Abstract P3040: Development Of A Protein Synthesis Network At The Sarcoplasmic Reticulum In Adult Cardiomyocytes

Abstract

Introduction: The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. In striated muscle cells, a specialized membrane system known as the sarcoplasmic reticulum (SR) sequesters Ca 2+ and carries key functions in excitation-contraction coupling. Textbooks often define the SR as the ER of muscle, but surprisingly, the biochemical and physiological relationship between the ER and SR and even whether they are distinct membrane networks or refer to the same structure remain unknown. Methods: To characterize the relationship between the ER and SR, we applied biochemical and imaging techniques, including immunofluorescence and election microscopy, fluorescence in situ hybridization, and STimulated Emission Depletion (STED) microscopy to ventricular myocytes, which allowed us to follow the differentiation of these membrane compartments in developing cardiomyocytes. Results: Using localization and expression levels of SERCA2, RyR2, KDEL and different markers of ribosomes, mRNA, and the secretory pathway, we found that the membrane compartment that exists in the early postnatal stage, resembles mostly the ER, and this membrane system decreases in postnatal development. The SR is the main membrane network that exists in the adult cardiomyocytes, replacing the ER, in all but a small perinuclear region. Both sites which appear to be interconnected, perform specialized tasks, with protein secretion through the classical ER-Golgi secretory pathway originating mainly from the perinuclear ER and Ca 2+ -dependent excitation-contraction coupling originating exclusively from the SR. Other functions, such as Ca 2+ intake or posttranslational protein processing occur in both compartments. Importantly, we show that in adult cardiomyocytes ribosomes are not only attached to the ER but also to the SR, where they actively perform mRNA translation and are major sites of protein synthesis. Conclusion: The ER and SR are two distinct coexisting membrane networks in cardiomyocytes that undergo molecular differentiation and structural organization during cardiac muscle development. Significant amounts of protein synthesis occur in adult cardiomyocytes at the SR and may serve localized mRNA translation near the sarcomere.