Input Data Requirements

the upregulation of ECM genes, e.g. collagen I, collagen III, MMP-2 and fibronectin (Lijnen et al., 2000). TGF-β1 mRNA expression was significantly increased in cmCNP WT mice following AAC (sham 1.000±0.063, AAC 1.580±0.137; p<0.05; n=5-6) (Figure 73). There is a trend towards to an increased TGF-β1 mRNA expression in cmCNP KO sham mice compared to WT sham (WT sham, 1.000±0.063; cmCNP KO, 1.356±0.137; p>0.05; n=6), but this did not elevate further in response to AAC (Figure 73). Again, these observations suggest cmCNP KO mice have intrinsic upregulation of pro-fibrotic gene expression that underlies the accentuated fibrotic burden in pre-clinical models I demonstrated earlier.

Upregulation of MMP-2 mRNA expression was also observed in cmCNP KO mice in both sham and AAC compared to WT (sham: WT 1.000±0.074 vs. cmCNP KO 1.415±0.083; AAC:

WT 1.268±0.174 vs. cmCNP KO 1.525±0.169; p<0.05; n=6) (Figure 74). A trend towards to an increased Col1a1 mRNA expression was also observed in cmCNP KO compared to WT following AAC (WT, 1.034±0.134; cmCNP KO, 1.463±0.215; p>0.05; n=6) (Figure 75).

Furthermore, Col1a1 mRNA expression was equivalently increased in WT and NPR-C KO mice following AAC (WT: sham 1.000±108 vs. AAC 1.606±0.155; NPR-C KO: sham 1.046±0.114 vs.± 1.788±0.160; p<0.05; n=5-6) (Figure 75). Whereas, fibronectin expression was not altered in response to AAC in all animals (Figure 76).

Comparison of the expression of the reference genes in response to abdominal aorta constriction

Figure 68. Comparison of the expression of the reference genes in response to abdominal aorta constriction.

Left ventricular mRNA expression of the two reference/housekeeping genes, β-actin (A) and RPL-19 (B) in WT mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=10-12. p>0.05 using unpaired t-test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 69. Effect of cmCNP, fbCNP and NPR-C deletion on ANP mRNA expression in pressure overload-induced heart failure.

ANP mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=3-6. *p<0.05, **p<0.01, ***p<0.001 using 2-way ANOVA followed by Bonferroni post-hoc test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 70. Effect of cmCNP, fbCNP and NPR-C deletion on α-MHC mRNA expression in pressure overload-induced heart failure.

α-MHC mRNA expression from left ventricles isolated from littermate WT, cmCNP KO (A), fbCNP KO (B) and NPR-C KO (C) mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=4-6.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 71. Effect of cmCNP, fbCNP and NPR-C deletion on β-MHC mRNA expression in pressure overload-induced heart failure.

β-MHC mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=3-6. **p<0.01 using 2-way ANOVA followed by Bonferroni post-hoc test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 72. Effect of cmCNP, fbCNP and NPR-C deletion on SERCA2a mRNA expression in pressure overload-induced heart failure.

SERCA2a mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=4-6. *p<0.05 using 2-way ANOVA followed by Bonferroni post-hoc test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 73. Effect of cmCNP, fbCNP and NPR-C deletion on TGF-β1 mRNA expression in pressure overload-induced heart failure.

TGF-β1 mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=4-6. *p<0.05 using 2-way ANOVA followed by Bonferroni post-hoc test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 74. Effect of cmCNP, fbCNP and NPR-C deletion on MMP-2 mRNA expression in pressure overload-induced heart failure.

MMP-2 mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=4-6. *p<0.05 using 2-way ANOVA followed by Bonferroni post-hoc test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 75. Effect of cmCNP, fbCNP and NPR-C deletion on Col1a1 mRNA expression in pressure overload-induced heart failure.

Col1a1 mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=4-6. ***p<0.001 using 2-way ANOVA followed by Bonferroni post-hoc test.

Effect of cmCNP, fbCNP and NPR-C deletion on cardiac hypertrophic gene profile in pressure overload-induced heart failure

Figure 76. Effect of cmCNP, fbCNP and NPR-C deletion on fibronectin mRNA expression in pressure overload-induced heart failure.

Fibronectin mRNA expression from left ventricles isolated from littermate WT, cmCNP KO, fbCNP KO and NPR-C KO mice subjected to sham or abdominal aortic constriction (AAC) for 6 weeks. Data are represented as the mean±SEM. n=4-6.

4.8 Summary of key findings

The production of CNP from cardiomyocytes and cardiac fibroblasts in unison contributes to cardiac protection during HF. The cardiac dysfunction and morphology observed in cmCNP KO and fbCNP KO were replicated by the loss of NPR-C signalling, which was worse, per se, than each individual cell-specific CNP deletion. More importantly, administration of CNP rescued the deterioration of cardiac function and structure in WT mice, but this protective effect was absent in NPR-C KO mice. This indicates the importance of NPR-C signalling in CNP-mediated cardiac protection.

Chapter 5 – Discussion

Chapter 5 – Discussion

5 Discussion

5.1 Summary of key findings

In the first part of my thesis, I investigated the role of endogenous CNP in the coronary vasculature. My data showed the coronary reactivity is attenuated in ecCNP KO mice in response to endothelium-dependent vasodilators and reperfusion-induced vasodilatation compared to WT animals. This indicates endothelium-derived CNP is involved in the regulation of coronary vascular function. However, the production of CNP in the endothelium did not protect against IR injury, whilst cardiomyocyte-derived CNP displayed cardioprotective effects. These data suggest CNP has dual functions in the heart, i.e.

regulates coronary vascular reactivity and protects against ischaemic myocardial damage.

The reduced vascular responses and aggravated IR injury observed in ecCNP KO and cmCNP KO mice, respectively, were replicated in NPR-C KO mice, indicating endogenous CNP mediates its biological activity, at least in part, via NPR-C activation.

In the second part of this thesis, I explored the role of endogenous CNP in cardiac function, with focus on cardiac stress. I have demonstrated that mice lacking cardiomyocyte CNP exhibit worse cardiac dysfunction, accompanied by greater cardiac hypertrophy and fibrosis in both ISO- and pressure overload-induced HF models. This indicates that cardiomyocyte-derived CNP regulates cardiac function during cardiac stress via anti-remodelling and anti-fibrotic actions. This exacerbated response to cardiac stress was replicated in NPR-C KO mice. However, the phenotype observed in NPR-C KO was worse than cmCNP KO. This may stem from incomplete deletion of CNP (my data suggest an approximate 80% reduction in CNP mRNA expression in cardiomyocytes), activation of NPR-C by another natriuretic peptide (i.e. ANP or BNP), or that another cellular course of CNP is important. To investigate the latter hypothesis, I generated mice with fibroblast-restricted CNP deletion. This genetic alteration resulted in similar cardiac dysfunction to that observed in cmCNP KO mice following AAC. This indicates the production of CNP from both cardiomyocytes and cardiac fibroblasts in concert contributes to the cardioprotective effects during the development of HF. In addition, exogenous CNP attenuated the increase of hypertrophy in response to Ang II in isolated neonatal cardiomyocytes. In line with this finding, infusion of CNP normalised cardiac dysfunction following AAC in WT mice.

However, this protective effect was lost in mice lacking NPR-C. Taken together, the data suggest endogenous CNP has cardioprotective effects in the setting of HF and these are mediated via NPR-C activation.