Regulation of IKs Potassium Current by Isoproterenol in Adult Cardiomyocytes Requires Type 9 Adenylyl Cyclase

Figures

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  Figure 1. AC9 association with Yotiao and KCNQ1. Heart extracts from WT, IKs or IKs-AC9KO mice were subjected to immunoprecipitation (IP) with IgG (rabbit) or anti-Yotiao (A) and goat IgG or anti-KCNQ1 (B). The resulting immunoprecipitate was stimulated with 300 nM Gαs to measure AC activity. Data are shown as mean +/− SD. A portion of the IP’s from each sample was subjected to WB analysis for Yotiao (A) or KCNQ1 (B) and are shown below. Statistics: Kruskal-Wallis Analysis of Variance followed by Dunn’s or Bonferroni’s comparison method. n = 6–8, *** p < 0.001.

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  Figure 2. AC9 is required for sympathetic stimulation of KCNQ1 phosphorylation in vivo. Membranes were prepared from IKs-WT versus IKs-AC9KO heart. (A) AC activity was measured under basal conditions and upon stimulation with 300 nM Gαs (n = 3, performed in duplicate or triplicate). Data are shown as mean +/− SD. Statistics: One Way Analysis of Variance followed by Tukey comparison method for Gαs-stimulated AC activity; * p < 0.05 for WT compared to IKs and IKs-AC9KO. (B) IKs and IKs-AC9KO mice were injected with saline or isoproterenol (2 μg/g body weight, IP). Animals were sacrificed 5 min later and heart tissue was harvested. Cardiac extracts were prepared in the presence of phosphatase inhibitors. Equal protein supernatants were subjected to WB analysis; the ratio of phosphoprotein to total KCNQ1 was quantitated and normalized to IKs-AC9KO control. Data are shown as mean +/− SEM, with individual experimental data points in red. Statistics: Two-way Analysis of Variance followed by a Bonferroni multiple comparison method. n = 5, ** p < 0.001.

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  Figure 3. Deletion of AC9 dampens isoproterenol stimulation of IKs. Representative traces of IKs recordings in ventricular myocytes isolated from IKs (A) and IKs-AC9KO mice (B) before (control) and after perfusion of isoproterenal and okadaic acid (ISO). Enlargement of IKs tail current in ventricular myocytes isolated from IKs (C) and IKs-AC9KO mice (D) before (control, black line) and after perfusion of isoproterenol and okadaic acid (ISO, red line). Increase in IKs current at the end of pulse at + 60 mV (E) and in the tail current (F) in myocytes from IKs and IKs-AC9KO in control and after 1 µM isoproterenol and 1 µM okadaic acid. Total IKs density at the end of the 2 sec pulse at + 60 mV (G) and IKs tail density (H) in IKs and IKs-AC9KO ventricular myocytes was unchanged. Statistics: t-test, * p < 0.05, (Mann-Whitney), number of cells is indicated on the figure for each condition.

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  Figure 4. AC9 deletion impairs isoproterenol-induced regulation of IKs. IKs currents in ventricular myocytes of IKs (A) and IKs-AC9KO (B) mice in before (control, left panels) and after perfusion of 1 µM isoproterenol and 1 µM okadaic acid (ISO, right panels). Currents were elicited by applying depolarizing potentials to various levels ranging from −80 to +80 mV for 2 s from a holding potential of −40 mV. I-V relationships for tail current in IKs (C) and IKs-AC9KO (D) before (control) and after 1 µM isoproterenol and 1 µM okadaic acid (ISO). (E) Shift of V50 induced by 1 µM isoproterenol and 1 µM okadaic acid perfusion of myocytes from IKs and IKs-AC9KO mice. Statistics: t-test, * p <0.05, (Mann-Whitney), number of cells is indicated on the figure for each condition.

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  Figure 5. Expression of catalytically inactive AC9 decreases isoproterenol-stimulated phosphorylation of KCNQ1. CHO cells stably expressing KCNQ1 were transfected with myc-tagged Yotiao for 24 h prior to infection with GFP control or catalytically inactive AC9-D399A (AC9d) adenoviruses for an additional 40 hours. Cells were treated with vehicle or isoproterenol (1 μM) for 5 min prior to cell lysis. The cell lysate was subjected to immunoprecipitation (IP) with IgG (rabbit) or anti-Yotiao. (A) The ratio of p-KCNQ1 to total KCNQ1 was measured by WB; quantitation is shown in (B). #, ** p < 0.05 t-test, n = 3, mean +/− SD. (C) Total cell lysates were probed for expression of myc-Yotiao, Flag-AC9d, and GFP. (D) CHO cells were transfected and infected as in (A) and treated for 5 min with 1 mM 3-isobutyl-1-methylxanthine (Basal) +/− 20 µM forskolin. cAMP was quantitated by enzyme immunoassay.

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  Figure 6. Validation of Yotiao Antibody. (A) Neonatal rat CMs were infected with Myc-Yotiao for 60 h and stained with Myc (red) and Yotiao (green). Separate channels are shown to the right of the composite. (B) Control cells were stained with myosin binding protein C 3 (MyBPC) (green), Myc (red), and phalloidin (blue). A separate image of Myc staining is shown to the right of the composite image. Note, non-specific nuclear staining of Myc (arrows). Scale bar represents 10 µm (A,B). Images are representative of >10 CMs per condition from ≥2 isolations. (C) Equal protein lysates from neonatal rat CMs were subjected to western blot analysis with anti-Myc, Yotiao, FLAG, and MyBPC3. M (marker).

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  Figure 7. AC9 co-localizes with N-cadherin at the plasma membrane and points of cell–cell contacts in neonatal rat cardiomyocytes. Neonatal rat CMs were infected with Flag-AC9d for 60 h then stained with N-cadherin (green), FLAG (red), and phalloidin (blue). Arrows point to co-localization of AC9 with N-cadherin at the plasma membrane or points of cell–cell contact. Individual channels are shown to the right of composite images. Scale bar represents 10 µm, n > 10 cells per condition from ≥2 CM isolations.

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  Figure 8. Endogenous Yotiao localizes to sarcolemmal membrane and intercalated discs in adult mouse cardiomyocytes. Wild-type adult mouse CMs were stained with Yotiao (green) and (A) N-cadherin (red) or (B) α-actinin (blue). Individual channels are shown to the right of composite images. Scale bar represents 10 µm, n > 10 CMs per antibody condition.