Figure 1.
Persistent PKA activation increased Na current amplitudes without increasing the total NaV1.5 protein level in ventricular myocytes. PKA myocytes had been incubated with 8CPT-cAMP (100 µM)/okadaic acid (100 nM) for 6–15 h before experiments. CON myocytes had been cultured for the same duration under the control conditions. (A) PKA activation is confirmed by the appearance of cAMP response element binding protein 1 with serine at position 133 phosphorylated (CREB1-S133P) and its nuclear entry. Left upper: Immunoblot (IB) images of WCLs from CON and PKA myocytes probed with antibodies specific for CREB1-S133P (top) and CREB1 (bottom). Left lower: PKA to CON ratio of CREB1-S133P band intensities (5.46 + 1.04, dotted line denotes value of 1). Right: CREB1-S133P immunofluorescence (red, nuclei stained blue) in CON and PKA myocytes. Images are presented in orthogonal view (XY, YZ, and XZ planes) to show that CREB1-S133P was within, instead of around, nuclei of the PKA myocyte. *: CREB1-S133P specific band in IB and CREB1-S133P signals within nuclei. (B) Top: Na current (INa) traces recorded using a cell-attached patch clamp with a pipette tip positioned on the top surface of the cell center or close to the cell end (within three sarcomeres) of CON and PKA myocytes. These exceptional traces were obtained on the same day (CON myocytes 5–6 pm, PKA myocytes 11 PM–midnight). During patch clamp recording, myocytes were superfused with nominally Ca-free (supplemented with 2 mM Mg) Tyrode’s without 8CPT-cAMP/okadaic acid. The pipette was filled with Ca-containing (2 mM) Tyrode’s solution. The tip resistance was (in MΩ): CON cell end 1.44 + 0.06, CON cell center 1.41 + 0.06, PKA cell end 1.33 + 0.05, and PKA cell center 1.33 + 0.06 (one-way ANOVA, P = 0.267). (C) Top: Bar graphs (mean + SE) and individual data points of maximal peak INa amplitudes. Bottom: Time constants (τ) of inactivation of maximal INa. Data were pooled from five independent experiments. The numbers of myocytes studied are shown in parentheses. (D) Airyscan images of NaV1.5 immunofluorescence from CON and PKA myocytes in XY and YZ planes. (E) Top: NaV1.5 immunoblot images of SDS extracts of CON and PKA myocytes in two independent experiments. Middle: Coomassie blue (CB) stain of the same gels to confirm even loading. Bottom: Average PKA:CON ratio of NaV1.5 band intensities (0.95 + 0.09), not different from 1 (dotted line). Information on the antibodies used in experiments shown in this and the following figures is listed in Table 1. The listed P values are from t tests against null hypothesis (A and E), or CON versus PKA myocytes (C). Source data are available for this figure: SourceData F1.

Persistent PKA activation increased Na current amplitudes without increasing the total Na V 1.5 protein level in ventricular myocytes. PKA myocytes had been incubated with 8CPT-cAMP (100 µM)/okadaic acid (100 nM) for 6–15 h before experiments. CON myocytes had been cultured for the same duration under the control conditions. (A) PKA activation is confirmed by the appearance of cAMP response element binding protein 1 with serine at position 133 phosphorylated (CREB1-S133P) and its nuclear entry. Left upper: Immunoblot (IB) images of WCLs from CON and PKA myocytes probed with antibodies specific for CREB1-S133P (top) and CREB1 (bottom). Left lower: PKA to CON ratio of CREB1-S133P band intensities (5.46 + 1.04, dotted line denotes value of 1). Right: CREB1-S133P immunofluorescence (red, nuclei stained blue) in CON and PKA myocytes. Images are presented in orthogonal view (XY, YZ, and XZ planes) to show that CREB1-S133P was within, instead of around, nuclei of the PKA myocyte. *: CREB1-S133P specific band in IB and CREB1-S133P signals within nuclei. (B) Top: Na current (INa) traces recorded using a cell-attached patch clamp with a pipette tip positioned on the top surface of the cell center or close to the cell end (within three sarcomeres) of CON and PKA myocytes. These exceptional traces were obtained on the same day (CON myocytes 5–6 pm, PKA myocytes 11 PM–midnight). During patch clamp recording, myocytes were superfused with nominally Ca-free (supplemented with 2 mM Mg) Tyrode’s without 8CPT-cAMP/okadaic acid. The pipette was filled with Ca-containing (2 mM) Tyrode’s solution. The tip resistance was (in MΩ): CON cell end 1.44 + 0.06, CON cell center 1.41 + 0.06, PKA cell end 1.33 + 0.05, and PKA cell center 1.33 + 0.06 (one-way ANOVA, P = 0.267). (C) Top: Bar graphs (mean + SE) and individual data points of maximal peak INa amplitudes. Bottom: Time constants (τ) of inactivation of maximal INa. Data were pooled from five independent experiments. The numbers of myocytes studied are shown in parentheses. (D) Airyscan images of NaV1.5 immunofluorescence from CON and PKA myocytes in XY and YZ planes. (E) Top: NaV1.5 immunoblot images of SDS extracts of CON and PKA myocytes in two independent experiments. Middle: Coomassie blue (CB) stain of the same gels to confirm even loading. Bottom: Average PKA:CON ratio of NaV1.5 band intensities (0.95 + 0.09), not different from 1 (dotted line). Information on the antibodies used in experiments shown in this and the following figures is listed in Table 1. The listed P values are from t tests against null hypothesis (A and E), or CON versus PKA myocytes (C). Source data are available for this figure: SourceData F1.

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