Distinct patterns of constitutive phosphodiesterase activity in mouse sinoatrial node and atrial myocardium

Abstract

Phosphodiesterases (PDEs) are critical regulators of cyclic nucleotides in the heart. In ventricular myocytes, the L-type Ca(2+) current (I(Ca,L)) is a major target of regulation by PDEs, particularly members of the PDE2, PDE3 and PDE4 families. Conversely, much less is known about the roles of PDE2, PDE3 and PDE4 in the regulation of action potential (AP) properties and I(Ca,L) in the sinoatrial node (SAN) and the atrial myocardium, especially in mice. Thus, the purpose of our study was to measure the effects of global PDE inhibition with Isobutyl-1-methylxanthine (IBMX) and selective inhibitors of PDE2, PDE3 and PDE4 on AP properties in isolated mouse SAN and right atrial myocytes. We also measured the effects of these inhibitors on I(Ca,L) in SAN and atrial myocytes in comparison to ventricular myocytes. Our data demonstrate that IBMX markedly increases spontaneous AP frequency in SAN myocytes and AP duration in atrial myocytes. Spontaneous AP firing in SAN myocytes was also increased by the PDE2 inhibitor erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA), the PDE3 inhibitor milrinone (Mil) and the PDE4 inhibitor rolipram (Rol). In contrast, atrial AP duration was increased by EHNA and Rol, but not by Mil. IBMX also potently, and similarly, increased I(Ca,L) in SAN, atrial and ventricular myocytes; however, important differences emerged in terms of which inhibitors could modulate I(Ca,L) in each myocyte type. Consistent with our AP measurements, EHNA, Mil and Rol each increased I(Ca,L) in SAN myocytes. Also, EHNA and Rol, but not Mil, increased atrial I(Ca,L). In complete contrast, no selective PDE inhibitors increased I(Ca,L) in ventricular myocytes when given alone. Thus, our data show that the effects of selective PDE2, PDE3 and PDE4 inhibitors are distinct in the different regions of the myocardium indicating important differences in how each PDE family constitutively regulates ion channel function in the SAN, atrial and ventricular myocardium.

Figure 1. Quantitative mRNA expression of PDE 2, 3 and 4 subtypes in mouse SAN, right atrium and right ventricle.

A. Expression of PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D are shown relative to GAPDH for the SAN, right atrium (RA) and right ventricular free wall (RV). B. SAN samples were distinguished from RA samples by the characteristic pattern of expression of HCN4 and ANP in these regions. SAN tissue shows high expression of HCN4 and low expression of ANP whereas the RA shows very low HCN4 expression and very high ANP expression. Note scale break on Y-axis. Data are means ± SEM; n = 5 SAN trials, 5 right atrial appendage trials and 3 right ventricular free wall trials; *P<0.05; ^† P<0.001 by two way ANOVA with Tukey's posthoc test.

Figure 2. Effects of IBMX on action potential firing in SAN and atrial myocytes.

A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of IBMX (100 µM) and after IBMX washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of IBMX on spontaneous AP frequency (B), DD slope (C) and APD₅₀ (D). E. Representative stimulated right atrial myocyte APs in control conditions, in the presence of IBMX (100 µM) and after IBMX washout. Dotted line is at 0 mV. F. Summary of the effects of IBMX on atrial AP duration (APD₅₀, APD₇₀, and APD₉₀). Summary data are means ± SEM; n = 11 SAN myocytes and 13 right atrial myocytes; *P<0.05 vs. control by one way ANOVA with Tukey's posthoc test.

Figure 3. Effects of the PDE2 inhibitor EHNA on action potential firing in SAN and atrial myocytes.

A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of EHNA (10 µM) and after EHNA washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of EHNA on spontaneous AP frequency (B), DD slope (C) and APD₅₀ (D). E. Representative stimulated right atrial myocyte APs in control conditions, in the presence of EHNA (10 µM) and after EHNA washout. Dotted line is at 0 mV. F. Summary of the effects of EHNA on atrial AP duration (APD₅₀, APD₇₀, and APD₉₀). Summary data are means ± SEM; n = 6 SAN myocytes and 7 right atrial myocytes; *P<0.05 vs. control by one way ANOVA with Tukey's posthoc test.

Figure 4. Effects of the PDE3 inhibitor milrinone on action potential firing in SAN and atrial myocytes.

A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of Mil (10 µM) and after Mil washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of Mil on spontaneous AP frequency (B), DD slope (C) and APD₅₀ (D). E. Representative stimulated right atrial myocyte APs in control conditions, in the presence of Mil (10 µM) and after Mil washout. Dotted line is at 0 mV. F. Summary of the effects of Mil on atrial AP duration (APD₅₀, APD₇₀, and APD₉₀). Summary data are means ± SEM; n = 9 SAN myocytes and 9 right atrial myocytes; *P<0.05 vs. control by one way ANOVA with Tukey's posthoc test.

Figure 5. Effects of the PDE4 inhibitor rolipram on action potential firing in SAN and atrial myocytes.

A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of Rol (10 µM) and after Rol washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of Rol on spontaneous AP frequency (B), DD slope (C) and APD₅₀ (D). E. Representative stimulated right atrial myocyte APs in control conditions, in the presence of Rol (10 µM) and after Rol washout. Dotted line is at 0 mV. F. Summary of the effects of Rol on atrial AP duration (APD₅₀, APD₇₀, and APD₉₀). Summary data are means ± SEM; n = 10 SAN myocytes and 7 right atrial myocytes; *P<0.05 vs. control by one way ANOVA with Tukey's posthoc test.

Figure 6. Effects of IBMX on L-type Ca²⁺ current in SAN and right atrial myocytes.

A. Representative I_Ca,L recordings in SAN myocytes in control conditions, in the presence of IBMX (100 µM) and following IBMX washout. B. Summary I–V relationships for the effects of IBMX on SAN myocyte I_Ca,L. C. Summary I_Ca,L conductance density plots for the effects of IBMX in SAN myocytes. D. Representative I_Ca,L recordings in right atrial myocytes in control conditions, in the presence of IBMX (100 µM), and following IBMX washout. E. Summary I–V relationships for the effects of IBMX on right atrial myocyte I_Ca,L. F. Summary I_Ca,L conductance density plots for the effects of IBMX in right atrial myocytes. Summary data are means ± SEM; n = 8 SAN myocytes and 12 right atrial myocytes; *P<0.05 vs. control by paired Student's t-test.

Figure 7. Effects of the PDE2 inhibitor EHNA on L-type Ca²⁺ current in SAN and right atrial myocytes.

A. Representative I_Ca,L recordings in SAN myocytes in control conditions, in the presence of EHNA (10 µM) and following EHNA washout. B. Summary I–V relationships for the effects of EHNA on SAN myocyte I_Ca,L. C. Summary I_Ca,L conductance density plots for the effects of EHNA in SAN myocytes. D. Representative I_Ca,L recordings in right atrial myocytes in control conditions, in the presence of EHNA (10 µM), and following EHNA washout. E. Summary I–V relationships for the effects of EHNA on right atrial myocyte I_Ca,L. F. Summary I_Ca,L conductance density plots for the effects of EHNA in right atrial myocytes. Summary data are means ± SEM; n = 6 SAN myocytes and 10 right atrial myocytes; *P<0.05 vs. control by paired Student's t-test.

Figure 8. Effects of the PDE3 inhibitor milrinone on L-type Ca²⁺ current in SAN and right atrial myocytes.

A. Representative I_Ca,L recordings in SAN myocytes in control conditions, in the presence of Mil (10 µM) and following Mil washout. B. Summary I–V relationships for the effects of Mil on SAN myocyte I_Ca,L. C. Summary I_Ca,L conductance density plots for the effects of Mil in SAN myocytes. D. Representative I_Ca,L recordings in right atrial myocytes in control conditions, in the presence of Mil (10 µM), and following Mil washout. E. Summary I–V relationships for the effects of Mil on right atrial myocyte I_Ca,L. F. Summary I_Ca,L conductance density plots for the effects of Mil in right atrial myocytes. Summary data are means ± SEM; n = 6 SAN myocytes and 12 right atrial myocytes; *P<0.05 vs. control by paired Student's t-test.

Figure 9. Effects of the PDE4 inhibitor rolipram on L-type Ca²⁺ current in SAN and right atrial myocytes.

A. Representative I_Ca,L recordings in SAN myocytes in control conditions, in the presence of Rol (10 µM) and following Rol washout. B. Summary I–V relationships for the effects of Rol on SAN myocyte I_Ca,L. C. Summary I_Ca,L conductance density plots for the effects of Rol in SAN myocytes. D. Representative I_Ca,L recordings in right atrial myocytes in control conditions, in the presence of Rol (10 µM), and following Rol washout. E. Summary I–V relationships for the effects of Rol on right atrial myocyte I_Ca,L. F. Summary I_Ca,L conductance density plots for the effects of Rol in right atrial myocytes. Summary data are means ± SEM; n = 6 SAN myocytes and 12 right atrial myocytes; *P<0.05 vs. control by paired Student's t-test.