Effects of aging and diet on neprilysin activity
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Neprilysin (NEP) is an endopeptidase with several vasoactive substrates, including natriuretic peptides. A NEP inhibitor combined with an angiotensin receptor blocker (LCZ696) improves outcomes in patients with heart failure with reduced ejection fraction (HFrEF); however, it has not been tested in patients with HF with preserved ejection fraction (HFpEF). Two common forms of HFpEF are caused by aging and metabolic syndrome (MetS)/type 2 diabetes. Therefore, the goal of this study was to determine whether NEP activity is increased in mice with diastolic dysfunction due to aging or MetS. C57BL/6J mice were fed normal chow diet (NCD) for the first two months of their life, before being switched to either control diet (CD) or a high fat, high sucrose (HFHS) diet for 4 or 16 months, which allowed for determination of the effects of both age and diet on NEP activity. NEP activity was measured in plasma based on the generation of a fluorescent product, methoxy-2-naphthylamine (MNA). Samples were incubated +/- NEP inhibitor thiorphan to distinguish NEP activity from activity of other non-specific endopeptidases. The difference in fluorescence of MNA was then measured, and samples incubated with thiorphan were subtracted from samples incubated without thiorphan to give plasma NEP activity. Baseline NEP activity values (BL, 5.0±2.9) were derived from 2 month old mice fed Normal Chow Diet (NCD). Several mice were not switched to CD or HFHS, but were instead kept on NCD for an additional 4 months to act as an age-matched control against the CD cohort, for determination as to whether CD had a differential effect on NEP activity relative to NCD. The mice switched to CD had increased NEP activity at 6 months compared to NCD-fed mice (14.8±6.4; p<0.0002 vs. BL). For this reason, mice were fed CD for all subsequent experiments. NEP activity in CD-fed mice increased from 4 months to 16 months (14.8±6.4 vs. 65.3±16.4; p= 0.0028). NEP activity in HFHS-fed mice increased from 4 months to 16 months as well (20.0±7.6 vs. 43.8±20.9; p= 0.0031) Comparison of the age-matched effect of HFHS diet relative to CD revealed that there was a trend towards significance at 4 months (20.7±7.6; p=0.06 vs. CD) but not at 16 months (43.8±20.9; p=ns vs. CD). This study revealed that both CD and HFHS increase NEP activity compared to NCD, perhaps as a result of the lard (saturated fat) as the principal source of fat in those diets. More importantly, it revealed that NEP activity increases with aging, and that aging exerts a greater influence on NEP activity than diet (MetS) does. Finally, it was shown that LCZ696 can effectively lower plasma NEP activity. Mice fed CD and HFHS diet for 16 months that received one week of 100 mg/kg LCZ696 therapy had 78% inhibition (p= 0.0286) and 85% inhibition (p= 0.0006 ) of their plasma NEP (sNEP) activity, respectively. This strongly suggests the utility of LCZ696 as a promising treatment option for patients with aging and MetS-related HFpEF.
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