Development of a protein synthesis network at the sarco/endoplasmic reticulum in adult cardiac myocytes
IntroductionThe endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which constitute a large fraction of the total protein output of a mammalian cell. Striated muscle cells contain a specialized membrane system known as the sarcoplasmic reticulum (SR) that controls calcium homeoastasis and contraction, however the biochemical and physiological relationship between the ER and SR and how both compartments participate in protein synthesis remains incompletely understood. MethodsProtein quantification and imaging of selected marker proteins for ER and SR functions was performed to characterize the relationship of the ER and SR and its individual involvement in protein synthesis in neonatal and adult cardiac myocytes. Superresolution microscopy was used to examine the interaction of ribosomes and the SR in adult cardiac myocytes. ResultsQuantification of ER and SR-associated proteins of isolated ventricular cardiac myocytes showed that relative expression of ER/SR resident protein quality elements, as well as relative ribosome levels are decreased in adult cells, whereas SR-associated Ca2+ handling proteins increase. Immunocytoflourescence revealed that the membrane compartment that exists in early postpartum resembles mostly the ER and decreases in postnatal development. The SR is the main membrane network that exists in the adult cardiac myocytes, replacing the ER, in all but the perinuclear region. Immunocytoflourescence staining further indicated that both networks perform overlapping but distinct, specialized functions, such as localization of excitation-contraction coupling exclusively to the SR or initiation of secretion via the classical secretory pathway mainly from the ER. Ribosomes and mRNA were localized both in close proximity to the ER and the SR of adult ventricular cardiac myocytes. Superresolution microscopy confirmed that both the ER as well as the developed SR bind ribosomes and are direct sites of protein synthesis and protein homeostasis in adult cardiac myocytes. ConclusionOur findings suggest molecular differentiation and structural organization of the ER/SR in cardiac muscle development, resulting in the development of a protein synthesis network at the sarco/endoplasmic reticulum in adult cardiac myocytes.