Chen, Horng H.

Introduction: Insulin-like growth factor-binding protein 7 (IGFBP7) was previously found to be associated with diastolic function in heart failure (HF) with reduced ejection fraction (EF), but it is unclear whether such associations exist in HF with preserved EF (HFpEF), where diastolic function is of pivotal importance. Hypotheses: Baseline (BL) and change (Δ) in IGFBP7 will be significantly associated and correlated with BL and Δ in diastolic indices as well as peak oxygen consumption (VO2 max ) in patients with HFpEF. IGFBP7 will decrease with sildenafil therapy. Methods: At BL and 6 months (6M), IGFBP7, imaging studies, and VO2 max were obtained and compared in 160 HFpEF patients randomized to sildenafil or placebo. Results: Patients with supramedian BL IGFBP7 concentrations (>219 ng/mL) were more likely to be older, to have more congestion and worse renal function, and to have elevated concentrations of several emerging or established HF biomarkers including amino-terminal pro-B-type natriuretic peptide, cystatin C, and highly sensitive troponin (all P<0.05). Higher BL IGFBP7 was correlated with indices of worse diastolic function, including higher E velocity (ρ=0.395, P<0.001), E/e’ (ρ=0.398, P<0.001), left atrial volume index (ρ=0.386, P<0.001), right ventricular systolic pressure (RVSP; ρ=0.413, P<0.001), and transmitral E/A (ρ=0.258, P=0.006). IGFBP7 was also inversely correlated with aortic distensability (ρ= -0.301, P=0.02). There was no association or correlation with measures of systolic function. Notably, in analyses adjusted for respective baseline variables, ΔIGFBP7 was significantly correlated with Δ in E (ρ=0.114), E/A (ρ=0.225), E/e’ (ρ=0.174), and RVSP (ρ=0.282; all P<0.03). Elevated BL IGFBP7 was associated with lower BL VO2 max (13.2 versus 11.1 mL/min/kg; P<0.001) and ΔIGFBP7 was inversely correlated with ΔVO2 max (ρ=-0.19, P=0.01). Subjects receiving sildenafil had a decrease in IGFBP7 over 6M in contrast to placebo-treated patients (median ΔIGFBP7 -1.5 versus +13.6 ng/mL; P<0.001). Conclusions: In patients with HFpEF, concentrations of IGFBP7 have unique relationships with measures of diastolic dysfunction and VO2 max . Rise in IGFBP7 may reflect worsening diastology and deteriorating VO2 max .

Introduction: In vitro, ST2 is a marker of myocyte stress and inflammation but in vivo, there is no trans-cardiac ST2 concentration step-up to support cardiac ST2 activation in human heart failure (HF) calling into question the source of ST2 in HF. While ST2 is predictive of adverse outcomes in HF, the relationship between ST2 and key pathophysiologic domains in HF with preserved ejection fraction (HFpEF) is not well characterized. Hypothesis: ST2 levels increase in proportion to the severity of left ventricular (LV) remodeling and dysfunction in HFpEF. Methods: At enrollment, patients (n=174) from the RELAX trial testing sildenafil in HFpEF had serum ST2 levels measured and underwent assessment of pathophysiologic domains including congestion, cardiac structure and function, exercise performance (peak oxygen consumption (VO2)) and biomarkers of neurohumoral activation (NT-proBNP, aldosterone, endothelin-1), renal dysfunction (cystatin-C), fibrosis (pro-collagen III N-terminal peptide [NTP], C-telopeptide for type I collagen [CITP]), inflammation (high-sensitivity CRP [hsCRP]) and myocardial necrosis (high-sensitivity troponin-I [hsTnI]). Analysis adjusted for sex, a covariate associated with ST2 levels in normal persons. Results: See table. ST2 was associated with greater congestion, worse right ventricular (RV) pressure overload and dysfunction, lower peak VO2 and biomarkers of neurohumoral activation, renal dysfunction, fibrosis and myocardial necrosis and tended to be associated with systemic inflammation (hsCRP). ST2 was not associated with LV structure or LV systolic or diastolic function. Conclusions: In HFpEF, ST2 was associated with measures of right sided congestion, RV overload and dysfunction, exercise intolerance and other systemic markers of more advanced HF but not with LV structure or LV systolic or diastolic function. These data suggest that mechanisms distinct from LV stress stimulate ST2 activation in HFpEF.

Introduction: While left ventricular assist devise (LVAD) support improves the prognosis of end-stage heart failure (HF) patients, the long-term outcomes remains suboptimal. Improved outcomes may occur with organ protection including reverse myocardial remodeling. Cenderitide (CDNP) is a Mayo engineered designer natriuretic peptide (NP) that co-activates particulate guanylyl cyclase A and B receptors and functions via the second messenger cGMP. We reported a NP deficient state in the heart with LVAD support which lays the rationale for NP replacement therapy with Cenderitide which has cardiorenal protective actions in experimental HF. We sought to determine if Cenderitide co-therapy would be safe and activate cGMP in patients with LVAD support. Hypothesis: Acute single dose of subcutaneous (SQ) Cenderitide will be safe and activate cGMP in patients with LVAD support. Methods: Stable HF patients with LVAD support (Heartmate II, all males) were enrolled in a cross-over design comparing SQ placebo and low (Low-C, 5 ug/kg, n=8) or high dose (High-C, 10 ug/kg, n=8) Cenderitide (Capricor Therapeutics) administration. Vital signs, plasma C(D)NP, cGMP, Aldosterone (Aldo), and NT-proBNP levels were measured before or after drug administration over 24 hours. All parameters in Cenderitide group were adjusted by those in placebo. Results: Both Low-C and High-C group demonstrated significantly increased plasma C(D)NP and cGMP levels for 2 hours after drug injection and those in High-C group showed more prolonged and higher increases than Low-C group. Mean arterial pressure (MAP) in Low-C did not show any changes but in High-C group tended to be lower for 18 hours but this was not significant. Plasma Aldo and NT-proBNP levels in Low-C group did not show any change but those in High-C group tended to be lower after drug injection s similar to the change in MAP. No adverse events were observed in the study. Conclusions: SQ Cenderitide was successfully absorbed, activated cGMP and well tolerated in patients with LVAD support. High dose Cenderitide may have cardiac unloading effects suggested by decreases in NT-proBNP. Further clinical trial for efficacy is warranted to clarify cardiorenal protective effects of Cenderitide in the LVAD population to improve outcomes.

Introduction: As importantly underscored by the SPRINT Trial, innovative blood pressure (BP) lowering and multiorgan protective drugs are needed for treating hypertension (HTN). We engineered a Mayo Clinic designer atrial natriuretic peptide (ANP)-based drug, ZD100, which possesses a unique 12 amino acid (AA) carboxy-terminus (CT) extension while maintaining the N-terminus (NT) of ANP. ZD100 is more potent than ANP in reducing BP, inducing natriuresis and inhibiting aldosterone via pGC-A and its second messenger cGMP. To date, the in vivo actions of ZD100 in a model of essential HTN are undefined. Further, in attempt to enhance and/or minimize pGC-A activation, the importance of specific AAs in the CT and NT of ZD100 is unknown and is critical to define. Hypothesis: We hypothesize that ZD100 is a potent anti-HTN drug activating pGC-A/cGMP system and that specific AA variations in the CT and/or NT of ZD100 will modify the ability of activating pGC-A. Methods: Spontaneously hypertensive rats (SHRs) received intravenous (IV) ZD100 or vehicle and BP, plasma and urinary cGMP and the natriuretic effects were determined. We then engineered 15 strategic ZD100 variants (Vs) consisting of mutated peptides in the CT or NT or both. We stimulated HEK293 cells overexpressing pGC-A with ZD100 and the 15 Vs (10-8 M) and measured intracellular cGMP generation. Results: In SHRs, acute IV ZD100 reduced BP, increased plasma and urinary cGMP generation and induced natriuresis demonstrating efficacy in HTN. In vitro, we found two more potent pGC-A activators (+100% cGMP activation) only among the CT-only mutated ZD100 Vs. Importantly, key mutations of either the CT or NT resulted in 6 loss-of-function Vs identifying importance of peptide-receptor dependency. Conclusions: We demonstrate for the first time in a genetic model of HTN that ZD100 reduces BP, activates systemic and renal pGC-A/cGMP and induces natriuresis. Enhanced or reduced pGC-A activation with targeted AA mutations in the CT and NT of ZD100 provides key structure function insights into ZD100 which may lead to next generation ZD100 therapeutic peptides. These findings support ZD100 as a novel peptide therapeutic strategy for HTN and will help guide the selection of best-in-class ZD100 analogues.

Background: Left ventricle assist device (LVAD) support, a well established therapy in advanced CHF, improves renal function assessed by creatinine. While renal function in large numbers of continuous flow (CF) LVADs vs pulsatile flow (PF) LVADs has been evaluated at 3 mo., long term studies are needed with large numbers of CF vs PF devices. To date longer term analysis comparing renal function in CF vs PF devices have relied on a small number of CF devices. We evaluated renal function assessed by creatinine in a large number of CF (HM II) (n=119) devices and compared this to renal function in a group of subjects treated with PF devices (HM XVE) (n=8) at 6 mo. post implant. Our aim was to evaluate change in creatinine between 3 and 6 months in these groups. Methods and Results: Plasma creatinine was evaluated at 3 and 6 months in two groups: subjects with CF HM II (n=119) and PF LVAD (HM XVE, n=8). The data were collected from two centers in patients undergoing implant for destination or bridge to transplant therapy. Baseline creatinine was higher in the HM XVE group at baseline (BL), and decreased at 3 mo. (1.7 [1.1-2.1] vs 1.1 [0.8-1.5] mg/dL, p<0.05). BL creatine in the HM II group decreased at 3 mo as well (1.2 [1-1.5] vs 1.1 [0.9-1.3] mg/dL, p,0.05). Between 3 and 6 months there was no difference in degree of creatinine change in CF vs PF, however both groups had an increase in Creatinine in this time frame: PF (1.1 [0.8-1.5] vs 1.35 [1.05-1.9] mg/dL, p<0.05); CF (1.1 [0.9-1.3] vs 1.2 [0.92-1.4] mg/dL, p<0.05). Conclusion: This is the first study to demonstrate with large numbers of CF LVAD that creatinine does not incrementally increase between 3 and 6 mo. in comparison to PF LVAD. Further studies are required with large numbers of continuos flow LVADs evaluating the comparative effect of long term continuous flow vs pulsatile flow. Additionally, it remains important to identify surrogates of pulsatility in subjects with continuous flow devices and evaluate prognostic impact on renal function long term.

Background: B-type natriuretic peptide (BNP1–32) is a 32- amino acid cardiac peptide secreted in response to cardiac stress and plays an important compensatory role in cardiorenal homeostasis. Studies have recently reported that a mRNA encoding an alternatively spliced variant of BNP (ASBNP) exists in the human heart and like BNP1–32 is increased in the failing heart. While ASBNP and BNP1–32 have identical ring structures and amino termini, ASBNP and BNP1–32 differ in their carboxy terminus (34 vs. 6 amino acids, respectively). Studies have also reported that ASBNP activates cGMP, the second messenger of BNP1–32, in isolated glomeruli but not in endothelial cells. Also, unlike BNP1–32 ASBNP does not vasorelax isolated arteries or veins. The cardiorenal actions in vivo of ASBNP are unknown. We hypothesized that ASBNP in vivo would have selective renal actions yet as suggested by studies in isolated vessels would be devoid of blood pressure effects. Methods: We therefore determined for the first time the cardiorenal actions of three subsequent intravenous doses of ASBNP (10, 50, and 150 pmol/kg/min) in 5 healthy canines in an acute study under anesthesia in 30-minute clearances at baseline, during, and following infusion. Values provided are mean±SEM for baseline vs. 150 pmol/kg/min. *p<0.05. Results: ASBNP dose-dependently increased plasma levels of cGMP (8.6±1.0 vs. 11.5±1.3* pmol/mL), and the same was true for urinary cGMP excretion (878±191 vs. 2028±551* pmol/min). Mean arterial pressure (p=0.83) and heart rate (p=0.43) remained unchanged. Urine flow increased (0.38±0.05 vs. 1.32±0.20* mL/min) as did urinary sodium excretion (38±15 vs. 133±38* μ Eq/min). Renal blood flow (p=0.51) and glomerular filtration rate (p=0.99) remained unchanged. BNP-immunoreactivity as measured by the Biosite BNP assay increased (5±4 vs. 3807±573* pg/mL). Conclusion: ASBNP, an alternatively spliced form of BNP produced in the human heart, activates cGMP and has diuretic and natriuretic actions but is without blood pressure lowering actions. Thus, ASBNP represents a novel renal selective, non-hypotensive, natriuretic, and diuretic peptide.

Background: C-type natriuretic peptide (CNP) is of endothelial cell origin and represents the most potent anti-fibrotic peptide of the natriuretic peptide family and is important for extracellular matrix homeostasis including mediating bone growth in the young. Cardiorenal aging is associated with altered cardiac and renal structure and function that may contribute to the development of heart and kidney failure, particularly in the elderly. The impact of aging on circulating CNP and associated changes in cardiorenal structure and function are undefined. Hypothesis: We hypothesized that a decrease in endogenous plasma CNP occurs with aging and is associated with an increase in cardiorenal fibrosis and altered left ventricular (LV) and renal structure and function. Methods: Studies were performed in 2, 11 and 20 month old male Fischer rats (n = 8 –10/group). Echo was used to assess LV structure and function. Blood pressure (BP), urinary protein excretion, plasma B-type natriuretic peptide (BNP) and CNP were measured. Kidneys and LV were harvested for gross and histology analysis. Mean±SE, *p<0.05. Results: Aging from 2 to 20 months was associated with progressive reductions in plasma CNP (30±3 to 21±1* to 9±1*pg/ml). Significant and progressive cardiac and renal fibrosis were observed with aging (LV: 9±0.3 to 15±0.7* to 21±1*% and kidney: 1±0.3 to 8±2* to 17±1* %). In contrast to CNP, plasma BNP was modestly increased from the 2 to 20 month old group (21±2 to 26±1*pg/ml). The reduction in CNP and increase in BNP, the latter a biomarker for LV dysfunction, were associated with decreased EF (88±1 to 80±1*%) and increased LVEDd (6.61±0.09 to 7.48±0.09*mm). BP and urinary protein excretion were mildly elevated with aging (91±1 to 101±3* mmHg and 8±0.3 to 20±3* mg/day, respectively). Conclusion: We report for the first time that aging is associated with a progressive decline in circulating CNP and progressive cardiorenal fibrosis, systolic dysfunction and proteinuria. Further studies are warranted to explore the hypothesis that a mechanism of cardiorenal aging with altered cardiorenal structure and function may include a decrease in the bioavailability of CNP.

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.

Abstract The discovery of the heart as an endocrine organ resulted in a remarkable recognition of the natriuretic peptide system (NPS). Specifically, research has established the production of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) from the heart, which exert pleiotropic cardiovascular, endocrine, renal, and metabolic actions via the particulate guanylyl cyclase A receptor (GC-A) and the second messenger, cGMP. C-type natriuretic peptide (CNP) is produced in the endothelium and kidney and mediates important protective auto/paracrine actions via GC-B and cGMP. These actions, in part, participate in the efficacy of sacubitril/valsartan in heart failure (HF) due to the augmentation of the NPS. Here, we will review important insights into the biology of the NPS, the role of precision medicine, and focus on the phenotypes of human genetic variants of ANP and BNP in the general population and the relevance to HF. We will also provide an update of the existence of NP deficiency states, including in HF, which provide the rationale for further therapeutics for the NPS. Finally, we will review the field of peptide engineering and the development of novel designer NPs for the treatment of HF. Notably, the recent discovery of a first-in-class small molecule GC-A enhancer, which is orally deliverable, will be highlighted. These innovative designer NPs and small molecule possess enhanced and novel properties for the treatment of HF and cardiovascular diseases.