Beta-adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca2+ channel

Abstract

As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.

Fig. 1.

Visualization of single LCC-activated Ca²⁺ sparks. (A) Representative loose-patch confocal images (Middle) and their time profiles (Lower): 50-mV depolarizations from RP (i.e., RP + 50 mV; Upper) evoke Ca²⁺ sparks in a stochastic manner in control group (Left) and during βAR stimulation by ISO (1 μM; Right). (B) Distribution of spark amplitude in control (Upper) and ISO (Lower) groups. (C-E) The amplitude (C), time to peak (D), and full width at half maximum (FWHM) (E) of Ca²⁺ sparks compared between control (white) and ISO (black) groups. Data represent the average of >90 sparks from at least 4 animals. **, P < 0.01 vs. control.

Fig. 2.

Effect of βAR stimulation on the kinetics of LCC-RyR coupling. (A) A confocal image (Upper, example from the ISO group) and its time profile (Lower) illustrate measurement of the coupling latency from the onset of an LCC Ca²⁺ sparklet (blue arrowhead) to the takeoff of a triggered RyR Ca²⁺ spark (red arrowhead). (B) Distributions (bars) and exponential fits (lines) of coupling latency in control group (Upper, 102 sparks from 8 animals) and during βAR stimulation by ISO (Lower, 149 sparks from 8 animals). (C) Comparison of the time constant (τ_L) of coupling latency (L) between control and ISO groups. The τ_L and its SE were determined by exponential fitting of the data in B with the formula N = N₀exp(-L/τ_L), where N is the number of observations and N₀ is a fitting parameter. **, P < 0.01 vs. control.

Fig. 3.

Effect of ISO on LCC-RyR coupling fidelity. (A) Representative loose-patch confocal images (Middle) and their time profiles (lower): LCC Ca²⁺ sparklets (blue arrowheads) activate RyR Ca²⁺ sparks (red arrowheads) in a probabilistic manner during depolarizations of RP + 50 mV (Upper); note the first Ca²⁺ sparklet (Left) failed to trigger a spark, whereas the Ca²⁺ spark (Right) was apparently triggered by the first sparklet (although the coupling latency was too short to visualize the sparklet per se). (B) Effect of ISO on LCC-RyR coupling fidelity as indexed by the percentage of first detectable Ca²⁺ sparklets that successfully activated Ca²⁺ sparks. The percentages were calculated first for each cell, and then averaged among at least 10 cells from 3 animals in each group. (C) Representative records of unitary LCC currents elicited by depolarizations of RP + 50 mV in control (Left) and ISO (Right) groups. (D) Comparison of the delays from depolarization to the first LCC opening (D_LCC, marked in C) and to the first spark (D_spark, marked in A) in control (white) and ISO (black) groups. Data represent the average of >350 D_LCCs or >50 D_sparks from at least 3 animals. *, P < 0.05 vs. control.

Fig. 4.

Role of PKA in βAR stimulation by ISO in modulation of Ca²⁺ sparklet-induced Ca²⁺ sparks. Before stimulation with ISO, the cell was pretreated for 30 min with a PKA antagonist, either 100 μM Rp-8-CPT-cAMP (Rp+ISO) or 10 μM H89 (H89+ISO). Amplitude of Ca²⁺ sparks (A), the τ_L (B), and the coupling fidelity (C) were compared among control (white), ISO (black), Rp+ISO (light gray), and H89+ISO (dark gray) groups. Data represent the average of at least 15 cells in 3 animals. **, P < 0.01 vs. control.

Fig. 5.

Role of SR Ca²⁺ content in βAR stimulation modulation of LCC-RyR signaling. (A) Representative time course of indo-1 fluorescence ratio (Lower) showing the measurement of SR Ca²⁺ content by application of 10 mM caffeine with 0 Na⁺ and 0 Ca²⁺ (Upper). (B) SR Ca²⁺ content, indexed by the amplitude of caffeine-induced Ca²⁺ transients in control (white) and ISO (black) groups. The SR Ca²⁺ content was adjusted back to the control level when cells were bathed with 0.25 mM extracellular Ca²⁺ (ISO+lowCa, light gray) or pretreated for 30 min with 4 μM CPA (ISO+CPA, dark gray). Data are the average of at least 46 cells from 10 animals. Time constant τ_L (C) and coupling fidelity (D) in control, ISO, ISO+lowCa, and ISO+CPA groups. Data represent measurements from at least 60 cells in 12 animals. *, P < 0.05, **, P < 0.01 vs. control.

Fig. 6.

The effect of ISO treatment on the Ca²⁺ release flux of Ca²⁺ sparks (I_spark). (A) Representative confocal image (Upper) and its time profile (Lower, black) illustrate measurement of I_spark. The rising phase of the Ca²⁺ spark (Lower, red) was fit to the equation Δ[Ca²⁺] = C_∞II_spark/(1 - exp(-t/τ)), with C_∞ = 62 nM/pA and τ = 9.7 ms, determined by fitting a standard long LCC sparklet (23). (B) I_spark in control, ISO, ISO+lowCa and ISO+CPA groups (see Fig. 5). Data represent the average of >120 sparks from 10 animals. (C) Ratios of I_spark and SR content in control, ISO, ISO+lowCa, and ISO+CPA groups (see Fig. 5). *, P < 0.05, **, P < 0.01 vs. control.