Abstract Body (Do not enter title and authors here): Introduction: Distribution of newly synthesized proteins is vital for the maintenance of healthy cardiomyocytes (CMs), but is poorly understood. We previously reported that multiple newly made sarcoplasmic reticulum (SR) proteins first concentrated in the perinuclear endoplasmic reticulum (ER) puncta of CM, before their transition to transversely oriented SR z-tubules (zSR), aligned with T-tubules (TTs) (the NEST pathway). We now study the initial trafficking steps of newly made sarcolemma (SL) proteins Na, K-ATPase (NKA) and its phosphoprotein regulator phospholemman (PLM), which we recently reported to have a unique uneven steady state distribution to lateral, transverse SL TTs, and intercalated discs (IDs), key to cardiac conduction.
Hypothesis: Newly synthesized SL proteins sort through NEST pathway in CMs.
Methods: Confocal immunofluorescence assay was performed on adult rat CMs 12-48h post infected with adenoviruses encoding dog isoform of NKA-Flag, PLM (dPLM) ± SR protein dSERCA2a (> 10 CMs at each condition), as indicated by corresponding affinity purified dog–specific monoclonal antibodies, which do not detect endogenous rat proteins.
Results: Fig. A, newly made NKA-Flag proteins were concentrated in a punctate pattern within perinuclear ER at ~2µm intervals (18h, NE), and then spread distally from perinuclear ER puncta into zSR 24h post infection (zSR). Fig. B, Dual immunostaining also showed overlapping fluorescence of dPLM and co-expressed dSERCA2a in these perinuclear ER puncta and into zSR. Thus, morphologically, the same pathways were initially used for the trafficking of newly made SR protein SERCA2a, SL proteins dPLM and NKA. dPLM and NKA ultimately diverge from SERCA2a to localize to IDs, which occurs at 24 h and 30 h post infection, respectively. Anti-dPLM B8 and anti-Flag fluorescence intensity ratios of ID/TT were 2.6 ± 0.1 and 2.2 ± 0.5 at 30 h post infection, respectively, paralleling its steady state distribution in heart tissue.
Conclusions: Newly made SL proteins PLM and NKA co-localize with SR protein SERCA2a in a series of organized perinuclear ER puncta, revealing a key CM-specific trafficking step that appears to “presort” SR and membrane proteins by aligning their transport with zSR.