Effect of LKB1 deficiency on mitochondrial content, fibre type and muscle performance in the mouse diaphragm

Abstract

Aim: The liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signalling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm.

Methods: Two methods of AMPK activation were used to characterize LKB1/AMPK signalling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation of the diaphragm. Diaphragms were excised 60 min post-AICAR injection and immediately after electrical stimulation.

Results: AMPK phosphorylation increased with AICAR and electrical stimulation in control but not KO mice. Acetyl CoA carboxylase phosphorylation increased with AICAR in control but not KO mice, but increased in both genotypes with electrical stimulation. While the majority of mitochondrial protein levels were lower in KO diaphragms, uncoupling protein 3, complex I and cytochrome oxidase IV protein levels were not different between genotypes. KO diaphragms have a lower percentage of IIx fibres and an elevated percentage of IIb fibres when compared with control diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover.

Conclusion: LKB1 regulates AMPK phosphorylation, mitochondrial protein expression, fibre type distribution, as well as recovery of the diaphragm from fatigue.

Fig. 1. Effect of LKB1 deficiency upon AMPK and ACC phosphorylation in mouse diaphragm after 1hr post-AICAR injection or a 5-min direct electrical stimulation

Western blots show LKB1 protein levels (A) and phosphorylation states of AMP-activated protein kinase (pAMPK; Thr172) and Acetyl CoA Carboxylase (pACC1 & pACC2; Ser 79 & Ser221, respectively), with electrical stimulation (B & C) or AICAR (D & E) treatments. Values are means ± SE (n = 5–8). *Significantly different from control sham/saline groups. †Significantly different from sham KO group (P < 0.05).

Fig. 2. Protein content of peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1) and mitochondrial proteins in LKB1-deficient mouse diaphragms

Western blots showing protein levels of PGC-1, Complex II, Complex 3 Core 2 (Core 2), Cytochrome C, Cytochrome Oxidase I (COX1), ATP Synthase α-subunit (ATP Synthase), Long Chain Acyl Dehydrogenase (LCAD). Values are means ± SE (n = 13–15). *Significantly different from control mice (P < 0.05).

Fig. 3. Citrate synthase (CS) activity in LKB1-deficient mouse diaphragm

Spectrophotometric determination of CS activity based on the conversion of oxaloacetate and acetyl CoA to citrate. Values are means ± SE (n = 5–6). *Significantly different from control mice (P < 0.05).

Fig. 4. Myosin heavy chain-isoform (MHC) percentage distribution in mouse diaphragms lacking LKB1

Electrophoresis of homogenized diaphragms followed by silver staining. Molecular weight and MHC-isoform standards were used to identify the various isoforms. Top: Representative image of the MHC bands. KO = muscle-specific LKB1-knockout mice; C = littermate control mice. Bottom: Mean MHC distribution ± SE (n=13–15/group), expressed as the percentage of the total MHC signal comprised by each MHC isoform. *Significantly different from control mice(P < 0.05).

Fig. 5. 10-minute In vitro stimulation of LKB1-deficient diaphragms

Rate of fatigue was determined by stimulating a diaphragm strip (~2mm) at 200 Hz every 2 seconds (100 m sec train duration) for ten minutes. Values are means ± SE (n = 7–10). *Significantly different from control mice (P < 0.05).

Fig. 6. Force production of LKB1-deficient diaphragms at 0 min, 1 min, and 5 min after 10-minute in vitro stimulation

After the ten minute fatigue protocol, diaphragms were stimulated for 5 additional pulses at 1-min and 5-min to determine ability to recover force production. Values are means ± SE (n = 7–10). *Significantly different from corresponding genotype at 0 min (P < 0.001). † Significantly different from control at corresponding time point (P < 0.05).