Abstract 11506: Roles of Mechanical Overload and the Microtubule Network in Mediating T-Tubule Remodeling

Abstract

Background: Mechanical overload is an established contributor to the remodeling of T-tubules (TT) and associated dyssynchronous Ca 2+ release leading to abnormal excitation-contraction coupling in cardiomyopathies. We examined the induction of pathologic TT remodeling in isolated cardiomyocytes and the involvement of microtubules in this process. Methods: Normal adult rat ventricular myocytes (aRVMs) were cultured for 48hrs on MREs at either physiological (10kPa) or pathological (50kPa) stiffness. Variable in vitro load application was achieved using custom magnetorheologic elastomers (MREs) with tunable stiffness. Cells were stained with Di-8-ANEPPS and Fluo-4-AM and imaged using Airyscan confocal microscopy. Microtubule dependency was investigated by treating cells with 1μM nocodazole, a microtubule depolymerization agent.Structural changes in TT networks were quantified in terms of regularity, luminal dimensions, density, and directionality. Field stimulation of cells allowed for acquisition of [Ca 2+ ] i transients via line scans and subsequent analysis of key [Ca 2+ ] i transient parameters. Changes in protein expression were determined via Western blotting. Results: Compared to the physiological stiffness group, aRVMs subjected to pathologically increased stiffness exhibited decreased density and regularity of the TT system, and increased luminal dimension. Direction-specific density analysis showed that pathologically loaded cells have more longitudinal tubule elements and fewer transverse tubule elements. This remodeling was attenuated in aRVMs treated with nocodazole in terms of regularity, luminal diameter, and direction-specific density. The observed changes in both Ca 2+ handling parameters and key protein expression resembled those typical of the in vivo myopathic phenotype. Conclusion: The results suggest that load-induced TT remodeling occurs via a cardiomyocyte autonomous, microtubule-dependent mechanism.