Phosphodiesterases 3 and 4 Differentially Regulate the Funny Current, If, in Mouse Sinoatrial Node Myocytes

Abstract

Cardiac pacemaking, at rest and during the sympathetic fight-or-flight response, depends on cAMP (3',5'-cyclic adenosine monophosphate) signaling in sinoatrial node myocytes (SAMs). The cardiac "funny current" (I_f) is among the cAMP-sensitive effectors that drive pacemaking in SAMs. I_f is produced by hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels. Voltage-dependent gating of HCN channels is potentiated by cAMP, which acts either by binding directly to the channels or by activating the cAMP-dependent protein kinase (PKA), which phosphorylates them. PKA activity is required for signaling between β adrenergic receptors (βARs) and HCN channels in SAMs but the mechanism that constrains cAMP signaling to a PKA-dependent pathway is unknown. Phosphodiesterases (PDEs) hydrolyze cAMP and form cAMP signaling domains in other types of cardiomyocytes. Here we examine the role of PDEs in regulation of I_f in SAMs. I_f was recorded in whole-cell voltage-clamp experiments from acutely-isolated mouse SAMs in the absence or presence of PDE and PKA inhibitors, and before and after βAR stimulation. General PDE inhibition caused a PKA-independent depolarizing shift in the midpoint activation voltage (V_1/2) of I_f at rest and removed the requirement for PKA in βAR-to-HCN signaling. PDE4 inhibition produced a similar PKA-independent depolarizing shift in the V_1/2 of I_f at rest, but did not remove the requirement for PKA in βAR-to-HCN signaling. PDE3 inhibition produced PKA-dependent changes in I_f both at rest and in response to βAR stimulation. Our results suggest that PDE3 and PDE4 isoforms create distinct cAMP signaling domains that differentially constrain access of cAMP to HCN channels and establish the requirement for PKA in signaling between βARs and HCN channels in SAMs.

Keywords: HCN channel; cardiac pacemaking; cardiomyocyte; cell compartmentalization; cyclic AMP (cAMP); funny current (If); ion channel; patch clamp; phosphodiesterases; sinoatrial node.

Figure 1

General PDE inhibition activates I_f in SAMs at rest via a PKA-independent mechanism. (A,B) Average (±SEM) normalized conductance-voltage plots for I_f in control (black), IBMX (100 µM in extracellular solution; red), PKI (10 µM in patch pipette; grey), or IBMX plus PKI (dark red). Numbers in parentheses in the legends indicate the number of cells in each dataset. Insets show representative I_f current families. Red traces show currents elicited by voltage steps to −120 mV to illustrate shifts in voltage dependence. Scale bars, 200 pA, 500 ms; and (C) average (±SEM) V_1/2 for I_f for the indicated conditions. Asterisks indicate p < 0.05 versus control; one-way ANOVA with Holm-Sidak post-test.

Figure 2

PDE4 inhibition activates I_f in SAMs at rest via a PKA-independent mechanism. (A,B) Average (±SEM) normalized conductance-voltage plots for I_f in control (black), rolipram (10 µM in the extracellular solution; green), PKI (10 µM in the patch pipette; grey), or PKI plus rolipram (dark green). Numbers in parentheses in the legends indicate the number of cells in each dataset. Insets show representative I_f current families. Red traces elicited by voltage steps to −120 mV illustrate shifts in the voltage dependence. Scale bars, 100 pA, 500 ms. for control, 500 pA, 500 ms for PKI; and (C) shows the average (±SEM) V_1/2 for I_f for indicated conditions. Asterisks indicate p < 0.05 versus control; one-way ANOVA with Holm-Sidak post-test.

Figure 3

Effects of PDE3 inhibition on I_f at rest. (A,B) Average (±SEM) normalized conductance-voltage plots for I_f in control (black), milrinone (50 µM in the extracellular solution; blue), PKI (10 µM in the patch pipette; grey), or milrinone plus PKI (dark blue). Numbers in parentheses in the legends indicate the number of cells in each dataset. Insets show representative I_f current families. Red traces elicited by voltage steps to −120 mV illustrate shifts in the voltage dependence. Scale bars, 100 pA, 500 ms for control, 500 pA, 500 ms for PKI; and (C) shows the average (±SEM) V_1/2 for I_f for indicated conditions. Asterisk indicates p < 0.05 versus control; one-way ANOVA with Holm-Sidak post-test.

Figure 4

Effects of PDE inhibition on the ability of ISO to shift the voltage-dependence of I_f. The shift in V_1/2 of I_f in response to ISO (ΔV_1/2-ISO) was determined for individual cells by measuring V_1/2 values before and after wash-on of ISO in (A) control Tyrode’s extracellular solution (black) or Tyrode’s solution with PKI (10 µM) in the patch pipette (grey); (B) IBMX (100 µM in the extracellular solution; red) or IBMX with PKI in the pipette (dark red); (C) rolipram (10 µM in the extracellular solution; light green) or rolipram with PKI in the pipette (dark green); and (D) milrinone (50 µM; blue) or milrinone with PKI in the pipette (dark blue). Dashed lines indicate the ΔV_1/2-ISO shift in control conditions for comparison. Asterisks indicate p < 0.05 compared to the corresponding condition without PKI; t-tests. Double daggers indicate a significant shift in response to ISO (p < 0.05 versus a hypothetical shift of 0 mV; one-sample t-tests). Insets represent the hyperpolarization-activated currents elicited by single voltage steps to near the midpoint activation voltage for each condition from individual cells before (black) and after (red) wash-on of ISO in the absence (left) or presence (right) of PKI. Scale bars: Tyrode’s, 200 pA; PKI, 200 pA; IBMX, 50 pA; IMBX + PKI, 100 pA; rolipram, 100 pA; rolipram + PKI, 200 pA; milrinone, 100 pA; milrinone + PKI, 200 pA. All time scale bars, 500 ms.