The tubulin code during cardiac hypertrophy and failure [IV]

A proliferated and post-translationally modified microtubule network underlies cellular growth in cardiac hypertrophy and contributes to contractile dysfunction in heart failure. Yet how the heart achieves this modified network is poorly understood. Determining how the “tubulin code” – the permutations of tubulin isoforms and post-translational modifications - is rewritten upon cardiac stress may provide new targets to modulate cardiac remodeling. Further, while tubulin can autoregulate its own expression, it is unknown if autoregulation is operant in the heart or tuned in response to stress. Here we use heart failure patient samples and murine models of cardiac remodeling to interrogate transcriptional, autoregulatory, and post-translational mechanisms that contribute to microtubule network remodeling at different stages of heart disease. We find that autoregulation is operant across tubulin isoforms in the heart and leads to an apparent disconnect in tubulin mRNA and protein levels in heart failure. We also find that within 4 hours of a hypertrophic stimulus and prior to cardiac growth, microtubule detyrosination is rapidly induced to help stabilize the network. This occurs concomitant with rapid transcriptional and autoregulatory activation of specific tubulin isoforms and microtubule motors. Upon continued hypertrophic stimulation, there is an increase in post-translationally modified microtubule tracks and anterograde motors to support cardiac growth, while total tubulin content increases through progressive transcriptional and autoregulatory induction of tubulin isoforms. Our work provides a new model for how the tubulin code is rapidly rewritten to establish a proliferated, stable microtubule network that drives cardiac remodeling, and provides the first evidence of tunable tubulin autoregulation during pathological progression. For animal experiments: 8wks old C57/Bl6 mice were used. total RNA was isolated from mouse heart tissue harvested after 4 hours or 4 days (day 0 and day 2 injection) post 10mg/kg phenylephrine or 5mg/kg isoproterenol injections. Isolated cardiomyocytes treated with 6h of DMSO or 10uM colchicine or 10uM tax were used as positive control in the tubulin autoregulation panel. (Replicates: 4h Ctrl, 4h PE, 4h Iso = 6 and 4d Ctrl = 7, 4d PE = 7, 4d Iso = 6) For human experiment: total RNA was isolated from post-mortem heart tissue. (Replicates: non-failing = 12, failing = 23) The total RNA were analyzed using custom Nanostring nCounter panels.