Adaptation for Positron Physics

1.9.1 CNP in cardiac pathologies

Early studies detected expression of all three types of NPR (NPR-A, -B and -C) in both rat and human hearts (Nunez et al., 1992). Subsequently, CNP mRNA was also found in rat hearts (Vollmar et al., 1993), confirming the production of CNP in the myocardium and hinting that it may contributes to the regulation of cardiac function. By measuring the difference in plasma levels of CNP between the aortic root and coronary sinus in patients with CHF, it has been confirmed that failing hearts produce CNP (Kalra et al., 2003, Del Ry et al., 2006a), and that plasma levels of CNP are related to disease severity (Del Ry et al., 2005). The expression of CNP mRNA is also significantly increased in ischaemic cardiomyopathy and inversely associated with LV function (Tarazon et al., 2014). Similarly, CNP mRNA expression is elevated in the fibrotic area of the infarct and border regions of the lesion (Soeki et al., 2005), indicating local CNP acts in an autocrine manner and may play an important part in cardiac fibrosis. In in vivo studies, CNP levels decrease as collagen deposition increases in aging rat (Ichiki et al., 2014, Sangaralingham et al., 2011), suggesting a reduction in CNP may contribute to fibrosis. These observations indicate CNP production and secretion in the heart may play an important role in pathophysiological processes, and may have a compensatory or potentiating effect on other cardioprotective mediators, such as NO and alternate natriuretic peptides. In addition, higher levels of CNP and NPR-B expression are observed in leukocytes of HF patients with respect to control subjects (Cabiati et al., 2012), indicating CNP may also influence cardiovascular disorders via anti-inflammatory activity (Wang et al., 2007).

Nevertheless, the physiological function of CNP in cardiac function remains to be elucidated due to lack of selective pharmacological tools and cell-specific gene deletion, since global CNP KO is lethal before mice reach adulthood (Chusho et al., 2001).

1.9.2 CNP in ischaemia-reperfusion injury

Numerous studies have shown a protective effect of pGC/cGMP/PKG activity in IR injury (Abdallah et al., 2005, Burley et al., 2007, Inserte et al., 2000, Jin et al., 2014), implying the activation of NPR-B by CNP is myocardial protective. However, cardiomyocytes respire anaerobically during ischaemia that results in intracellular acidosis, which blunts cGMP production via pGC signalling in this environment (Agullo et al., 2003). This suggests that a pGC/cGMP-independent protective pathway might be involved. The Hobbs’ lab has

demonstrated in the isolated Langendorff heart that infusion of CNP prior to or following an ischaemic insult results in a 30-50% reduction in infarct size. The NPR-C agonist, cANF

4-23, mimicked this beneficial effect of CNP, indicating that NPR-C activation contributes to the CNP-mediated protective mechanism against IR injury (Hobbs et al., 2004).

Furthermore, it has been suggested that the activation of NPR-C/Gi coupling exerts its biological action by recruiting PI3K/Akt cascade that in turn stimulates eNOS and NO production (Anand-Srivastava, 2005). This pathway is known to be protective in IR injury (Schulz et al., 2004). Thus, both cGMP-dependent (i.e. B) and –independent (i.e. NPR-C) mechanisms might underlie the cardioprotective effect of exogenous CNP.

1.9.3 CNP in the regulation of cardiac remodelling

Cardiac remodelling and fibrosis is a hallmark of end-stage HF that involves cardiac fibroblast proliferation, and changes in myocardial morphology and function (Wu et al., 2017b). Thus, to slow cardiac hypertrophy and fibrosis is a sought-after therapeutic option in CHF. To date, it is well established that ANP and BNP possess hypertrophic and anti-fibrotic properties and improve cardiac function in the diseased heart (Tamura et al., 2000, Rosenkranz et al., 2003). However, their potent diuretic and natriuretic properties are a huge drawback to patients with unstable haemodynamics, dropping BP and impairing renal function (Vaduganathan et al., 2013). Interestingly, it has been reported that CNP has more potent anti-hypertrophic and anti-fibrotic actions than ANP (Horio et al., 2003), suggesting CNP may be a better pharmacological tool for CHF. Studies of CNP pharmacology have demonstrated that CNP attenuates an increase in cardiac hypertrophy and fibrosis, reduces LV enlargement in response to pressure-induced or ischaemia-induced HF (Soeki et al., 2005, Izumiya et al., 2012). In concert with these studies, mice with cardiac-restricted CNP-overexpression do not develop cardiac hypertrophy and fibrosis after MI, but no difference in infarct size was observed compared to WT (Wang et al., 2007). This indicates that CNP moderates the adverse cardiac remodelling process post-ischaemia. The protective mechanism of CNP is proposed to be attributed to CNP/NPR-B signalling as down-regulation of NPR-B signalling exhibits progressive cardiac hypertrophy without altering BP (Langenickel et al., 2006, Del Ry et al., 2008a). Yet, the ventricular expression of NPR-B is reduced in HF (Del Ry et al., 2008a), limiting the therapeutic potential of targeting NPR-B signalling. In addition, there is no evidence of increased fibrosis in animals with down-regulated NPR-B, while loss of NPR-C results in structural remodelling and fibrosis (Egom et al., 2014). These observations suggest that the anti-fibrotic action of CNP might be conveyed via NPR-C.

1.9.4 CNP in the control of heart rate

All three NPRs are expressed in the sinoatrial node (SAN) and atrium (Springer et al., 2012), suggesting natriuretic peptides have the ability to modulate HR and the cardiac conduction system. Early in vivo studies showed that CNP has positive chronotropic and inotropic effects via NPR-B signalling (Beaulieu et al., 1997, Hirose et al., 1998). Correspondingly, a recent study has shown that CNP increases HR via increasing L-type Ca²⁺ current (ICa(L)) and hyperpolarisation-activated current (If) (Springer et al., 2012). It is proposed that CNP regulates HR and conductivity by activating GC-linked NPR-B that increases cGMP concentration and inhibits PDE3 activity (Springer et al., 2012). Blockade of PDE3 would be expected to result in an increase of cAMP, which has positive chronotropic effect.

However, Herring et al. (2001) reported a bradycardia effect of CNP via a cGMP/PDE3 dependent pathway, causing an increase of cAMP PKA-dependent phosphorylation of presynaptic N-type calcium channels. This leads to ACh release into the synapse and activates M2 receptor on pacemaker cells that results in an enhanced vagal tone (Herring et al., 2001).

Rose et al. (2004) have demonstrated a negative chronotropic effect of CNP mediated by activation of NPR-C through Gi protein coupling in isolated SAN cells that leads to inhibition of L-type Ca²⁺ current (Rose et al., 2004). This group has also demonstrated a dual role of CNP in HR and SAN function by stimulating NPR-B and/or NPR-C in response to different levels of sympathetic drive (Azer et al., 2012). Under basal condition, CNP is able to increase HR, but this effect is not replicated by cANF^4-23, which suggests NPR-B is responsible for the positive chronotropic activity. In sharp contrast, CNP causes a reduction in HR in the presence of isoprenaline (ISO) and this effect is enhanced by NPR-B blockade, but abolished in NPR-C KO mice. These observations provide a definitive role for CNP in the SAN function and demonstrated CNP acts via NPR-B to increase HR under basal conditions but that this appears to switch to NPR-C signalling, probably via decrease in cAMP level, during sympathetic hyperactivity. In addition, NPR-C KO mice have elevated heart rate with reduction in parasympathetic activity and enhanced sympathetic activity, confirming NPR-C signalling modulates autonomic function (Moghtadaei et al., 2017). Furthermore, CNP can also reduce cardiac sympathetic neurotransmission by inhibiting the release of noradrenaline (Buttgereit et al., 2016). These biological actions of CNP on the sympathetic system may represent an important therapeutic target in CHF patients as damping sympathetic activity improves survival (Gheorghiade et al., 2003, Butler et al., 2006).

1.9.5 CNP in the control of cardiac contractility

Patients with CHF have elevated plasma levels of CNP that parallels clinical severity (Del Ry et al., 2005). Furthermore, clinical studies also reported a negative correlation between CNP and dP/dt in CHF patients, indicating CNP has a possible role in cardiac contractility (Del Ry et al., 2008b). In cardiac muscle preparations and isolated cardiomyocytes, CNP displays a positive lusitropic effect associated with a negative inotropic effect (Brusq et al., 1999, Nir et al., 2001, Zhang et al., 2005a). Whereas in the isolated perfused working heart, CNP exhibits a biphasic action with an immediate increase in inotropy and lusitropy, followed by a slowly developing negative inotropic effect (Pierkes et al., 2002, Wollert et al., 2003). These actions associate with PLB phosphorylation and activation of SERCA, and are mimicked by a cGMP-analogue (Pierkes et al., 2002), suggesting a cGMP-dependent pathway contributes to CNP bioactivity. In addition, cGMP-dependent protein kinase I (PKG I) overexpression enhances CNP-mediated cell shortening, systolic Ca²⁺ levels and accelerates Ca²⁺ decay (Wollert et al., 2003). Thus, it is plausible that CNP modulates cardiac contractility via the NPR-B/cGMP cascade.