PMC

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.)

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).

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.

Loss of DDR response to ISO in AC3-I SAN cells. (A) Expanded AP tracings from A 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.

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.

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.