Role of advanced glycation end products in titin mechanics

Abstract

Post-translational modifications (PTMs) are common changes that occur to proteins by, for example, addition of small molecules. Those modifications have a wide range of effects and are able to modulate protein stability, localization, function or even their mechanical properties, which has been linked to the pathophysiology of various conditions, including cancer, neurodegeneration or cardiovascular diseases. One type of PTM is non-enzymatic glycation, which is a hallmark of aging and diseases like diabetes. It has been shown that glycation leads to formation of intermolecular crosslinking advanced glycation end products (AGEs) in extracellular proteins, which contribute to cardiac tissue stiffening observed in both situations. Nevertheless, the alteration of extracellular proteins cannot explain why increased rigidity is observed in diabetic or aged cardiomyocytes. Here, we have studied whether titin, the largest protein encoded by the human genome and a key contributor to cardiomyocyte mechanics, is the target of glycation that can affect its mechanical properties. We focused on glycation induced by methylglyoxal (MG), a major glycating agent in diabetes. Specifically, we have used singlemolecule nanomechanical profiling by Atomic Force Microscopy (AFM) to examine how MG-induced glycation affects the mechanics of titin domains. Our results demonstrate that MG leads to prominent formation of intradomain crosslinks in titin, which causes increased protein stiffness due to the reduction of its contour length and faster folding kinetics. We also show that MG increases cardiomyocyte passive tension, a parameter that is directly related to titin mechanical properties. By running Monte Carlo simulations, we were able to provide a mechanistic explanation for the increased titin-based cardiomyocyte stiffness, pointing to the fundamental contribution of contour length reduction in titin random coil regions upon glycation. We have also obtained evidence that titin glycation is increased in aged myocardium, suggesting that our observations can have pathophysiological relevance. We propose that the presence of intradomain crosslinks in proteins can contribute to changes in the mechanical properties of diabetic and aged tissues