The effect of exercise on the protein profile of rat knee joint intra- and extra-articular ligaments

Purpose: Ligaments are vital to normal knee joint function, however they are commonly damaged due to age-related rupture or sports-related incidents. Injuries to the intra-articular anterior cruciate ligament (ACL) together with the extra-articular medial collateral ligament (MCL) result in significant pain, immobility for the affected individual, joint instability and development of knee osteoarthritis. Moderate endurance type exercise has been shown to increase ligament strength in the rat, however little is known on the effect of a more strenuous exercise regime such as treadmill training on knee joint ligaments and whether it may be beneficial to ligament structure, in particular the extracellular matrix proteins. This study aimed to identify the effect of strenuous exercise on the proteome of the rat ACL and MCL using mass spectrometry. Methods: Sprague Dawley male rats were split into equal groups of control and exercised animals. The training programme for the exercised group involved one hour treadmill running a day with a speed of 17m/min for 4 weeks, whilst in the control group, the rats were allowed to move freely in standard cages. The ACLs and MCLs were harvested from five animals limb knee joints. Proteins from the ACLs and MCLs were extracted using 4M guanidine-HCL, subjected to in-solution trypsin digestion and then analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins were identified with PEAKS® and were analysed using label-free quantitative analysis with ProgenesisQI LC-MS software. Identification of proteins with two or more peptides, greater than 2 fold abundance and FDR adjusted p-value <0.05 were considered as significant. Gene ontology and pathway analysis was performed using bioinformatics tool String and Ingenuity pathway analysis (IPA). Results: A total of 381, 473, 589 and 451 proteins were identified in ACL control, MCL control, ACL exercised and MCL exercised groups respectively. 124 significant proteins were found to be more abundant in ACL control than ACL exercised group. However no difference in any protein was identified between MCL control and MCL exercised group. The majority of abundant protein that were differentially expressed in ACL exercised compared to ACL control groups were cytoskeletal, ribosomal and enzymes. Several abundant matrisomal proteins, such as collagen type IX alpha-3 chain, collagen type XVIII alpha-1 chain, collagen type XIV alpha-1 chain, asporin, lumican, periostin and TGFβ were also found to be more abundant in ACL exercised group compared to the control group. The main principal gene ontology processes of upregulated proteins in the ACL exercised group when compared to the ACL control group were identified as metabolic (p=7.08e-16) and cellular (p=2.05e-11). Significant pathway analysis identified networks that were related to metabolic, cell signalling, and protein synthesis. Conclusions: The results of this study suggests that exercise has an impact on ACL protein expression, with the majority of differential expressed proteins being cellular proteins such as actins, ribosomal and heat shock proteins, indicative of an intracellular response. Further study is necessary to determine the impact of these changes on ligament function.