Calmodulin kinase II is required for fight or flight sinoatrial node physiology

Abstract

The best understood "fight or flight" mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by beta-adrenergic receptor (betaAR) agonist stimulation. HCN4 conducts an inward "pacemaker" current (I(f)) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I(f)-independent pathways are critical to SAN fight or flight responses. The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) is a downstream signal in the betaAR pathway that activates Ca(2+) homeostatic proteins in ventricular myocardium. Mice with genetic, myocardial and SAN cell CaMKII inhibition have significantly slower HRs than controls during stress, leading us to hypothesize that CaMKII actions on SAN Ca(2+) homeostasis are critical for betaAR agonist responses in SAN. Here we show that CaMKII mediates ISO HR increases by targeting SAN cell Ca(2+) homeostasis. CaMKII inhibition prevents ISO effects on SAN Ca(2+) uptake and release from intracellular sarcoplasmic reticulum (SR) stores that are necessary for increasing DDR. CaMKII inhibition has no effect on the ISO response in SAN cells when SR Ca(2+) release is disabled and CaMKII inhibition is only effective at slowing HRs during betaAR stimulation. These studies show the tightly coupled, but previously unanticipated, relationship of CaMKII to the betaAR pathway in fight or flight physiology and establish CaMKII as a critical signaling molecule for physiological HR responses to catecholamines.

Fig. 1.

HRs from AC3-I mice are slower than controls during stress in vivo, but not during rest or ex vivo. (A) HRs recorded in unanesthetized, physically restrained mice during echocardiography. In vivo HRs were significantly (P <0.001) slower in AC3-I mice than in controls (n = 9–12/group). (B and C) HRs recorded from ECG telemetered mice at rest (B) and after ISO injection (C) (0.4 mg/kg i.p.). In vivo HRs were significantly (P <0.05) slower in AC3-I mice compared with controls (n = 4–7/group) after ISO, but not at rest (P = 0.1). (D) Langendorff-perfused hearts from AC3-I and control mice (n = 5–6/group) beat at equivalent rates in the absence of ISO (P = 0.318). (E) ECGs recorded from Langendorff-perfused hearts at baseline and after 1 μM ISO. (F) ISO-HR response relationship in Langendorff-perfused hearts (n = 5–6/group). *, P <0.05 for AC3-I versus control hearts.

Fig. 2.

SAN cell CaMKII inhibition reduces ISO rate responses. (A) Example recordings of APs in a WT SAN at baseline (black) and after 100 nM ISO (red). (B) SAN cell AP frequencies in response to a range of ISO concentrations. AC3-I SAN AP frequencies were significantly (*, P <0.05, **, P <0.01, ANOVA) slower than controls at each ISO concentration (n = 6–10 per data point).

Fig. 3.

Representative immunofluorescence micrographs show ISO activates CaMKII in SAN cells isolated from WT (A) and AC3-C (B), but not from AC3-I mice (C) with SAN CaMKII inhibition. Columns are as follows: 1, eGFP (expressed in AC3-C and AC3-I SAN cells); 2, Thr 287 autophosphorylated, activated CaMKII (pCaMKII, red); 3, merge; 4, magnified images from column 2. (Scale bar, 10 μm.)

Fig. 4.

Loss of DDR response to ISO in AC3-I SAN cells. (A) Expanded AP tracings from Fig. 2A show DDR increase after ISO (red) compared with baseline (black). (B) The relationship between DDR and ISO concentration. Control SAN cells have significant DDR increases with ISO (n = 5–7/group), but AC3-I SAN cells do not increase DDR with ISO. *, P <0.05 comparing all genotypes at each ISO concentration by ANOVA. (C) Summary data showing the maximum diastolic membrane potential (MDP) does not change during ISO (1 μM) increases in DDR and is not different between genotypes. Data for MDP and DDR in C are from 8–24 cells per group and include the cells in (B). **, P <0.01 for ISO versus baseline.

Fig. 5.

AC3-I SAN cells fail to increase diastolic Ca²⁺ spark frequency or DDR with ISO. (A–C) Representative line scan confocal images of Rhod-2 fluorescence and simultaneously recorded spontaneous APs (Upper) and spatially averaged Ca²⁺ transients (Lower) at baseline and after ISO (1 μM). (Insets) Superimposed DDR for each genotype, before and after ISO, corresponding to the dashed boxes marking the confocal images. (D–G) Summary data for DDR (D), Ca²⁺ spark frequency (E), Ca²⁺ transient (F), and diastolic Ca²⁺ (G) before and after ISO (1 μM) (n = 13–21 per group). *, P <0.05; **, P <0.01 compared with baseline.

Fig. 6.

SR Ca²⁺ content is reduced in AC3-I SAN cells. (A and B) Representative I_NCX recordings from an AC3-C (A) and AC3-I (B) SAN cells in response to a caffeine spritz (at arrow). SR Ca²⁺ content is calculated from the integral of I_NCX (see Materials and Methods for details). (C) Summary data for SAN SR Ca²⁺ content at baseline and after 1 μM ISO (n = 6–9/group). †, P <0.05 and ††, P <0.01 compared with WT and AC3-C; *, P <0.05 and ***, P <0.001 compared with basal.