Assessment of Heart Rate and SAN Function in Wild-Type and NPR-C^−/− Mice Treated With Ang II
(A) Representative ECG recordings obtained during intracardiac programmed stimulation in anesthetized wild-type and NPR-C^−/− mice treated with saline or Ang II to measure SNRT. (B) Summary of heart rate in wild-type and NPR-C^−/− mice treated with saline or Ang II. (C) Summary of cSNRT in wild-type and NPR-C^−/− mice treated with saline or Ang II. *p < 0.001 versus saline; ⁺p < 0.001 versus wild type by 2-way analysis of variance with Tukey post hoc test; n = 28 mice for wild-type/saline treatment, 29 mice for wild-type/Ang II treatment, 19 mice for NPR-C^−/−/saline treatment, and 19 mice for NPR-C^−/−/Ang II treatment. Ang II = angiotensin II; cSNRT = corrected SNRT; ECG = electrocardiography; NPR-C= natriuretic peptide receptor C; SAN = sinoatrial node; SNRT = sinoatrial node recovery time.

Assessment of Electrical Conduction in the SAN in Wild-Type and NPR-C^−/− Mice Treated With Ang II
(A) Atrial preparation used in optical mapping. Dashed line indicates the location of the crista terminalis. The SAN is located in the intercaval region of the right atrial posterior wall adjacent to the crista terminals. The yellow box illustrates the area that was typically mapped. (B) Representative activation maps in atrial preparations from wild-type and NPR-C^−/− mice treated with saline or Ang II. Bars = 1 mm. (C) Summary data for cycle length in wild-type and NPR-C^−/− mice treated with saline or Ang II. *p < 0.001 versus saline; ⁺p = 0.048 versus wild type by 2-way analysis of variance (ANOVA) with Tukey post hoc test. (D) Summary data for SAN CV in wild-type and NPR-C^−/− mice treated with saline or Ang II. *p < 0.001 versus saline; ⁺p < 0.001 versus wild type by 2-way ANOVA with Tukey post hoc test; n = 16 hearts for wild type/saline, 24 hearts for wild type/Ang II, 13 hearts for NPR-C^−/−/saline, and 12 hearts for NPR-C^−/−/Ang II. (E) Representative spontaneous SAN optical APs in wild-type and NPR-C^−/− mice treated with saline or Ang II. Dashed lines run through the maximum diastolic potential. AP = action potential; CV = conduction velocity; IAS = interatrial septum; IVC = opening of inferior vena cava; SVC = opening of superior vena cava; other abbreviations as in .

Effects of Ang II on AP Morphology and Hyperpolarization-Activated Current (I_f) in Isolated SAN Myocytes From Wild-Type and NPR-C^−/− Mice
(A) Representative spontaneous AP recordings in wild-type and NPR-C^−/− SAN myocytes treated with saline or Ang II. (B, C) Summary data illustrating cycle length (B) and DD slope (C) in wild-type and NPR-C^−/− mice treated with saline or Ang II. *p < 0.001 versus saline; p = 0.557 for AP cycle length and p = 0.517 for DD slope between genotypes within treatment group; n = 16 cells for wild type/saline, 11 cells for wild type/Ang II, 10 cells for NPR-C^−/−/saline, and 13 cells for NPR-C^−/−/Ang II for AP data. See for additional AP parameters. (D) Representative I_f recordings in SAN myocytes isolated from wild-type and NPR-C^−/− mice treated with saline or Ang II. (E) (F) Summary I_f IV curves for wild-type (E) and NPR-C^−/−(F) mice treated with saline or Ang II. *p < 0.001 versus saline at each membrane potential by 2-way repeated-measures analysis of variance with Tukey post-hoc test. (G, H) I_f activation curves for wild-type (G) mice and NPR-C^−/−(H) mice treated with Ang II. See for additional I_f kinetic analysis. n = 17 cells for wild type/saline, 13 cells for wild type/Ang II; 13 cells for NPR-C^−/−/saline, and 16 cells for NPR-C^−/−/Ang II. IV = current voltage relationship; pA = picoamperes; pF = picofarads; other abbreviations as in and .

Effects of Ang II on Interstitial Fibrosis in the SAN of Wild-Type and NPR-C^−/− Mice
(A) Sections through the SAN in wild-type and NPR-C^−/− mice treated with saline or Ang II. For each treatment group, the top image illustrates the HCN4-positive zone (green) used to identify the SAN region, and the bottom image illustrates the picrosirius red staining from an adjacent section used to identify fibrosis (red). Bars = 50 μM. (B) Summary of SAN fibrosis measured in the HCN4-positive zone for wild-type and NPR-C^−/− mice treated with saline or Ang II. *p < 0.001 versus saline; ⁺p = 0.014 versus wild type by 2-way analysis of variance with Tukey post hoc test; n = 5 hearts for each group.

Assessment of Heart Rate and SAN Function in Wild-Type Mice Co-Treated With Ang II and the NPR-C Agonist cANF
(A) Representative ECG recordings and SNRT measurements during intracardiac programmed stimulation in wild-type mice treated with saline, Ang II. or Ang II plus cANF (0.07 or 0.14 mg/kg/day). (B) (C) Summary of heart rate (B) and cSNRT (C) in wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). (B) *p < 0.001 versus saline; ⁺p = 0.009 versus Ang II; ˆp = 0.006 versus Ang II plus cANF (0.07). (C) *p < 0.001 versus saline; ⁺p < 0.001 versus Ang II; ^∧p = 0.006 versus Ang II plus cANF (0.07). Data were analyzed by 1-way analysis of variance with Tukey post hoc test; n = 28 mice for saline, 29 mice for Ang II, 20 mice for Ang II plus cANF (0.07 mg/kg/day) and 22 mice for Ang II plus cANF (0.14 mg/kg/day). cANF = NPR-C agonist; other abbreviations as in .

Assessment of Electrical Conduction in the SAN in Wild-Type Mice Co-Treated With Ang II and the NPR-C Agonist cANF
(A) Maps show wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). (B) (C) Summary data for cycle length (B) and SAN CV (C) in wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). (B) *p = 0.013 versus saline; ⁺p < 0.001 versus Ang II. (C) *p < 0.001 versus saline; ⁺p < 0.001 versus Ang II. Data were analyzed by 1-way analysis of variance with Tukey post hoc test; n = 16 hearts for saline, 24 hearts for Ang II, 8 hearts for Ang II + cANF (0.07 mg/kg/day), and 8 hearts for Ang II plus cANF (0.14 mg/kg/day). (D) Representative spontaneous SAN optical APs in wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). Dashed lines run through the maximum diastolic potential. Abbreviations as in , , and .

Effects of the NPR-C Activator cANF on Ang II-Mediated Alterations in AP Morphology and Hyperpolarization-Activated Current (I_f) in Isolated SAN Myocytes
(A) Representative spontaneous AP recordings obtained in wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). (B) (C) Summary of cycle length (B) and DD slope (C) illustrate the effects of cANF co-treatment on Ang II-mediated changes in SAN AP morphology. (B) *p < 0.001 versus saline; ^#p = 0.006 versus saline; ⁺p = 0.046 versus Ang II; ^$p = 0.008 versus Ang II. (C) *p < 0.001 versus saline; ⁺p = 0.014 versus Ang II; ^$p < 0.001 versus Ang II; ^∧p = 0.024 versus Ang II plus cANF (0.07). Data were analyzed by 1-way analysis of variance with Tukey post hoc test; n = 16 saline, 11 Ang II, 13 Ang II plus cANF (0.07), and 10 Ang II plus cANF (0.14) for AP data. See for additional AP parameters. (D) Representative I_f recordings from wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). (E) (F) Summary IV curves for the effects of Ang II and cANF (0.07 and 0.14 mg/kg/day) on I_f. (G) (H) I_f activation curves illustrating the effects of Ang II and cANF (0.07 and 0.14 mg/kg/day) in SAN myocytes. (E) *p < 0.001 versus saline; ⁺p = 0.002 versus Ang II. (F) *p < 0.001 versus saline; ⁺p < 0.001 versus Ang II. Data were analyzed by 2-way repeated-measures analysis of variance with Tukey post hoc test; n = 17 cells for saline, 13 cells for Ang II, 14 cells for Ang II+cANF (0.07), and 13 cells for Ang II plus cANF (0.14). See for additional I_f kinetic analysis. Abbreviations as in , , , and .

Effects of the NPR-C Agonist cANF on Ang II-Mediated Alterations in SAN Fibrosis
(A) SAN immunohistochemistry for HCN4 (top) and picrosirius red staining of fibrosis (bottom) for wild-type mice treated with saline, Ang II, or Ang II plus cANF (0.07 or 0.14 mg/kg/day). Bars = 50 μM. (B) Summary of the effects of cANF on Ang II-mediated fibrosis in the SAN. *p < 0.001 versus saline; ^#p = 0.037 versus saline; ⁺p < 0.001 versus Ang II; ^∧p = 0.004 versus Ang II plus cANF (0.07) by 1-way analysis of variance with Tukey post hoc test; n = 5 hearts for each group.