Cyclic AMP reverses the effects of aging on pacemaker activity and If in sinoatrial node myocytes

Abstract

Aerobic capacity decreases with age, in part because of an age-dependent decline in maximum heart rate (mHR) and a reduction in the intrinsic pacemaker activity of the sinoatrial node of the heart. Isolated sinoatrial node myocytes (SAMs) from aged mice have slower spontaneous action potential (AP) firing rates and a hyperpolarizing shift in the voltage dependence of activation of the "funny current," I_f Cyclic AMP (cAMP) is a critical modulator of both AP firing rate and I_f in SAMs. Here, we test the ability of endogenous and exogenous cAMP to overcome age-dependent changes in acutely isolated murine SAMs. We found that maximal stimulation of endogenous cAMP with 3-isobutyl-1-methylxanthine (IBMX) and forskolin significantly increased AP firing rate and depolarized the voltage dependence of activation of I_f in SAMs from both young and aged mice. However, these changes were insufficient to overcome the deficits in aged SAMs, and significant age-dependent differences in AP firing rate and I_f persisted in the presence of IBMX and forskolin. In contrast, the effects of aging on SAMs were completely abolished by a high concentration of exogenous cAMP, which restored AP firing rate and I_f activation to youthful levels in cells from aged animals. Interestingly, the age-dependent differences in AP firing rates and I_f were similar in whole-cell and perforated-patch recordings, and the hyperpolarizing shift in I_f persisted in excised inside-out patches, suggesting a limited role for cAMP in causing these changes. Collectively, the data indicate that aging does not impose an absolute limit on pacemaker activity and that it does not act by simply reducing the concentration of freely diffusible cAMP in SAMs.

Figure 1.

Slower AP firing rates in aged SAMs persist during maximal stimulation of endogenous cAMP production. (A) Representative spontaneous APs from perforated-patch recordings from SAMs isolated from young (black) or aged (red) mice under control conditions (left) or in 100 µM IBMX plus 10 µM forskolin (Forsk; right). Scale bar: 250 ms, 50 mV. Dashed lines indicate 0 mV. (B) Mean AP firing rates (±SEM) from young and aged SAMs in control or IBMX plus forskolin. Numbers in parentheses indicate numbers of cells. *, P < 0.05 versus corresponding condition in young cells (t tests); ‡, P < 0.05 versus control conditions for the same age before wash on of IBMX/forskolin (paired t tests). (inset) Schematic illustration of perforated-patch recording configuration. Lightning bolts represent amphotericin B, and red asterisks represent endogenous cAMP remaining in the cell during the recordings.

Figure 2.

cAMP rescues the slower AP firing rate in aged SAMs. (A) Representative spontaneous APs from whole-cell recordings from SAMs isolated from young (black) or aged (red) mice under control conditions (left) or with 1 mM cAMP in the patch pipette (right). Scale bar: 250 ms, 50 mV. (B) Mean (±SEM) AP firing rates from whole-cell recordings from SAMs isolated from young (black) or aged (red) mice. Firing rates were recorded in control conditions (filled bars) or with 1 mM cAMP in the patch pipette (hatched bars). Numbers in parentheses indicate numbers of cells. P-values are from unpaired t tests. (inset) Schematic illustration of whole-cell recording configuration. Red asterisks represent cAMP introduced into the cell via the patch pipette in whole-cell recordings.

Figure 3.

Different effects of endogenous and exogenous cAMP on the voltage dependence of I_f in young and aged SAMs. (A and B) Normalized mean (±SEM) conductance-voltage relationships for I_f in whole-cell recordings from young (A) and aged (B) SAMs in control conditions (black and red symbols), in the presence of IBMX plus forskolin (white and pink) or with 1 mM cAMP in the patch pipette (gray and dark red). (insets) Representative whole-cell I_f current families. Scale bars: 500 pA, 500 ms (A) and 200 pA, 500 ms (B). (C) Mean (±SEM) V_1/2 values for I_f in whole-cell recordings in control conditions (filled), with IBMX and forskolin in the bath (open), or with 1 mM cAMP in the patch pipette (hatched). *, P < 0.05 versus young SAMs in the same conditions (t tests); ‡, P < 0.05 versus control for the same age (ANOVAs with Holm-Šidák post-tests).

Figure 4.

Age-dependent changes in I_f in perforated-patch recordings. (A) Normalized mean (±SEM) conductance-voltage relationships for I_f in SAMs isolated from young (black) or aged (red) mice in amphotericin B perforated-patch recordings. (insets) Schematic illustration of perforated-patch recording configuration and representative perforated-patch I_f current families in young (black) or aged (red) SAMs. Scale bars: 500 pA, 500 ms. (B) Mean (±SEM) V_1/2 values for I_f in young (black) and aged (red) SAMs in perforated-patch (PP, spotted bars) and whole-cell (WC, filled bars) recordings. P-values are comparisons between PP and WC for the same age (t tests); ‡, P < 0.05 compared with young in the same recording configuration (t tests).

Figure 5.

Age-dependent hyperpolarizing shift in the voltage dependence of I_f persists in excised inside-out patches but is rescued by cAMP. (A) Normalized mean (±SEM) conductance-voltage relationships for I_f from excised inside-out patches in SAMs isolated from young (black) and aged (red) mice in control conditions (filled) or upon wash-on of cAMP (open). (insets) Schematic illustration of excised patch recording configuration and representative I_f current families recorded from patches from young (black) and aged (red) SAMs. (B) Mean (±SEM) V_1/2 values for I_f in young (black) and aged (red) SAMs in control conditions (filled bars) and in the presence of 100 µM cAMP (hatched bars). *, P < 0.05 compared versus young control (t tests).