Novel insights into the mechanisms mediating the local antihypertrophic effects of cardiac atrial natriuretic peptide: role of cGMP-dependent protein kinase and RGS2

Abstract

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on beta-adrenergic versus Angiotensin II (Ang II)-dependent (G(s) vs. G(alphaq) mediated) modulation of Ca(2+) (i)-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca(2+) currents and Ca(2+) (i) transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca(2+) currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, beta-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca(2+)-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca(2+) (i)-dependent hypertrophic growth response to Ang II, but not to beta-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT(1) signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for beta-adrenergic Ca(2+) (i)-stimulation in adult myocytes.

Fig. 1

Ca²⁺ _i transients (Indo-1 ratio_405/495 nm, systolic–diastolic a, b) and simultaneously recorded cell length (L _max–L _min, expressed as percent from L _max c, d) in field-stimulated myocytes at baseline and during superfusion with either Ang II (10 nM) or ISO (100 nM) in the presence or absence of ANP (100 nM, 10 min pretreatment). Top original tracings of Ca²⁺ _i transients. Bottom mean ± SEM, n = 4–6 cells from four mice; asterisks denote a significant difference versus basal (B) (P < 0.05)

Fig. 2

L-type Ca²⁺ channel activity was analyzed in ventricular myocytes by whole-cell patch-clamp recordings. Left original L-type Ca²⁺ current traces of two cells at baseline and upon superfusion with 10 nM Ang II (a) or 100 nM ISO (c). Right change in L-type Ca²⁺ channel current density (I) in percent b after application of 10 nM Ang II, 100 nM ANP or ANP and Ang II (n = 7 cells from 3 mice) and d after application of 100 nM ISO, 100 nM ANP or ANP and ISO (n = 5 cells from 3 mice); *P < 0.05 versus B, basal

Fig. 3

Ca²⁺ _i transients (Indo-1 ratio_405/495 nm, systolic–diastolic) in field-stimulated cardiomyocytes at baseline and during superfusion with Ang II in the presence or absence of ANP. In respective control cardiomyocytes (with unaltered protein expression levels) Ang II (10 nM) increased systolic Ca²⁺ _i levels and the peak amplitude of Ca²⁺ _i transients. This Ca²⁺ _i responses to Ang II were fully prevented in the presence of ANP (100 nM, pretreatment during 10 min). This inhibitory effect of ANP on the Ca²⁺ _i responses to Ang II was abolished a in GC-A-deficient (GC-A^−/−), b in PKG I-deficient (PKG I^−/−) and c in RGS2-deficient (RGS2^−/−) myocytes. b The inhibitory effect of ANP on the responses to Ang II was also abolished after pharmacological blockade of PKG I with Rp-8-Br-PET-cGMP (10 μM, 30 min pretreatment). c Note that in RGS2^−/− myocytes the Ca²⁺ _i-stimulating effects of Ang II (1 nM) were significantly greater when compared with the effects of Ang II (10 nM) on control cells; n = 4–6 cells (4 mice per genotype); *P < 0.05 versus basal; ^# P < 0.05 versus controls

Fig. 4

HEK293 cells coexpressing GC-A, PKG Iα and RGS2 were incubated with 10 nM ANP during the indicated time periods or remained untreated (vehicle). Protein expression levels in cytosolic and membrane fractions were analyzed by Western blotting. Top representative experiment. Bottom cytosolic and membrane levels of RGS2 (n = 3; *P < 0.05 vs. vehicle)

Fig. 5

Effect of chronic treatment with Ang II or ISO on a systolic blood pressure (SBP), b left ventricular weight (LVW)—to body weight (BW) ratios, and c myocyte diameters of floxed GC-A (controls) and CM GC-A KO mice; (n = 8–12 per group) asterisks denote a significant difference versus vehicle (P < 0.05), crosses denote a significant difference from control mice (P < 0.05)

Fig. 6

Effect of chronic Ang II (a) or ISO infusion (b) on the cardiac expression and autophosphorylation of CaMKII. Top representative Western blots for the left ventricular levels of autophosphorylated CaMKII, total CaMKII and GAPDH. Bottom levels of phosphorylated CaMKII were normalized to the total levels of CaMKII and calculated as X-fold respective vehicle-treated control mice; (n = 8; *P < 0.05 when compared with vehicle; ^# P < 0.05 compared with controls)

Fig. 7

Ca²⁺ _i transients (Indo-1 ratio_405/495 nm, systolic–diastolic) in field-stimulated cardiomyocytes at baseline and during superfusion with either Ang II or ISO. The stimulatory effects of Ang II were abolished in myocytes with genetic (TRPC3^−/−/C6^−/−) (a) or pharmacological inhibition of TRPC channels (2 μM BTP 2, pretreatment during 15 min) (c). The effects of ISO were preserved (b, d). Asterisks denote a significant difference versus basal (B) (P < 0.05). For each condition, six cells from four mice were tested

Fig. 8

Stimulation of L-type Ca²⁺ channel activity by Ang II and ISO in wild-type myocytes (controls) and myocytes from TRPC3^−/−/C6^−/− mice. Left representative L-type Ca²⁺ current traces at baseline and upon superfusion either with 10 nM Ang II (a) or with 100 nM Iso (c). Right stimulation of L-type Ca²⁺ channel current density (I, in percent) in cells of wild-type (controls) or TRPC3^−/−/TRPC6^−/− mice b after application of 10 nM Ang II (n = 6 cells from 3 mice) and d after application of 100 nM ISO (n = 5 cells from 2 mice; *P < 0.05 vs. basal, B)