Phospholamban (PLB) regulates the cardiac sarcoplasmic reticulum Ca-ATPase: phosphorylation of PLB at Ser16 or Thr17, by protein kinase A and Ca-calmodulin-dependent protein kinase II (CaMKII), respectively, stimulates the pump. Previous work (Drago et al. 1998) has shown that PLB phosphorylated at Thr17 is localised at the Z-lines, adjacent to the t-tubules. Because Ca influx pathways are concentrated in the t-tubules, it seemed possible that this pattern of phosphorylation was due to local Ca2+ entry. We therefore investigated the pattern of phosphorylation in detubulated myocytes.
Ventricular myocytes were isolated from the hearts of Wistar rats, which were killed humanely by a Schedule 1 method, and detubulated as described previously (Kawai et al. 1999). Control and detubulated myocytes were stimulated at 0.5 Hz for 5 min in control perfusate or perfusate plus 1 mM isoproterenol (ISO) at room temperature. The cells were then fixed, permeabilised and PLB location determined using primary antibodies against phospholamban (A1), phospholamban phosphorylated at Ser16 (PS-16) or Thr17 (PT-17) and FITC-labelled secondary antibodies, which were visualised using confocal microscopy. Data are presented as means ± S.E.M. of n cells. Statistical significance was determined using Student’s t test or Mann-Whitney test as appropriate.
A1 antibody showed regular transverse striations in control and detubulated cells, at intervals of 1.65 ± 0.02 mm (n = 10) and 1.65 ± 0.04 mm (n = 9, P > 0.05), respectively, with no significant difference in mean fluorescence between control and detubulated cells. PT-17 antibody showed similar transverse striations in control and detubulated cells in the absence and presence of ISO, although the intensity of staining was significantly greater in the presence of ISO; these striations occurred at intervals of 1.68 ± 0.02 mm (n = 15) and 1.67 ± 0.02 mm (n = 17, P > 0.05) in control and detubulated cells, respectively, in the presence of ISO. A similar pattern of staining and response to ISO was observed using the PS-16 antibody in control and detubulated cells, although some staining of the nuclear envelope was also visible. Specificity of antibody staining was confirmed by competition with the appropriate peptide.
These data suggest that PLB is localised close to the Z-lines. This distribution, and the pattern of phosphorylation, is unchanged by detubulation. Thus it appears likely that phosphorylation of Thr17 is not due to local Ca signalling at the t-tubule, but to the distribution of PLB and an increase in global [Ca] within the cell.
This work was supported by The Wellcome Trust.
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