Dual acting RAAS blockade and neprilysin inhibition has been

Dual-acting RAAS blockade and neprilysin inhibition has been evaluated in several clinical HF trials. In PARADIGM-HF [6,7], LCZ696 was superior to enalapril in reducing mortality and HF hospitalizations in symptomatic patients with HF with reduced EF. Augmented benefits on maladaptive cardiac remodeling and reductions in cardiac biomarkers with LCZ696 over single acting RAAS blockade have also been reported in HF with preserved EF [22]. Here we provide further mechanistic insight into the putative underlying pathophysiological mechanisms, in particular myocardial hypertrophy and fibrosis. At 5 weeks post-MI, LV posterior wall thickness, LV mass and tissue weight, as well as cardiomyocyte cross-sectional area in the non-infarct and border regions were reduced with both LCZ696 and perindopril compared to MI-vehicle. This confirms the well-proven efficacy of stand-alone RAAS inhibition with ACEi or ARB to attenuate hypertrophy following MI [13,23]. The addition of the neprilysin component in LCZ696 augments plasma levels of natriuretic peptides such as ANP which have been reported to reduce demonstrate anti-hypertrophic effects in cardiomyocytes and myocardium [24,25]. However, when LV function begins to deteriorate, the myocardium expresses natriuretic peptides and this feature is used as a diagnostic and prognostic indicator of CHF [26]. It been demonstrated in the MI setting that increased LV wall stress and LV filling pressures elevate ANP cell culture supplement in the heart at 1 and 6 weeks post-MI [27]. In the current study, gene expression of ANP was significantly reduced with both treatments, and further reduced by LCZ696 compared to perindopril, which may reflect reduced LV loading [28]. Furthermore, natriuretic peptides have been shown to inhibit RAAS by reducing renin secretion and angiotensin II production in experimental studies [29], this could explain the increased attenuation of ANP expression with LCZ696 treatment. Combined ACE and neprilysin inhibition (by vasopeptidase inhibitors) has been shown to reduce LV adverse remodeling post-MI more than ACEi alone [30]. By contrast, an earlier study suggests that single-acting inhibition of neprilysin alone does not appear to modulate cardiac hypertrophy in this model [31]. We have previously shown incremental benefits of adding a neprilysin inhibitor to valsartan on angiotensin-II mediated cardiac cellular hypertrophy and fibrosis [9]. Similar reductions in ANP gene expression have been observed with LCZ696 compared to valsartan treatment in mice with diabetes [18]. Thus, efficient RAAS blockade seems to be a critical requirement to fully utilize the benefits of neprilysin inhibition. Further to hypertrophic changes in the myocardium, increased interstitial tissue fibrosis plays an important role in cardiac remodeling and dysfunction. Here, interstitial cardiac fibrosis, and collagen I deposition were reduced with LCZ696 in the border and non-infarct regions. The anti-fibrotic effect observed with perindopril was similar to that previously reported by others [32,33]. RAAS blockade comparing the effect of valsartan with the ACEi fosinopril have realized similar reductions in collagen synthesis and myofibroblast activation [34]. As well, combined ACE and neprilysin inhibition has been demonstrated to reduce tissue collagen content in the LV [30]. TIMP2, an inhibitor of matrix metalloproteases that prevents matrix degradation was reduced with LCZ696 suggesting a potential mechanism for enhanced resolution of fibrosis. Data from animal studies supports a role for TIMP2 in protecting the extracellular matrix from proteolysis [35,36], and post-MI this may be through inhibition of MMP14 activity [36]. In addition to changes in TIMP2, MMP9 was elevated in MI animals, this increase has been reportedly linked with inflammation, extracellular matrix degradation and synthesis and cardiac dysfunction [35]. In spontaneous hypertensive heart failure rats both TIMP2 and MMP9 gene and protein expression levels were found to be elevated [37]. Similarly in volume overload rats TIMP2 protein expression was increased after 8 weeks [38]. The work described in this study provides further evidence regarding the functional relevance of TIMP2 by significant correlations between TIMP2 and indices of LV dilatation, diastolic dysfunction and chamber stiffness (see Supplemental Table 1).