Major findings
The present studies in vivo and in isolated myocytes show essentially that the ANP/GC-A/cGMP pathway counter regulates cardiac Ang II, but not β-adrenergic stimulation of calcium handling, of the calcium-dependent prohypertrophic CaMKII pathway and of cardiac hypertrophy. The mechanism behind this differential regulation of the Ang II versus ISO responses by ANP involves PKG I and the regulator of G protein signalling (RGS) 2, a major target of PKG I, because the inhibitory effects of ANP on the Ca
2+
i responses to Ang II were abolished in PKG I- or RGS2-deficient myocytes. Supporting these findings, in transfected HEK293 cells ANP, via GC-A/PKG I, markedly induced the expression of RGS2 and its translocation to the plasma membrane. We conclude that ANP, via GC-A/cGMP/PKG I signalling and activation of RGS2, specifically counter regulates the hypertrophic effects of agonists linked to Gαq-protein coupled receptors, such as Ang II. In contrast, ANP does not affect β-adrenergic modulation of cardiac calcium handling and myocyte growth. Lastly, our study extents and strengthens previously published observations in neonatal cardiomyocytes [
34] demonstrating for the first time that transient receptor potential canonical TRPC3/C6 channels are indispensable for the calcium responses of adult myocytes to Ang II and are not involved in calcium responses to β-adrenergic stimulation.
Differential regulation of cardiac Ang II, but not β-adrenergic responses by ANP
Our results are in accordance with previous studies showing that cardiac remodeling in mice with global (not cardiomyocyte specific) GC-A disruption is markedly inhibited by genetic or pharmacological blockade of the AT
1 receptor [
27]. As a corollary, Ang II-induced cardiac remodeling was suppressed in mice overexpressing brain natriuretic peptide (BNP) in the circulation, a member of the natriuretic peptide family which also activates the GC-A receptor, although with less affinity than ANP [
43]. However, these studies could not distinguish between systemic and cardiac interactions of ANP/BNP and Ang II. Our study adds an important piece of information, because the conditional, cardiomyocyte-restricted disruption of the GC-A gene in mice allowed us to specifically dissect the local cardiac from the systemic interactions between the ANP/GC-A and Ang II/AT
1 systems. Furthermore, it characterizes PKG I and RGS2 as molecular effectors mediating the cross-talk between these pathways in cardiac remodeling.
Our observation of a differential regulation of Ang II- but not β-adrenergic stimulation of myocyte calcium handling by ANP is in agreement with a study by Takimoto et al. [
44] which also observed that β-adrenergic calcium and contractile responses of adult myocytes and intact hearts are unaffected by ANP/GC-A-elicited cGMP production at the plasma membrane. In contrast, nitric oxide (NO)-stimulated cGMP production in the cytosol (which is mediated by the soluble guanylyl cyclase, sGC), markedly blunted the cardiac contractile responses to ISO [
44,
50]. Extending these observations, cardiac overexpression of endothelial NO synthase in transgenic mice attenuated cardiac hypertrophy induced by chronic ISO infusion [
36]. Hence, these studies indicate that the NO/sGC/cGMP system acts as a negative modulator of both the cardiac contractile and hypertrophic responses to β-adrenergic stimulation [
36,
44]. In contrast, the ANP/GC-A/cGMP system does not regulate these responses (present study and [
44]). In conclusion, our observations support the notion that cGMP synthesized by sGC (in response to NO) and membrane-bound GC-A (in response to ANP) does not feed a common cGMP pool in cardiac myocytes, but instead remains spatially and functionally compartmentalized to modulate different targets and thereby different myocyte functions.
Counteraction of the cardiac Ang II effects by ANP/GC-A/cGMP involves PKG I and RGS2
At least two cGMP-stimulated proteins (as third messengers) are expressed in myocytes: PKG I; and phosphodiesterase (PDE) 2, a dual substrate esterase, which appears to hydrolyze cGMP under resting conditions [
2], but targets cAMP in the presence of β-adrenergic stimulation [
33]. In cultured endothelial cells, the ANP/GC-A/cGMP-signalling pathway stimulates PDE2 activity, decreases intracellular cAMP concentrations and thereby increases endothelial barrier functions [
42]. However, in cardiomyocytes, the lack of effects of ANP on the calcium and inotropic responses to ISO (see present study and [
44]) suggests that PDE 2 activity is not modulated by the pool of cGMP formed after ANP/GC-A stimulation. This could be due to spatial compartmentalization or could be related to different concentrations of cGMP reached after GC-A stimulation in myocytes (expressing low levels of GC-A) when compared with endothelia (with very dense GC-A expression) [
25,
26].
Instead, using both a genetic and a pharmacological approach, our study demonstrates that PKG I is a downstream target activated by the ANP/GC-A/cGMP system in cardiomyocytes. As illustrated, the counter regulation of the Ca
2+ responses of isolated adult myocytes to Ang II by ANP was absent in PKG I-deficient myocytes and also in wild-type myocytes pre-treated with the selective PKG I inhibitor, Rp-8-Br-PET-cGMP. However, the in vivo relevance of these in vitro findings has still to be demonstrated. Mice with a global deletion of PKG I show severe gastrointestinal dysfunction and high mortality before 6 weeks of age [
38]. To circumvent these limitations, smooth muscle rescue mice were generated, in which PKG I expression is restored selectively in smooth muscle, but not in other cell types of PKG I null mice [
49]. We used these rescue mice not only to isolate adult myocytes for our ex vivo calcium studies, but also to study the impact of PKG I deletion on the cardiac hypertrophic responses to chronic Ang II treatment. Ang II was administered for 14 days to eight 20-week-old male PKG I rescue mice and eight litter-matched control mice (300 ng Ang II/kg BW/min with osmotic minipumps). Interestingly, the cardiac hypertrophic responses to Ang II were attenuated in the PKG I mutants when compared with control mice (data not shown). However, it is important to note that this group of rescue mice also showed a systemic phenotype. Unfortunately, these animals had anemia and splenomegalia [
7]. In addition, and in contrast to the normal blood pressure of 10-week-old PKG I rescue mice [
49], we observed in the “older” rescue mice pronounced arterial hypotension (decreases in SBP by ~20 mmHg measured by the tail cuff method, both before and during Ang II administration, data not shown). Thus, it is likely that the attenuated hypertrophic response of the PKG I mutants to Ang II is related to their systemic phenotype, especially to arterial hypotension. Hence, although our own and published data support an important protective role for myocardial PKG I in pathological cardiac hypertrophy in vivo [
45], a final proof is lacking.
At least in vitro several proteins centrally involved in myocyte calcium handling have been shown to be regulated by PKG I, such as the L-type Ca
2+ channel (inhibition), phospholemman (inhibition) and phospholamban (inhibition, resulting in enhanced SR calcium uptake by SERCA) [
6]. Our observation that the ANP/GC-A/cGMP/PKG I pathway selectively inhibits the myocyte Ca
2+-responses to Ang II, but not to ISO, suggested a selective modulation of G
αq/11-coupled receptor signalling. The regulator of G protein signalling (RGS)-2 is a selective and negative regulator of G
q/11 proteins in the cardiovascular system which is activated by PKG I [
51]. For instance, in vascular smooth muscle cells the nitric oxide/sGC/cGMP/PKG I pathway regulates the degradation of RGS2 and promotes its association with the plasma membrane by a mechanism requiring its PKG I phosphorylation sites [
35]. By regulating RGS2 plasma membrane association and degradation, PKG I therefore may control its inhibitory effect on Gq/11-coupled receptors [
35]. RGS2-deficient mice (RGS2
−/−) are hypertensive, exhibit increased vasoconstrictory responses to vasopressin and other hormones activating G
q/11-coupled receptors and diminished vasodilating responses to nitric oxide/cGMP/PKG I signalling [
14,
15,
35,
46]. Consistent with these observations, we found in the present study that the calcium responses of RGS2
−/− myocytes to Ang II were enhanced, whereas responses to β-adrenergic stimulation were unaltered. Notably, the inhibitory effect of ANP on calcium responses to Ang II was absent in RGS2
−/− myocytes. Concomitantly, in transfected HEK293 cells ANP, via GC-A/PKG I signalling, dramatically enhanced RGS2 expression and membrane localization. We conclude from these experiments that ANP, via GC-A/cGMP/PKG I, enhances the expression and/or phosphorylation of RGS2 and thereby counteracts AngII/AT1 signalling. In turn, inhibition of ANP/GC-A signalling (in CM GC-A KO mice) enhances the cardiac hypertrophic responses to Ang II. Unfortunately for technical reasons, we could not complement these experiments with analyses of RGS2 expression and/or phosphorylation in wild type when compared with GC-A- or PKG I-deficient myocytes. There are no phospho-specific antibodies available. Worse, Western-blot analyses revealed that commercially available anti-RGS2 antibodies unspecifically recognize an immunoreactive protein of 28 kDa (the MW of RGS2) in tissues from both wild-type and RGS2-deficient mice (not shown). Hence, in our hands commercially available antibodies were unsuitable to study RGS2 expression in native tissues.
Collectively, our findings are compatible with the notion that PKG I is the downstream target activated by the ANP/GC-A/cGMP-signalling pathway in cardiac myocytes. cGMP/PKG I-mediated modulation of RGS2 and subsequent inhibition of AT1/Gq-signalling appear to mediate the specific counter regulation of the calcium responses of myocytes to Ang II by ANP.
TRPC3/C6 channels are involved in the calcium responses of adult myocytes to Ang II
Studies in neonatal rat cardiomyocytes have indicated that Ang II induces TRPC3/C6 activation through Gαq-phospholipase C (PLC) signalling pathways [
34]. DAG, generated by PLC activation, directly activates TRPC3/C6 [
17]. In neonatal myocytes, this causes slow depolarization of the membrane potential and concomitantly increases the frequency of spontaneous firing due to activation of L-type Ca
2+ channel [
34]. Indeed, in our study with adult murine ventricular myocytes, the effects of Ang II both on Ca
2+ transients and on L-type Ca
2+ currents were abolished in TRPC3/C6-deficient myocytes. In addition, BTP2, which inhibits the activity of TRPC channels [
13], abolished the calcium responses to Ang II. Taken together these experiments demonstrate that TRPC channels are indispensable for the calcium responses of adult ventricular myocytes to Ang II. In contrast, β-adrenergic stimulation of Ca
2+ currents and Ca
2+
i handling was not altered by genetic or pharmacological blockade of TRPC channels. However, intriguingly, in whole-cell current-clamp recordings, we did not observe differences in the resting membrane potential of wild-type and TRPC3/C6-deficient myocytes or changes of the membrane potential in response to Ang II (data not shown). These unexpected findings point towards a novel mechanism of TRPC3/C6 activity regulating L-type Ca
2+ channel (LTCC) activity in response to Ang II which we will try to elucidate in our future studies. One hypothesis is that TRPC-mediated Ca
2+ entry activates CaMKII, which has been shown to be associated with cardiac LTCC complexes and increases channel open probability to dynamically increase Ca
2+ current by a process called facilitation [
10].