The Role of Oxidative Stress in Remodeling the Cardiac Microtubule Cytoskeleton

Abstract

Microtubules are cylindrical cytoskeletal polymers composed of α/β-tubulin heterodimers that make up an ordered tubulin lattice. In cells, microtubules form a network that is a key component of the cellular cytoskeleton. Under pathological conditions of oxidative stress, we and others have found that cardiomyocytes, the contractile cells in the heart, display a denser microtubule cytoskeleton, which may lead to the progressive structural and functional cellular changes associated with myocardial ischemia and systolic dysfunction. This reorganization of the microtubule network occurs despite only small increases in tubulin expression, suggesting that alterations to microtubule length regulation and stability are involved. Using biophysical reconstitution experiments and live-cell imaging, we found that oxidative stress may synergistically increase the density of microtubules inside of cells by simultaneously increasing the length of dynamic, short-lived microtubules, while fostering the longevity of stable, long-lived microtubules. We found that microtubules subjected to oxidative stress undergo cysteine oxidation, and our electron and fluorescence microscopy experiments revealed that the locations of oxidized tubulin subunits within the microtubule had structural damage within the cylindrical tubulin lattice, consisting of holes and lattice openings. For dynamic microtubules, incorporation of stabilizing GTP-tubulin into these damaged lattice regions led to an increased frequency of rescue events (the transition from shortening to growth), and thus longer microtubules. For long-lived microtubules, these same structural defects facilitate entry of the enzyme αTAT1 into the microtubule lumen, where it catalyzes the acetylation of α-tubulin. This intraluminal acetylation has been shown to increase the lifetime of stable microtubules by conferring mechanical stability to the microtubule lattice. In this way, oxidative stress triggers a dramatic, pathogenic shift from a sparse microtubule network into a dense, longitudinally aligned microtubule network inside of cardiac myocytes, likely contributing to increased cellular stiffness and contractile dysfunction. Our results provide insight into myocardial changes in ischemic heart disease by describing a mechanism for the dramatic remodeling of the microtubule cytoskeletal network within cardiac myocytes subjected to oxidative stress.