Abstract 15484: Distinct Subcellular Localizations of Non-Muscle Myosin IIB and IIC in Cardiomyocytes

Abstract

Introduction: Non-muscle myosin (NMII) contraction plays a role in generating cellular tension and its role in mediating myocardial hypertrophy is emerging. Non-muscle myosin is present in cardiomyocytes in two isoforms NMIIB and NMIIC. Prior literature suggests that NMIIB and NMIIC are found in the intercalated disc, while NMIIB is found in the Z discs. Recently, PKC was implicated in medating stiffness-dependent cardiomyocyte hypertrophy through NMII. Moreover, PKC and NMII were found to be upregulated at the costameres in heart disease. The respective role of NMIIB and NMIIC in mediating cardiac hypertrophy remains unknown. Methods: Cardiac tissues from 10w male C57BL6/J wildtype and Myh14-GFP mice were fixed in BE40 solution (70% ethanol, 1% glycerol, 0.5% glcial acetic acid, and 28.5% 0.5 x PBS) for 3 days. Tissues were washed in 70% ethanol and made into paraffin embedded sections. Sections were heated for 10 min at 50C, deparaffinized, washed in ethanol and rinsed in PBS. Antigen retrieval was performed by heating for 10 min in TET buffer (Tris-EDTA, pH9.0 and 0.05% Tween 20). After PBS rinse, sections were incubated in blocking buffer (3% NGS, 1% BSA, 0.15% Triton X 100, PBS) for 1 hour at RT. Then sections are incubated in primary antibodies in the blocking buffer for 1 hour at RT. After PBS rinse, sections were incubated in secondary antibodies in the blocking buffer for 1 hour at RT. After PBS rinse, sections were mounted in Vectashield with DAPI. Images were captured using a Nikon A1 confocal microscopy with 60X oil lens and 2X zoom. Results: NMIIB is expressed in a subset of intercalated discs and Z lines. NMIIC is expressed in a subset of intercalated discs and costameres. Phospho-Myosin light chain (P-MLC) partially co-localizes with NMIIC in the intercalated discs but is not found in costameres. Conclusions: NMIIB and NMIIC display distinct subcellular localization, including within the intercalated discs. Only NMIIB is found at Z lines. Only NMIIC is found at costameres. P-MCL is only found in the intercalated discs. These findings suggest differential regulation and function of NMIIB and NMIIC. Further characterization of specific subcellular NMIIB and NMIIC function may elucidate their respective roles in tensional homeostasis in the cardiomyocyte.