Anthracene

1.10.1 Overview

cGMP and cAMP are second messengers conveying the biological activity of NO and natriuretic peptides, and the sympathetic system (Tsai and Kass, 2009). Different stimuli activating these cyclic nucleotides can mediates diverse cellular and physiological effects on the CVS depending on their concentration, localisation and duration of stimulation (Levy, 2013). For instance, acute elevation of cAMP increases intracellular Ca²⁺ that enhances contractility of the heart and cardiac output (Bobin et al., 2016). However, prolonged activation of cAMP activity due to sustained activation of adrenoceptors, as occurs in hypertension and HF, leads to maladaptive cardiac remodelling, apoptosis and arrhythmia (Bobin et al., 2016). Hence, modulation of cyclic nucleotide amplitude, duration and localisation is very important in determining the physiological responses. This is tightly regulated by the balance between the rate of cAMP and cGMP generation by AC and GC, respectively, and the rate of degradation by PDEs.

1.10.2 cAMP/PKA cascade in the heart

The stimulation of Gs protein coupled receptors, such as β-adrenoceptors, activates AC that syntheses cAMP, which in turn activates cAMP-dependent protein kinase A (PKA) (Bobin et

al., 2016). PKA phosphorylates various downstream proteins that regulate Ca²⁺ transient in cardiomyocytes in response to acute sympathetic stimulation, including sarcolemmal L-type Ca²⁺ channels, ryanodine receptors (RyR2), PLB (which controls the activity of SERCA2) and troponin I, leading to positive inotropic and lusitropic effects (Bobin et al., 2016).

However, chronic activation of PKA suppresses GSK3β that releases numerous transcription factors from chronic inhibition (see section 1.8.4.3), leading to protein synthesis and maladaptive hypertrophy (Dorn and Force, 2005). In addition to PKA, cAMP-activated guanine nucleotide exchange proteins (Epacs) may also play an important role in remodelling (Bobin et al., 2016). A study has shown that Epac1 expression is increased in response to pressure-overload, and activation of Epac1 triggers the calcineurin/CaMKII signalling pathway that results in hypertrophic growth (Metrich et al., 2008). Thus, inhibition of the production of cAMP, i.e. by activating Gi protein coupled receptors, or increasing its rate of degradation by PDEs could be beneficial in CVDs.

1.10.3 cGMP/PKG cascade in the heart

It is widely agreed that elevation of cGMP is cardioprotective. Activation of sGC and pGC by NO and natriuretic peptides, respectively, leads to cGMP production.

1.10.3.1 The effector molecules of cGMP and its physiological action in the cardiovascular system

Two types of effector molecules of cGMP predominate in the CVS: cGMP-dependent protein kinases (PKG), and PDEs. A third type of cGMP effector is the cGMP-gated cation channel, which is found in retinal and olfactory neuro-epithelium and nephrons.

1.10.3.1.1 cGMP-dependent protein kinase

There are three isoforms of PKG. PKG-type Iα (PKG-Iα) is expressed in cardiac myocytes, PKG-Iβ is predominantly found in the endothelial cells, while VSMC expresses both isoforms. The third isoform is PKG-type II (PKG-II) that is found in the kidney, brain and intestine. All three isoforms are homodimers and each subunit consist of three functional domains – N-terminal, regulatory domain and a kinase domain. When cGMP binds to specific sites in the regulatory domain, PKG undergoes a conformational change that leads to the release of the N-terminal inhibition of the kinase domain. Subsequently, the kinase domain catalyses the phosphorylation of a serine/threonine residue of the target proteins.

The action of cGMP is often viewed as opposing cAMP activity, meaning cGMP has negative inotropy, anti-hypertrophic and anti-fibrotic effects. In isolated cardiomyocytes, cGMP

analogues modulate the contraction of electrically stimulated myocardium from WT control mice, but have no effect in the myocardium from cardiomyocyte-specific PKG-I null mice (Wegener et al., 2002), demonstrating cGMP/PKG-I signalling negatively modulates cardiac contractility. Moreover, it has been shown that inhibition of L-type Ca²⁺ channel and troponin I phosphorylation is responsible for the negative inotropic effect mediated by PKG (Yang et al., 2007).

The anti-hypertrophic role of cGMP has been demonstrated in numerous studies with genetic disruption of the cGMP pathway. Mice lacking eNOS develop cardiac hypertrophy and dysfunction (Wenzel et al., 2007, Li et al., 2004a, Flaherty et al., 2007). However, local ANP signalling can compensate for the loss of NO and prevent the development of hypertrophy in eNOS-deficient mice (Bubikat et al., 2005). Likewise, disruption of the cGMP pathway in the heart by cardiac-specific deletion of NPR-A leads to maladaptive hypertrophy with enhanced fibrosis and marked deterioration in cardiac function (Holtwick et al., 2003, Kuhn et al., 2002). These observations are also seen in mice with cardiomyocyte-restricted deletion of PKG-I and accompanied by diminished expression of SERCA2a and PLB that affects myocardial Ca²⁺ homeostasis (Frantz et al., 2013). Taken together, these studies illustrate that one of the potential molecular mechanisms by which ANP and/or BNP exert anti-hypertrophic effects is via regulation of intracellular Ca²⁺

through NPR-A-cGMP-PKG-I signalling that can consequently inhibit the Ca²⁺ -calcineurin-NFAT hypertrophic pathway (see section 1.2.2.2).

An anti-fibrotic effect of natriuretic peptide/pGC pathway has also been identified. It has been shown that ANP-cGMP-PKG signalling induces phosphorylation of Smad3 at a different site from TGF-β1 (Li et al., 2008). The resultant pSmad3 cannot translocate into the nucleus, thus, PKG disrupts TGF-β1-induced pro-fibrogenic gene expression (Li et al., 2008). Moreover, mice with ANP deficiency exhibit accelerated dilated cardiomyopathy with enhanced fibrosis and systolic dysfunction (Wang et al., 2014). Interestingly, these mice have marked increases in CNP and NPR-C expression, which may indicates CNP/NPR-C signalling is upregulated to play a compensational role when ANP production is diminished.

It is known that activation of Gi protein coupled NPR-C can indirectly generate cGMP by activating eNOS via PI3K and Akt (Costa et al., 2006). Perhaps, CNP can exert cardioprotective effects via stimulating cGMP production and inhibiting excessive cAMP signalling through activation of NPR-B and/or NPR-C, respectively. Regardless, the patho/physiological signalling of CNP in cardiac function is still poorly understood.

1.10.3.1.2 Phosphodiesterases

cGMP and cAMP catabolism is regulated by PDEs. Since some non-selective PDEs can catabolise both cAMP and cGMP, the hydrolysis of one cyclic nucleotide can inhibit the activity of PDEs on the other cyclic nucleotide. This provides a cross-regulation of cAMP and cGMP signalling. For example, cGMP production following the activation of NPR-B binds to PDE3 and inhibits cAMP degradation, resulting in an increase of cAMP-mediated β-adrenoceptor signalling and contractility in both failing and non-failing hearts (Qvigstad et al., 2010, Meier et al., 2017). Whereas, PDE2 is stimulated by cGMP, leading to an increase in cAMP hydrolysis and protects against sympathetic over-stimulation (Mehel et al., 2013).

Indeed, one plausible mechanism by which cGMP/PKG signalling mediates anti-hypertrophic effects is by inhibiting the calcineurin pathway that can activated by cAMP/PKA cascade (Tsai and Kass, 2009). Furthermore, PDE5 is specific for cGMP degradation. In HF models, studies reported PDE5 inhibition blunts β-adrenergic stimulation (Senzaki et al., 2001), protects against IR injury by targeting mitochondrial KATP

channels (Ockaili et al., 2002) and reverses pre-established cardiac hypertrophy in pressure-overload model (Takimoto et al., 2005). Small scale clinical trials in HErEF patients have reported that PDE5 inhibitors improve cardiac function and exercise capacity (Lewis et al., 2007, Kim et al., 2015). However, the larger scale RELAX trial, in HFpEF patients showed neutral results with sildenafil administration (Redfield et al., 2013). One explanation could be that the natriuretic peptides levels are lower in HFpEF patients compared to HErEF (Bishu et al., 2012), which suggests limited natriuretic peptide activity. Thus, to enhance cGMP levels by PDE5 alone may not be adequate in HFpEF patients.