Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance.

Han-Zhong Feng,J.-P Jin

doi:10.3389/fphys.2016.00597

Abstract

Carbonic anhydrase III (CAIII) is a metabolic enzyme and a regulator for intracellular pH. CAIII has been reported with high level expression in slow twitch skeletal muscles. Here we demonstrate that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. Expressing similar fast type of myofilament proteins, CAIII-positive tibial anterior (TA) muscle exhibits higher tolerance to fatigue than that of CAIII-negative fast twitch extensor digitorum longus (EDL) muscle in in situ contractility studies. We further studied the muscles of CAIII knockout (Car3-KO) mice. The loss of CAIII in soleus and TA muscles in Car3-KO mice did not change muscle mass, sarcomere protein isoform contents, and the baseline twitch and tetanic contractility as compared with age-matched wild type (WT) controls. On the other hand, Car3-KO TA muscle showed faster force reduction at the beginning but higher resistance at the end during a fatigue test, followed by slower post fatigue recovery than that of WT TA muscle. Superfused Car3-KO soleus muscle also had faster total force reduction during fatigue test than that of WT soleus. However, it showed a less elevation of resting tension followed by a better post fatigue recovery under acidotic stress. CAIII was detected in neonatal TA and EDL muscle, downregulated during development, and then re-expressed in adult TA but not EDL muscles. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fiber contents due to the loss of slow troponin T remained high. Car3-KO EDL, TA, and soleus muscles showed no change in the expression of mitochondria biomarker proteins. The data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH in skeletal muscle and may be explored as a target for improving fatigue resistance and for the treatment of TNNT1 myopathies.

Highlights

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to H2CO3
The CP3 monoclonal antibody (mAb) that we previously developed by immunization using chicken smooth muscle calponin 1 (Jin et al, 1996) strongly reacts with purified bovine Carbonic anhydrase III (CAIII) included in the Sigma molecular weight marker set L70 (Figure 1)
While the specific epitope structure shared by calponin 1 and CAIII merits a future study, mAb CP3 provides a useful tool to study the expression of CAIII in skeletal muscles

Summary

INTRODUCTION

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to H2CO3. At least 16 CA isozymes have been identified in mammals with different tissue distribution and catalytic activity (Imtaiyaz Hassan et al, 2013). CAIII is an ∼30-kDa cytosolic protein (Carter et al, 1978) present at high levels in liver, adipocytes, and skeletal muscles (Sly and Hu, 1995) It is a low activity enzyme among CA isozymes (Koester et al, 1977, 1981) but is resistant to most sulfonamide inhibitors (Sanyal et al, 1982). To investigate the potential function of CAIII in skeletal muscle and in adaptation to the loss of slow fibers in TNNT1 myopathy, here we demonstrated that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fibers remained high These data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH and fatigue resistance in skeletal muscle cells

MATERIALS AND METHODS

RESULTS

DISCUSSION