Abstract 16997: Cardiomyocyte-Autonomous and Microtubule Dependent Mechanisms Are Sufficient to Mediate Load-Dependent T-Tubule Remodeling

Abstract

Background: Mechanical overload is an established contributor to T-tubule (TT) remodeling and associated dyssynchronous Ca 2+ release leading to abnormal excitation-contraction coupling in cardiomyopathies. We examined the induction of pathological TT remodeling in isolated cardiomyocytes and the involvement of microtubules (MT) in this process. Methods: Adult rat ventricular myocytes (aRVMs) were cultured with applied variable in vitro load for 48hrs at either physiological (10kPa) or pathological (50kPa) stiffness, using custom magnetorheological elastomers with tunable stiffness. Cells were stained with Di-8-ANEPPS and Fluo-4-AM and imaged using confocal microscopy. Field stimulation of cells allowed for Ca 2+ transient acquisition via line scans. Protein expression changes were determined via Western blotting. IF staining allowed for visualization of the MT network and relevant TT proteins (TCAP, JPH2, BIN1). MT and tyrosination state dependency of remodeling was investigated by 1μM nocodazole (MT depolymerizer), taxol (MT stabilizer), and tubulin tyrosine ligase (TTL) treatments to alter the MT network accordingly. Results: Compared to the physiological stiffness group, aRVMs subjected to pathologically increased stiffness exhibited a 14.7% decrease in density, 18.9% decrease in regularity, and a 26.6% increase in luminal dimension of the TT system, along with an increase in longitudinal elements (p<0.05). The changes in both Ca 2+ handling parameters and protein expression resembled those typical of the in vivo myopathic phenotype. This included a 21.7% decrease in amplitude, along with a 33.7%, 29.8% and 21.1% increase in time to peak (TTP), variance in TTP, and the signal decay tau, respectively (p<0.05). Treatment of pathologically loaded cells with taxol worsened TT remodeling, specifically with respect to TT density which decreased by 12.5% compared to nontreatment cells, and longitudinal elements which increased by 24%. Increased MT tyrosination through TTL treatment showed a 37% recovery in TT density in cells cultured at pathological stiffness compared to nontreatment cells (p<0.05). Conclusion: The results suggest that load-induced TT remodeling occurs via a cardiomyocyte autonomous, MT-dependent mechanism.