Figure 4.
Disruption of the AKAP18δ–PLN complex influences PLN-Ser 16 phosphorylation and Ca2+ re-uptake in the sarcoplasmic reticulum. (A) Immunofluorescent labelling of AKAP18δ (red) and α-actinin (green) in rat neonatal cardiac myocytes. Scale bar, 20 μm. (B) Disruption of the AKAP18δ–PLN interaction with peptide PLN-Arg 11. Adult cardiac myocytes were attached to laminin-coated glass coverslips and incubated with the AKAP18–PLN disruptor peptide PLN-Arg 11 or the corresponding control peptide pSer 16-PLN. AKAP18δ was detected by immunofluorescence microscopy. Scale bar, 20 μm. (C) Rat neonatal cardiac myocytes were treated with or without Arg 9-PLN; peptide (50 μM, 30 min) before stimulation with isoproterenol (iso; 0.1 μM, 5 min) as indicated and analysed for immunoreactive pSer 16-PLN (IB; immunoblots; top panel). Phosphorylated PLN peptide (Arg 9-pSer 16-PLN; right lane) was used as a negative control. Dotted lines indicate lanes excised/combined from a single gel. The histogram shows levels of phosphorylated Ser 16-PLN quantified by densiometry relative to calsequestrin levels (bottom panel). Bars represent the mean±s.e.m. from 3–6 independent experiments (*P<0.005, Student's t-test; NS, not significant). (D) Rat neonatal cardiac myocytes were transfected with the D1ER sensor. Responses to a 10 mM caffeine pulse (1 s, arrow) in control cells (red curves) or cells pretreated with PLN-Arg 11 peptide (25 μM, 40 min, green curves) with (open symbols) or without (filled symbols) treatment with norepinephrine (NE; 10 μM, 20 min) as indicated were recorded. Time constant averages (τ, mean±s.e.m.) were calculated (right). For each sample, more than 20 independent cells were examined (*P<0.025, by Student's t-test and one-way ANOVA for paired and independent samples, respectively). AKAP, A-kinase anchoring protein; PLN, phospholamban; SERCA2, sarcoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum.