Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Figures

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  Fig. 1. Effect of protein kinase G (PKG) activation with 8-Br-cGMP on store overload-induced Ca2+ release (SOICR) and dependence on RyR2 phosphorylation sites.Human embryonic kidney (HEK293) cells were transfected with luminal Ca2+ sensor D1ER and induced to express RyR2 16–18 h prior to experimentation. Cells were treated with 8-Br-cGMP (100 μM, 10 min) or equivalent volume vehicle control before superfusion with increasing extracellular Ca2+ concentrations ([Ca2+]O) to stimulate SOICR. Tetracaine (Tet, 2 mM) and caffeine (Caff, 20 mM) were used to block RyR2 and to deplete the internal Ca2+ store, respectively. Representative effect of 8-Br-cGMP in HEK293 cells expressing wild-type (WT) RyR2 is shown and panel A, with quantification of effect on SOICR threshold and store size shown in panels B and C, respectively. Panels D-F show equivalent results from HEK293 cells expressing RyR2 containing triple mutation (3A) at phosphorylation sites S2808, S2030, and S2814. For A-C, vehicle N = 5 replicates (from n = 58 cells), 8-Br-cGMP N = 5 replicates (from n = 62 cells). For D-F, Triple mutant + vehicle N = 4 replicates (from n = 69 cells), triple mutant + 8-Br-cGMP N = 4 replicates (from n =…

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  Fig. 2. Effect of single serine 2808 (S2808A) or serine 2030 (S2030A) cardiac ryanodine receptor (RyR2) mutation on response to protein kinase G activation with 8-Br-cGMP. Human embryonic kidney (HEK293) cells were transfected with luminal Ca2+ sensor D1ER prior to induction of RyR2 containing a mutation at either S2808A or S2030A. RyR expression was induced 16–18 h prior to experimentation and cells were treated with 8-Br-cGMP (100 μM) or equivalent volume vehicle control. Cells were either superfused with increasing extracellular Ca2+ concentrations ([Ca2+]O) to stimulate SOICR (representative traces are shown in panels A and E) or lysates were harvested for western blotting (D). For D1ER imaging Tetracaine (Tet, 2 mM) and caffeine (Caff, 20 mM) were used to block RyR2 and to deplete the internal Ca2+ store, respectively. Effects on SOICR threshold and store size of 8-Br-cGMP in S2808A and S2030A cells are shown in panels A–C and E-F, respectively. For D, RyR2 phosphorylation at S2808 following vehicle or 8-Br-cGMP treatment was determined by normalization to total RyR2 expression. For A-C, S2808A + vehicle N = 4 replicates (from n = 62 cells), S2808A + 8-Br-cGMP N = 3 replicates (from…

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  Fig. 3. Effect of PKG inhibition with KT 5823 on store overload-induced Ca2+ release (SOICR) and RyR2 S2808 phosphorylation. Human embryonic kidney (HEK293) cells were transfected with luminal Ca2+ sensor D1ER and induced to express RyR2 16–18 h prior to experimentation. Cells were treated with PKG inhibitor, KT 5823 (1 μM, 10 min), or equivalent volume DMSO vehicle control before superfusion with increasing extracellular Ca2+ concentrations ([Ca2+]O) to stimulate SOICR (representative trace shown in panel A). Tetracaine (Tet, 2 mM) and caffeine (Caff, 20 mM) were used to block RyR2 and to deplete the internal Ca2+ store, respectively. KT 5823 effects on SOICR threshold and store size and are shown in panels B and C, respectively. D, Lysates were harvested from HEK293 expressing WT RyR2 treated as for A-C and RyR2 phosphorylation at S2808 determined. E, Data normalized to total RyR2 expression. For A-C, vehicle N = 4 replicates (from n = 71 cells), KT 5823 N = 4 replicates (from n = 75 cells). For E, vehicle n = 4 lysates, KT 5823 n = 4 lysates. Differences determined by unpaired t-test. P values are as indicated in the figure. Data are mean ± SEM of replicates with values from all cells…

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  Fig. 4. Effect of endogenous PKG knockdown on RyR2 S2808 phosphorylation and store overload induced Ca2+ release (SOICR). RyR2-expressing human embryonic kidney (HEK293) cells were transfected with a silencing RNA targeting endogenous PKG (siPKG) or a scramble control (Scr). Confirmation of PKG knockdown (normalized to GAPDH expression) shown in A. B, Effect of PKG knockdown on total RyR2 expression (normalized to GAPDH expression) and RyR2 S2808 phosphorylation (normalized to total RyR2 expression). C, HEK293 cells with PKG knockdown were transfected with luminal Ca2+ sensor D1ER and induced to express RyR2 16–18 h prior to experimentation. Cells were superfused with increasing extracellular Ca2+ concentrations ([Ca2+]O) to stimulate SOICR and with tetracaine (Tet, 2 mM) and caffeine (Caff, 20 mM) to block RyR2 and to deplete the internal Ca2+ store, respectively (representative trace shown in panel C). PKG knockdown effects on SOICR threshold and store size are shown in panels D and E, respectively. For A, scramble and siPKG n = 6 lysates each. For B, scramble and siPKG n = 3 lysates each. For D & E, scramble N = 3 replicates (from n = 55 cells), siPKG N = 4 replicates (from n = 39…