Impaired regulation of heart rate and sinoatrial node function by the parasympathetic nervous system in type 2 diabetic mice

Figures

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  Figure 1. Effects of carbachol on heart rate in db/db mice in vivo. (A) Representative surface ECGs in baseline conditions and after application of CCh (0.1 mg/kg) in anesthetized wildtype and db/db mice. (B) Summary of heart rate at baseline and after CCh in wildtype (n = 9) and db/db (n = 13) mice. *P < 0.05 vs baseline; +P < 0.05 vs. wildtype by two-way ANOVA with Holm–Sidak posthoc test. (C) Change in heart rate after CCh application in wildtype and db/db mice. *P < 0.05 vs. wildtype by Student’s t-test. (D) Time course of changes in heart rate after application of CCh and atropine (10 mg/kg) in wildtype (n = 5) and db/db (n = 6) mice. (E) Magnified region of data from panel D illustrating the changes in heart rate upon application of atropine in wildtype and db/db mice. For panels (D) and (E) *P < 0.05 vs. wildtype by two-way repeated measures ANOVA with Holm–Sidak posthoc test. (F) Recovery time for heart rate after application of atropine. *P < 0.05 vs. wildtype by Student’s t-test.

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  Figure 2. Effects of carbachol on sinoatrial node recovery time in db/db mice in vivo. (A) Representative recordings illustrating assessment of corrected sinoatrial node recovery time (cSNRT) at baseline and after CCh (0.1 mg/kg) in wildtype and db/db mice. (B) Summary of cSNRT at baseline and after CCh in wildtype (n = 7) and db/db (n = 11) mice. *P < 0.05 vs baseline; +P < 0.05 vs. wildtype by two-way ANOVA with Holm–Sidak posthoc test. (C) Change in cSNRT after CCh application in wildtype and db/db mice. *P < 0.05 vs. wildtype by Student’s t-test.

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  Figure 3. Effects of carbachol on spontaneous action potential firing in isolated sinoatrial node myocytes from db/db mice. (A) Representative spontaneous APs at baseline and after CCh (50 nM) in wildtype and db/db SAN myocytes. Dashed lines are at 0 mV. (B) Summary of spontaneous AP frequency at baseline and after CCh in wildtype and db/db SAN myocytes. (C) Summary of DD slope at baseline and after CCh in wildtype and db/db SAN myocytes. (D) Summary of maximum diastolic potential (MDP) at baseline and after CCh in wildtype and db/db SAN myocytes. For panels (B–D) n = 14 wildtype and 13 db/db SAN myocytes; *P < 0.05 vs baseline; +P < 0.05 vs wildtype by two-way ANOVA with Holm–Sidak posthoc test.

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  Figure 4. IKACh properties in isolated SAN myocytes from db/db mice. (A) Representative IK recordings during a voltage ramp from + 50 mV to − 120 mV (holding potential = − 80 mV) at baseline, at the peak of the CCh (10 µM) response and 2 min after the peak CCh response in wildtype and db/db SAN myocytes. (B) Peak IKACh I–V relationship in wildtype (n = 28) and db/db (n = 25) SAN myocytes. P = 0.998 for wildtype vs. db/db by two-way repeated measures ANOVA with Holm–Sidak posthoc test. (C) Time course of CCh stimulated IKACh, measured at − 100 mV, in wildtype and db/db SAN myocytes. (D) Summary of IKACh desensitization in wildtype and db/db SAN myocytes. (E) Summary of IKACh recovery time during CCh washout in wildtype and db/db SAN myocytes. For panels (D,E) *P < 0.05 vs. wildtype by Student’s t-test.

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  Figure 5. Effects of carbachol on the hyperpolarization-activated current (If) in db/db SAN myocytes. (A) Representative If recordings at baseline and after CCh (10 µM) application in wildtype and db/db SAN myocytes. Voltage clamp protocol shown at bottom of recordings. (B) If IV curves at baseline and after CCh application in wildtype SAN myocytes (n = 15). (C) If IV curves at baseline and after CCh application in db/db SAN myocytes (n = 16). For panels (B) and (C) *P < 0.05 vs. baseline by two-way repeated measures ANOVA with Holm–Sidak posthoc test. (D) If density at − 120 mV at baseline and after CCh application in wildtype and db/db SAN myocytes. *P < 0.05 vs baseline by two-way ANOVA with Holm–Sidak posthoc test. (E) If steady-state activation curves at baseline and after CCh application in wildtype SAN myocytes (n = 15). (F) If steady-state activation curves at baseline and after CCh application in db/db SAN myocytes (n = 16). (G) If V1/2(act) at baseline and after CCh application in wildtype and db/db SAN myocytes. *P < 0.05 vs. baseline by two-way ANOVA with Holm–Sidak posthoc test. Refer to Supplementary Table 1 for additional analysis of If steady-state activation.

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  Figure 6. Effects of RGS4 inhibition on IKACh kinetics in db/db SAN myocytes. (A) Representative IK recordings illustrating peak and desensitized responses to CCh (10 µM) in wildtype and db/db SAN myocytes pre-treated with the RGS4 inhibitor CCG-4986 (10 µM). (B) Time course of CCh stimulated IKACh, measured at − 100 mV, in wildtype and db/db SAN myocytes in the presence of CCG-4986. (C) Summary of peak IKACh at − 100 mV in wildtype and db/db SAN myocytes in the presence of CCG-4986. (D) Summary of IKACh desensitization in wildtype and db/db SAN myocytes in the presence of CCG-4986. (E) Summary of IKACh recovery time during CCh washout in wildtype and db/db SAN myocytes in the presence of CCG-4986. For panels (C–E) n = 16 wildtype and 18 db/db SAN myocytes; data analyzed by Student’s t-test.

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  Figure 7. RGS4 gene and protein expression in the SAN in db/db mice. (A) mRNA expression of rgs4 in wildtype (n = 5) and db/db (n = 5) SAN. *P < 0.05 vs wildtype by Student’s t-test. (B) Representative Western blot and summary protein expression for RGS4 in the SAN of wildtype (n = 6) and db/db (n = 6) mice. *P < 0.05 vs. wildtype by Student’s t-test. Uncropped Western blot provided in Supplementary Fig. S6.

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  Figure 8. Effects of PIP3 on IKACh kinetics in db/db SAN myocytes. (A) Representative IK recordings illustrating peak and desensitized responses to CCh in wildtype and db/db SAN myocytes dialyzed with PIP3 (1 µM). (B) Time course of CCh stimulated IKACh, measured at − 100 mV, in wildtype and SAN myocytes dialyzed with PIP3. (C-E ) Summary of peak IKACh at − 100 mV (C), IKACh desensitization (D) and IKACh recovery time during CCh washoff (E) in wildtype (n = 16) and db/db (n = 19) SAN myocytes dialyzed with PIP3. Data in panels (C–E) analyzed by Student’s t-test. (F) Representative IK recordings illustrating peak and desensitized responses to CCh in wildtype and db/db SAN myocytes dialyzed with PIP2 (1 µM). (G) Time course of CCh stimulated IKACh, measured at − 100 mV, in wildtype and SAN myocytes dialyzed with PIP2. (H–J) Summary of peak IKACh at − 100 mV (H), IKACh desensitization (I) and IKACh recovery time during CCh washoff (J) in wildtype (n = 18) and db/db (n = 19) SAN myocytes dialyzed with PIP2. For panels (H–J) *P < 0.05 by Student’s t-test.