Reginauld, Shawn

Introduction: Previously, miR-425 have been implicated in the inhibition of the cardiac natriuretic peptide (NP) ANP gene expression with their role on BNP undefined. The mechanism by which miR-425 modulates NP expression remains to be explored. In heart failure (HF), adrenergic and neurohormone pathways are aberrantly activated. Our goal was to investigate the role of miR-425 and its downstream target Cofilin 2 (CFL2) on ANP and BNP expression in human CV disease (CVD). Hypothesis: We hypothesized miR-425, via CFL2, supresses NPs expression in vitro. MiR-425, CFL2 and NP levels were also determined in CVD patients. Methods: In vitro, human cardiomyocytes (HCM) were treated with vehicle (Veh), catecholamine isoproterenol (ISO) and cytokine endothelin (ET), miR-425 mimic. Cells were transfected with CFL2 plasmid or CFL2 siRNA. Normal (NM, n=100), Hypertension (HTN, n=30) and Acute HF (AHF, n=74) cohorts were studied. A confirmatory cohort was recruited. Plasma NT-proBNP, miR-425 were measured. In left ventricles (LV) from NM or HF (n=5 each), NP, CFL2 and miR-425 expression were assessed. Data expressed as means±SEM. * p<0.05 vs NM or Veh. Results: In vitro in HCM, treatment with ISO or ET in HCM reduced miR-425 and elevated NP mRNA levels. Furthermore, miR-425 mimic reduced Nppa (Veh: 1.0±0.03, mimic: 0.7±0.02*) and Nppb levels (Veh: 1.0±0.03, mimic: 0.5±0.01*). We showed that a top predicted target of miR-425, CFL2, was also reduced by the mimic. Further, NP mRNA was elevated by CFL2 overexpression and reduced by the knockdown of CFL2. In humans, MiR-425 was lower in HTN and AHF patients (NM: 0.15±0.03, HTN: 0.03±0.005*, HF: 0.01±0.003*) than NM. MiR-425 negatively correlates with NT-proBNP in NM and AHF cohorts. Downregulation of miR-425 was validated in a second AHF cohort. Similarly, in HF LV tissues, miR-425 (NM: 2.5±0.5, HF: 1.0±0.5*) was lower than NM group, while CFL2 and NP gene and protein expression were increased. Conclusion: In vitro, cardiac stress hormones downregulate miR-425 and increase NP gene expression, potentially through a novel target, CFL2. In CVD patients, miR-425 is lower, accompanied by an elevation of CFL2 and NP. Our findings implicate the pathway of stress hormones/miR-425/CFL2/NP production in CVD.

Introduction: Atrial natriuretic peptide (ANP) is the most potent endogenous activator of the guanylyl cyclase A receptor (GC-A) mediating cardiorenal protective actions by increasing cGMP production. To test the hypothesis that acute decompensated heart failure (ADHF) is associated with increased circulating ANP but with reduced GC-A potency, we developed an ex-vivo precision medicine assay to quantify the potency of circulating ANP in plasma from healthy and ADHF subjects. Further, we assessed the ex-vivo potency of MANP, a novel ANP analog with greater cGMP activation than ANP, also using plasma of healthy and ADHF subjects. Methods: For the potency assay, we engineered HEK293 cells to overexpress human GC-A. Plasma from individual healthy (n=4) and ADHF (n=4) subjects was incubated in the assay and cGMP was assessed. The endogenous ANP derived cGMP was evaluated against the cGMP response of equimolar synthetic ANP (S ANP ) to assess potency of ANP from each cohort. MANP mediated cGMP generation was also assessed in the assay using healthy and ADHF plasma. Results: Healthy plasma, in which plasma ANP was 26±5 pg/mL, generated 10.3±0.7 pmol/mL of cGMP. S ANP added to the assay to mimic endogenous ANP levels in healthy subjects produced 7.8±0.5 pmol/mL of cGMP. ADHF plasma with markedly elevated ANP (350±57pg/mL) generated 23.5±3.1 pmol/mL of cGMP. S ANP mimicking plasma ANP in ADHF produced 59.7±13.8 pmol/mL of cGMP, which was greater than cGMP generated from ADHF patient-derived plasma (p<0.05), consistent with reduced potency of endogenous ANP in ADHF. Low treatment dose of MANP (LD MANP ; 10 -8 M) in healthy plasma increased cGMP to 45±5.5 pmol/ml while high treatment dose (HD MANP ; 10 -6 M) increased cGMP to 167.1±14.1 pmol/mL. In markedly elevated ANP ADHF plasma, LD MANP increased cGMP to 60.4±9.7 pmol/mL while HD MANP increased cGMP to 200.8±5.4 pmol/mL (p<0.03 for both) demonstrating similar cGMP production by MANP in healthy and ADHF plasma. Conclusions: Employing a novel ex-vivo precision medicine ANP/GC-A/cGMP assay, we conclude that ADHF is characterized by elevated ANP yet with impaired potency in GC-A activation and cGMP generation. MANP overcomes this impairment and serves as a potential efficacious GC-A activating therapeutic in heart failure.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII–NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Introduction: Therapeutic strategies dual-targeting renin-angiotensin system (RAS) and natriuretic peptide system (NPS), such as Sacubitril/Valsartan, have been proven promising in treating cardiorenal disease (CRD). However, mechanisms behind the crosstalk between RAS and NPS remain to be fully elucidated. While angiotensin II (ANGII), the central RAS hormone, is thought to interact with NPS at multiple levels, whether and how it may influence the actions of particulate guanylyl cyclase A (pGC-A), the receptor activated by ANP and BNP to increase its second messenger cGMP, has not been investigated. Hypothesis: We hypothesize ANGII naturally suppresses pGC-A activity and inhibition of this ANGII mediated action can be a new strategy to combat CRD. Methods: Studies consist of biomarker analyses in plasma from healthy humans, drug infusions in normal rats, molecular characterization in human HEK293 cells, and in vitro assessment of CRRL290, an innovative dual-functional peptide designed to activate pGC-A and inhibit protein kinase C (PKC). Results: In 128 healthy humans, plasma cGMP was positively associated with ANP or BNP in low (≤ 4.5 pg/mL) but not those with high (> 4.5 pg/mL) ANGII levels. Infusion of ANP (300 pmol/kg/min) in normal rats increased plasma and urinary cGMP, however ANP mediated cGMP generation was reduced in the presence of ANGII. Interestingly, suppression effect of ANGII on pGC-A was only recapitulated in HEK293 cell overexpressing both pGC-A and AT1R, but not in HEK293 cell overexpressing pGC-A alone. We also found PKC, a known downstream target of AT1R, was a key mediator as the suppression effect of ANGII was largely ablated by Go6983, a pan PKC inhibitor. Accordingly, we designed and synthesized CRRL290. Notably, CRRL290 had superior potency in cGMP generation compared to the currently best-in-class ANP-analog, MANP, in HEK293 cells. Conclusions: We demonstrate that ANGII is a natural suppressor of pGC-A via AT1R and PKC signaling. We also report for the first time that CRRL290, a novel bioengineered peptide that activates pGC-A receptor while inhibiting PKC signaling, potently generates cGMP. Our study provides new insights into the RAS-NPS interaction, and highlights a novel and mechanism-based therapeutic avenue for treating CRD.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones secreted as compensatory measures to preserve cardiorenal homeostasis. Treating heart failure (HF) with sacubitril/valsartan increased ANP and not BNP, suggesting the ANP system may confer therapeutic benefit. Thus, the presence of an ANP deficient subgroup of HF patients possibly posing higher risk for HF severity, underscores a need to characterize both, the biologically active and inactive (NT-proANP and NT-proBNP) natriuretic peptides (NPs)(latter resistant to degradation), to highlight the extent of NP production in HF. Characterizing neprilysin (NEP) activity may also provide insights into the degree of NP degradation in HF. This study aims to both highlight the cardiac NP response in acute decompensated HF(ADHF) by measuring molecular forms of ANP and BNP together with defining circulating soluble NEP activity as to assess mechanisms impairing the NP response in HF. We recruited 47 healthy individuals with neither cardiovascular nor metabolic disease and compared ANP, BNP, NT-proANP, NT-proBNP, and NEP activity levels with 107 recruited ADHF patients. In ADHF, compared to a healthy setting, median ANP, BNP, NT-proANP, and NT-proBNP were all elevated (15.7 vs. 118.1 pg/mL , p<0.0001; 23 vs. 515 pg/ml, p<0.0001; 60.1 vs. 651.6 pg/mL, p<0.0001; 46 vs. 3330 pg/mL, p<0.0001, respectively). However, circulating NEP activity was significantly lower in ADHF than in healthy circulation (32 vs. 12.3 nM/mL/min, p<0.0001). NPs did not correlate with NEP activity in healthy circulation. However, only ANP (r = 0.4788, p<0.0001) and NT-proANP (r = 0.6819, p<0.0001) correlated with NEP activity in ADHF. The presence of an impaired ANP response in ADHF, underscored by insufficient increases in NT-proANP than NT-proBNP, suggests decreased ANP production may induce not only an ANP deficient ADHF state, but be characteristic of ADHF. The presence of NEP activity in ADHF circulation, despite lower NEP activity compared to healthy individuals, suggests ANP degradation may second impaired production, further lowering ADHF ANP levels. Our studies support augmenting the ANP system in ADHF through either endogenous stimulation or exogenous supplementation as optimal therapeutic strategies.