Injuries to the intra-articular knee joint ligament (anterior cruciate ligament (ACL)) together with the extra-articular medial collateral ligament (MCL) result in significant joint instability, pain and immobility for the affected individual. Moderate endurance type exercise has been shown to increase ligament strength, however little is known on the effect of short-term high intensity exercise regimes such as treadmill training on the ACL and MCL and whether they may be beneficial to the extracellular matrix (ECM) structure of these ligaments. This study aimed to identify the effect of short-term high intensity exercise on the proteome of the rat ACL and MCL using mass spectrometry. Sprague Dawley male rats (n=12) were split into equal groups of control and exercise animals, which were subjected to high intensity training and followed by proteomic analysis of the ACL and MCL. Knee joint and ligament health was assessed using OARSI scoring or using a validated histological scoring system.Histopathological analyses demonstrated no significant changes in the ACL, MCL or cartilage of the knee joint, indicating that the exercise regime used in this study did not have substantial impact on tissue structure and health of several tissues within the rat knee joint. Some proteins were found to be significantly more abundant in the ACL in the exercised group than the control group. However, no proteins with a significantly different expression were identified between MCL control and MCL exercised groups. The majority of proteins expressed at higher levels in the ACL exercise group were cytoskeletal proteins, ribosomal proteins and enzymes. Several matrisomal proteins were also more abundant such as collagen proteins and proteoglycans in ACL exercise group. In conclusion, our results indicate that short-term high intensity exercise has an impact on ACL ECM protein expression, with the majority of differential expressed proteins being cellular proteins such as actins, ribosomal and heat shock proteins, indicative of metabolic and molecular responses. Further study is necessary to determine the impact of these short-term changes on ligament structure and function.

Injuries to the intra‐articular anterior cruciate ligament (ACL) and the extra‐articular medial collateral ligament (MCL) result in significant knee joint instability, pain, and immobility. Moderate endurance‐type exercise can increase ligament strength but little is known on the effect of short‐term regular bouts of high‐intensity exercise on the extracellular matrix (ECM) structure of knee ligaments. Therefore, this study aimed to identify the effect of short‐term regular bouts high exercise on the proteome of the rat ACL and MCL using mass spectrometry. Sprague‐Dawley male rats (n = 6) were split into control and exercise groups, and subjected to high‐intensity training for four 4 weeks followed by proteomic analyses of the ACL and MCL. Knee joint health status was assessed using OARSI and a validated histological scoring system. Histopathological analyses demonstrated no significant changes in either in cruciate, collateral ligaments, or cartilage between the control and exercised knee joints. However, significant proteins were found to be more abundant in the exercised ACL compared to ACL control group but not between the exercised MCL and control MCL groups. The significant abundant proteins in ACL exercise groups were mostly cytoskeletal, ribosomal and enzymes with several abundant matrisomal proteins such as collagen proteins and proteoglycans being found in this group. In conclusion, our results indicate that short‐term regular bouts of high‐intensity exercise have an impact on the intra‐articular ACL but not extra‐articular MCL ECM protein expression.

physiological oxygen levels. In our previous study, we found changes in the lipidome that signified mitochondrial changes. Several mitochondrial proteins were elevated (fold change >2) in high oxygen conditions, including proteins that are part of the electron transport chain complex III, IV and V, superoxide dismutase and proteins involved in cell metabolism. SDH is present in complex II of the electron transport chain and its protein levels were not altered by oxygen. However, its activity showed a negative correlation with oxygen levels in monolayer cultures of chondrocytes. MALDI-MSI followed by LDA was used to identify oxygen induced changes in metabolites. Amongst others, we could identify adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Conclusions: Using multimodal mass spectrometry approaches, we show that human chondrocytes have a distinct, oxygen-dependent molecular profile. These changes in the lipidome, metabolome and proteome signify changes in mitochondria and may be a sign of elevated levels of ROS in high oxygen conditions. These mitochondrial changes may explain why chondrocytes perform poorly and lose their phenotype in supraphysiological oxygen levels and cartilage degenerative disease Targeting these mitochondrial changes may restore the balance between anabolic and catabolic activities and thereby halt cartilage degeneration.