Figure 3.

Hypercapnia causes a decrease in skeletal muscle anabolism and expression of ribosomal RNA. Age-matched male C57BL/6 mice were exposed to 10% CO₂ (HC) for 60 days or maintained in room air (NC). RNA was isolated from these samples; qPCR was performed; and the amount of 45S pre-RNA/GAPDH ratio was determined in (A) soleus (n = 6) and (B) extensor digitorum longus (EDL) muscles (n = 6). After chronic high CO₂ or room air exposures and 30 minutes before mice were killed, 0.040 μmol/g of puromycin was intraperitoneally injected. Then, soleus and EDL muscles were procured, samples were immunoblotted with antipuromycin antibody, and the intensity of the resultant smear was scored. (C) Soleus muscle puromycin incorporation (n = 3). (D) EDL muscle puromycin incorporation (n = 3). (E) Tibialis anterior (TA) and gastrocnemius (GN) muscles from NC and HC mice were procured and immediately weighed (n = 3). (F) Soleus muscle average fiber size in NC and HC (n = 3). (G) EDL muscle average fiber size in NC and HC (n = 3). (H) Grip strength determinations in NC and HC mice (n = 3). (I) Fiber-specific cross-sectional area (CSA) in NC and HC (n = 3). (J) Cryosectioned samples from soleus or EDL muscles were immunostained with antipuromycin antibody and specific antibodies to type I, IIa, or type IIb/x fibers. Then, the amount of fiber-incorporated puromycin in the specific fiber types of soleus and EDL muscles was scored by grayscale scoring of room air–maintained (NC) CO₂ versus 10% CO₂ for 60 days (HC). NC and HC mice underwent a regime of endurance exercise during 1 month. Scale bar: 250 μm. (K and L) Then, fiber-specific CSA (K) and puromycin incorporation (L) were determined and scored. *P < 0.05 and **P < 0.01.