthetic CO(2) levels and show that levels exceeding 9% induce aberrant motility that is accompanied by age-dependent deterioration of body muscle organization, slowed development, reduced fertility and increased life span. These effects occur independently of the IGF-R, dietary restriction, egg laying or mitochondrial-induced aging pathways. Transcriptional profiling analysis shows specific and dynamic changes in gene expression after 1, 6, or 72 h of exposure to 19% CO(2) including increased transcription of several 7-transmembrane domain and innate immunity genes and a reduction in transcription of many of the MSP genes. Together, these results suggest specific physiological and molecular responses to hypercapnia, which appear to be independent of early heat shock and HIF mediated pathways.

Fig. 1.

Exposure of wild-type C. elegans (N2) to air containing 9%, 15% or 19% CO₂ caused reduced fertility. (A) Wild-type C. elegans (N2) were maintained at 20 °C (Left) or 25 °C (Right) on NGM plates and the number of laid eggs was scored daily. (B) DIC microscopy (Upper) and DAPI staining (Lower) of gonads of animals grown in air (Left) or in air containing 19% CO₂ from the time the eggs were laid until the adult stage (Right). In both cases, the morphology of gonads was normal. (C) Wild-type C. elegans (N2) were maintained on NGM plates at pH = 5.0, 6.0 or 7.0 at 20 °C (19 plates for each pH). The change in pH did not affect the progeny size. (D) ATP was measured using the MBL ApoSENSOR kit (n = 3). *, P < 0.05; ***, P < 0.0005.

Fig. 2.

Growth in air containing 19% CO₂ reduces motility and affects muscle morphology. (A) The average number of head movements of wild-type (N2) animals at the L4 stage grown in air (n = 22) or in air containing 19% CO₂ at 20 °C was scored either on NGM plates (Left, n = 21) or in a water drop (Right, n = 21). All measurements were performed after the animals were removed from the CO₂ chamber. The number of head movements/minute was divided by the average number of head movement of animals grown in air. (B) Thin-section electron micrographs demonstrating the gradual deterioration of body muscles in animals grown for 4, 8, or 12 days in air containing 19% CO₂ at 20 °C. Muscle morphology was normal in animals grown in air. The muscle of animals grown in air containing 19% CO₂ had deteriorated already at day 4 and muscle filaments were further disorganized at days 8 and 12. (Scale bars, 500 nm.)

Fig. 3.

Continuous exposure to air containing 19% CO₂ extends average life span. Survival plots of wild-type (N2) animals, or animals mutated in daf-2, daf-16, eat-2, clk-1 or glp-1 genes. Animals were grown at 20 °C in normal air (continuous line in A–E) or in air containing 19% CO₂ (broken lines in A-E), or at 25 °C in normal air (continuous line in F and continuous line with squares in A) or in air containing 19% CO₂ (broken line in F or broken line with squares in A). The average life span is shown in Table S2. There was a significant extension in life span (P < 0.0001) in all animals grown in air containing 19% CO₂.

Fig. 4.

Hypercapnia induces change in gene expression. Fold change in log₂ scale of gene expression during 1, 6 or 72 h of exposure to air containing 19% CO₂ of innate immunity (A), heat shock (B), 7 transmembrane domain (C), major sperm proteins (D), nuclear hormone receptor (E), and several other genes of interest (F). The data are taken from Table S3, Table S4, Table S5, and Table S6).

Fig. 5.

Aqp-2, rhr-1 and rhr-2 affect the hypercapnia-induced egg laying. (A) Animals homozygous for a deletion in a specific gene were maintained in air containing 15% or 19% CO₂ and the number of laid eggs was scored and divided by the number of eggs laid by animals of the same genetic background grown in normal air. *, P < 0.05; ***, P < 0.0005. (B) The effect of mutations in aqp-2, rhr-1, and rhr-2 on the rate of development in normal air and in air containing 19% CO₂.