Unique Localization of Phospholamban in Perinuclear Membranes of Cardiomyocytes from Several Species

Abstract

Phospholamban (PLB) regulates sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) in cardiomyocytes (CMs). In addition to their well-known location in SR membranes, only SERCA2a, but not PLB, is shown expressed in the perinuclear/nuclear membranes of CMs. Here we used confocal immunofluorescence microscopy to examine the intracellular distribution of PLB. As expected, in isolated mouse CMs, co-localized PLB and SERCA2a were detected in SR by the monoclonal antibodies 2D12 (against PLB) and 2A7-A1 (against SERCA2a), respectively. Importantly, in addition to 2A7-A1 binding signals, strong 2D12 binding signal was detected around the DAPI staining of the nuclei, indicating the expression of PLB in perinuclear/nuclear membranes. Further, we probed PLB expression in tissue sections from hearts of human, dog, or rabbit with use of a polyclonal (PLB9) and three monoclonal (2D12, 1G7, or 1F1) antibodies, which were produced against different epitope of PLB in a specie-dependent manner. In particular, 2D12 and PLB9 recognized all isoforms of PLB in these species and revealed PLB expression in SR as well as in the perinuclear/nuclear regions. In comparison, 1F1, the dog specie-specific antibody, recognized PLB expression in dog SR and in perinuclear/nuclear membranes, but is completely negative for human, mouse, or rabbit CMs. Moreover, 1G7, which is specie-specific for human, mouse, or rabbit isoform of PLB, recognized similar pattern of PLB expression in these species including in perinuclear membranes, but not in dog CMs. Finally, 2D12 binding signals were completely eliminated by co-incubation of a PLB peptide (residues 2-25). These results showed for the first time that in addition to its expression in SR, PLB is expressed in and around nuclear envelope of CMs.