Burnett, John C.

Introduction: ANP and BNP are cardiac hormones that have a natriuretic and diuretic role in heart failure (HF) through guanylyl cyclase receptor A (GC-A) and cGMP generation. C-type natriuretic peptide (CNP) is part of this hormone family and is produced in the heart and endothelium. The biologically active form, CNP-22, is known to have potent anti-remodeling properties through the activation of GC-B and cGMP, but lacks natriuretic actions. Recently we have identified that another larger CNP form, CNP-53, is elevated in the plasma of acute decompensated HF patients, however it unclear if CNP-53 possesses biological actions. Hypothesis: We hypothesized that CNP-53 would be biologically active in vivo and in vitro, and would stimulate GC-B specific cGMP production. Methods: HEK 293 cells over-expressing GC-A and GC-B and human cardiac fibroblasts (hCFs), in which GC-B are in abundance, were stimulated with CNP-53 for 10 minutes at a dose of (10-8M). Two groups of anesthetized rats (n=8) received a 75-minute infusion of normal saline vehicle (V) or CNP-53 (1 ug/kg/min) and then mean arterial pressure (MAP), sodium excretion and urinary and plasma cGMP were assessed. Results: CNP-53 significantly activated cGMP in GC-B HEK cells (75±9 vs. 0.4±0.1 pmol/well, p<0.05) and in hCFs (0.17±0.03 vs. 0.00±0.00 pmol/well, p<0.05) compared to no treatment. In contrast CNP-53 failed to generate cGMP in GC-A HEK cells. In vivo, CNP-53 decreased MAP compared to vehicle (V: 101±2, CNP-53: 94±2 mmHg, p<0.05). CNP-53 had a greater natriuretic effect compared vehicle (V: 0.86±0.11, CNP-53: 1.39±0.21 uEq/min, p<0.05). Urinary cGMP excretion (V: 33±6, CNP-53: 177±28 pmol/min, p<0.05) and plasma cGMP generation was also significantly higher with CNP-53 compared to vehicle (V: 9±2, CNP-53: 91±3 pmol/ml, p<0.05). Conclusions: CNP-53, a higher molecular form of CNP that was reported to be increased human HF plasma, is bioactive with GC-B specific cGMP generation in vitro. Moreover, infusion of CNP-53 generates significant urinary and plasma cGMP and has natriuretic actions with minimal hypotensive effects in vivo. This study advances our understanding of CNP biology and provides new insights into potential CNP therapeutic opportunities targeting the progression of HF.

Introduction: The cardiac atrium is an important endocrine organ which also releases natriuretic peptides (NPs). We reported that dysregulation of the proNP convertases, furin and corin, may disrupt NP progressing, causing atrial fibrosis in experimental heart failure (HF). Atrial fibrosis results in atrial arrhythmias and impairs the prognosis of human HF, therefore understanding mechanisms of atrial fibrosis may help in designing treatment strategies for HF. We sought to extend our studies into the entire NP system and global gene profiles including fibrotic and inflammatory pathways in experimental HF. Hypothesis: Components of the NP, pro-fibrotic and inflammatory system will be up-regulated in the atrium in HF. Methods: Experimental canine HF was induced by rapid right ventricular pacing at 240 bpm for 10 days. We examined hemodynamics, echo parameters, circulating neurohumoral factors, and global gene profiles using RT-PCR microarrays related to the NP system, fibrosis, growth factors, inflammation and cardiac hypertrophy in left atrium (LA) from normal and HF canines (n=4 of each). Results: Experimental HF was characterized by decreased % EF and LV radial strain, and increased circulating ANP, cGMP and aldosterone levels. HF LA weight significantly increased with fibrosis and interstitial edema compared to normals. Gene profile analyses showed increases in profibrotic cytokines and extracellular matrix related genes in LA. Increased ANP expression but also increased ANP degrading enzyme IDE and decreased proANP processing enzyme corin were also observed. Genes related to inflammatory cytokines such as TNF-alpha, members of the IL-6 family, MCP-1, and cell adhesion molecules were up-regulated in LA compared to normals. Conclusions: Experimental HF was characterized by on-going atrial fibrosis with increased circulating ANP levels. Although ANP gene expression increased in failing LA, impaired proANP processing and accelerated ANP degradation may impair any anti-fibrotic ANP effects. Further, atrium may be a source of inflammatory cytokines and pro-fibrotic growth factors which may advance organ damage in HF. Our studies suggest NP treatment, perhaps in a form less susceptible to IDE degradation, may be a novel therapeutic for HF.

Introduction: ProANP is a 108 amino acid (AA) prohormone which is produced in the atria and processed to the mature 28 AA ANP which activates the guanylyl cyclase receptor-A (GC-A) and its second messenger cGMP. ProANP is found in the human circulation but its bioavailability is undefined. Hypothesis: ProANP represents a novel cardiac peptide with more sustained natriuretic actions than either carperitide (ANP) or nesiritide (BNP). ProANP itself can activate GC-A in vitro and the proANP can be processed in human serum from normals and in patients with heart failure (HF). Methods: We examined the in vivo cardiorenal actions of proANP compared to ANP, BNP, or placebo in normal canines (667 pmol/kg, n=5/group). We determined cGMP activation of proANP, ANP and BNP in GC-A expressing HEK293 cells. ProANP processing and degradation was also defined in serum from normal subjects (n=13) and patients with HF (n=14) ex vivo. Results: ProANP had significantly greater diuretic and natriuretic properties with increased renal blood flow and sustained renal tubular actions compared to ANP or BNP in canines. ProANP also resulted in greater and more prolonged cardiac unloading than ANP, and much less hypotensive effects than BNP. The plasma half-life of proANP was 4.5 times longer than ANP and 3.8 times longer than BNP. ProANP itself stimulated cGMP generation in GC-A expressing HEK cells which was equal to ANP. ProANP was processed to ANP in fresh serum from normals as well as HF patients ex vivo and the processed peptide activated cGMP in GC-A expressing HEK cells. Conclusions: ProANP represents a novel GC-A activator with a longer half-life and greater natriuresis and diuresis than ANP and BNP and less hypotensive effects than BNP. The bioactivity of exogenous proANP may be through direct stimulation of GC-A, as well as through processing into ANP, as proANP is processed in the circulation of both normal and HF. As proANP circulates in the human circulation, proANP may represent a key circulating hormone in cardiorenal and blood pressure homeostasis as well as a potential innovative therapeutic beyond ANP (carperitide) or BNP (nesiritide) for cardiorenal diseases including HF.

Introduction: Plasma aldosterone (Aldo) increases Na and water reabsorption and mediates inflammation. Our recent findings in a general population cohort showed Aldo strongly associated with hypertension (HTN), obesity, chronic kidney disease (CKD) and lower natriuretic peptide levels (NPs) during a first visit (V1), even when Aldo was within the normal range. It is undefined whether Aldo itself (V1) and/or its increase after 4-year (second visit, V2) can predict new onset cardiovascular and metabolic diseases. Hypotheses: We hypothesized that: 1) Aldo-V1 predicts new onset disease at V2; 2) The increase of Aldo between V1 and V2 predicts new onset disease-V2; and 3) Aldo-V2 remains associated with cardiovascular and metabolic disease-V2. Methods: We defined the correlations between Aldo-V1 and Aldo-V2 with cardiometabolic diseases and medications at V2, in a general community cohort (N=1368) from Olmsted County, MN. Associations were adjusted for age, sex and BMI; Aldo was analyzed as continuous variable and according to tertiles. Results: After 4 years, Aldo-V1 continuous variable predicted new onset of central obesity (p=0.0113, OR=1.36, CI=1.07-1.73), HTN (p=0.0012, OR=1.36, CI=1.13-1.63) and use of antilipemic drugs (p=0.0119, OR=1.25, CI=1.05-1.48); while Aldo-V1 in the 3 rd tertile predicted new onset of type 2 diabetes (T2DM) (p=0.0392, OR=1.96, CI=1.03-3.70), use of antilipemic therapy (p=0.0162, OR=1.59, CI=1.09-2.31) and HTN (p=0.049, OR1.44, CI=1.00-2.08) at V2. Secondly, we observed that the increase in Aldo between V1 and V2 predicted new HTN-V2. Finally, we found higher Aldo-V2, but still within the normal range and Aldo-V2 remained associated with HTN, obesity and CKD, when analyzed as continuous variable and when in the 3 rd tertile. Conclusions: In the general community, Aldo-V1 predicts new onset HTN, central obesity, T2DM and use of antilipemic therapy. Importantly, the increase of aldosterone between V1 and V2 also predicts new onset HTN. Finally, Aldo-V2 correlates with HTN, obesity and CKD as we found at V1. Further studies are warranted to evaluate aldosterone as therapeutic target to delay disease progression, and to support its role as prognostic biomarker to identify high-risk subjects.

Background CD‐NP is a novel chimeric natriuretic peptide (NP) consisting of the full‐length human C‐type natriuretic peptide (CNP), a NP receptor (NPR)‐B agonist, and the C‐terminus of Dendroaspis NP (DNP), a NPR‐A agonist. In the first‐in‐human clinical trial, CD‐NP activated plasma and urinary cGMP, the second messenger for the NPs, and increased Na + excretion in normal subjects. Here we report for the first time plasma aldosterone (ALDO) and angiotensin II (ANG II) levels. Methods In the double‐blind, PLB‐controlled phase of this trial, adults received CD‐NP 17.5 ng/kg/min (n = 6) or PLB (n = 4) i.v. for 4 hours. Plasma ALDO and ANG II were quantified by RIA. Data were analyzed by 2‐tailed paired t‐test and signed‐rank (S‐R) test (SAS v9). Results In subjects who received CD‐NP, plasma ALDO (mean±SD) decreased from a baseline of 21.9±2.7 to 9.5±3.2 ng/dL (P<0.001 t‐test, P<0.05 S‐R test) at the end of infusion. Plasma ANG II tended to decrease (29.2±5.2 to 23.0±3.5 ng/L, P=0.086 t‐test, P=0.69 S‐R test). In the PLB group, ALDO (20.3±4.2 to 13.6±4.5 ng/dL) and ANG II (23.3±2.2 to 19.8±8.2 ng/L) did not change. Conclusion CD‐NP suppressed plasma ALDO in normal subjects, consistent with NPR‐A activation by a novel NP with dual NPR‐A and NPR‐B activating properties. CD‐NP may be of therapeutic potential in disease states that are characterized by ALDO activation, such as in heart failure warranting further studies. Sources of funding: Mayo Foundation and Nile Therapeutics, Inc.

Decreased renal function is associated with increased CV morbidity and mortality. The changes mild renal impairment may cause structurally/molecularly in the heart remain unclear. We hypothesized that mild renal dysfunction produced by unilateral nephrectomy(UNX)results in changes in ventricular structure and gene expression in the absence of hypertension or vol overload. Wistar rats were used[sham (S; n=10) and UNX; n=9]. At 4 weeks, GFR was determined by inulin clearance and RBF by PAH. Hearts and kidneys were harvested for histological analysis. Cardiac function assessed by echo. Genomewide microarray analysis was performed on LV myocardium (Affymetrix). Glomerular hypertrophy was observed in UNX. GFR tended to decrease with a reduction in RBF (S:8±1, Nx:5±1 ml/min, p<0.005). Blood pressure (BP), Na and water excretions were maintained without activation of PRA, aldo or BNP. LVEF and LV diameters were normal. Microarray analysis of LV revealed that 278 genes changed with UNX (1.5 fold, P<0.05) related to cell growth, bone remodeling and muscle contraction (Z value>2). Importantly, Pic Red staining in the LV of UNX revealed greater fibrosis (S:2±0.1, Nx:4±0.4 %, p<0.001). We conclude that mild renal insufficiency initiates myocardial gene responses and fibrosis independent of alterations in the circulating RAAS, BNP, BP, vol overload or systolic dysfunction.

Studies in human myocardial infarction (MI) suggest that even in the absence of heart failure (HF) alterations in renal function may occur and contribute to poor outcomes. We hypothesized that even without preexisting renal disease, renal functional, structural and molecular changes would be present following MI. Wistar rats were used, Sham(S;n=10) and MI(n=9). 3 weeks after MI, GFR was determined by inulin clearance. Blood was obtained for PRA and aldosterone. Cardiac function was assessed by echo. Genomewide microarray analysis was performed on kidney cortex (KC) and medulla (KM) (Affymetrix). EF decreased after MI (S:63±2, MI:43±6%, p<0.01) and LVEDd increased. PRA, aldosterone and blood pressure (BP) did not change. There was no HF as Na and water excretion was maintained. GFR tended to decrease. Pic Red staining in the KC and KM after MI showed greater fibrosis (KC S:1±0.2, MI:3±0.6%, p<0.001 and KM S:1±0.2, MI:19±6%, p<0.005). Microarray analysis showed that 303 genes changed in KM and 407 genes in KC after MI (1.5 fold, P<0.05) related to structure morphogenesis and metabolic processes(Z value>2). We conclude that experimental MI results in renal structural remodeling characterized by cortical and medullary fibrosis with a mild reduction in GFR and extensive modulation of molecular pathways related to renal growth and morphogenesis underscoring a heart‐kidney connection after MI.

Background CBB‐NP is a novel fully humanized designer natriuretic peptide (NP) integrating the 17‐amino‐acid ring of human C‐type NP (CNP) with the N‐ and the C‐termini of human B‐type NP (BNP). CBB‐NP exerted natriuretic, diuretic, renal‐preserving and cardiac‐unloading actions in anesthetized dogs. In this investigation, we compared for the first time the cGMP stimulating actions of CBB‐NP vs human atrial NP (ANP), BNP and CNP in human aortic endothelial cells (HAEC). Methods CBB‐NP was synthesized (SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH with a disulfide bond joining the C residues). HAEC were incubated with ANP, BNP, CNP or CBB‐NP for 10 min (10 −6 M). Cyclic GMP was quantified by RIA. Results CBB‐NP and CNP increased cGMP (pmol/mL) vs control (0.108±0.020 and 0.174±0.036 vs 0.001±0.001, P<0.01 and <0.001, respectively). CBB‐NP elicited a greater cGMP response vs ANP (0.027±0.002, P<0.05), whereas comparable responses were observed vs BNP and CNP. When the native NPs were compared, a greater cGMP stimulating response was detected with CNP vs ANP and BNP (P<0.001 and <0.01, respectively). Conclusions The novel designer NP, CBB‐NP, stimulates cGMP in human aortic endothelial cells to a comparable extent as compared to the native BNP and CNP. Moreover, its cGMP stimulating action is greater than that of ANP. The therapeutic potential of CBB‐NP in models of cardiovascular diseases warrants further investigation. Supported by NIH, HFSA and ASCPT

Background The 17‐amino‐acid (AA) ring of C‐type natriuretic peptide (CNP) is essential for activation of NP receptor (NPR)‐B and the 2 nd messenger cGMP. CNP induces minimal hypotension as NPR‐B is more abundantly found in veins vs arteries, but possesses limited renal properties. We designed a novel peptide fusing the CNP ring with AAs from ANP and BNP to test our hypothesis that this unique peptide, CAB‐NP, would exert enhanced renal actions without inducing excessive hypotension. Methods CAB‐NP was synthesized (SLRRSSCFGLKLDRIGSMSGLGCKVLRRH) and infused (21 pmol/kg/min iv) in 5 anesthetized dogs. GFR was assessed by inulin clearance. Mean±SE, P<.05*, <.01 † , BL vs 30 min infusion. Results CAB‐NP increased plasma cGMP (3.5±0.6 to 10.9±1 † pmol/mL), urinary cGMP excretion (980±96 to 4587±825* pmol/min), net renal cGMP generation (823±88 to 4091±824* pmol/min), urine flow (0.2±.03 to 1.2±.4* mL/min) and Na + excretion (19±7 to 191±55* †Eq/min). Proximal (85±3 to 68±1 † %) and distal (98±1 to 91±2 † %) FRNa were reduced; GFR was preserved (46±4 to 45±3 mL/min). MAP mildly decreased (121±4 to 111±5* mmHg). PCWP (6±1 to 4±1 † mmHg) and RAP (3±1 to 2±1* mmHg) were reduced. Plasma renin activity (11±1 to 2±0.3 † ng/mL/hr) and aldosterone (15±4 to 7±2* ng/dL) were suppressed. Conclusions CAB‐NP exerts favorable in vivo actions without excessively lowering BP. The therapeutic potential of CAB‐NP warrants further studies. Supported by the NIH, HFSA, and ASCPT.

Decreased renal function is associated with increased CV morbidity and mortality. However, the impact of mild renal dysfunction upon the heart remains to be fully elucidated. It is thought that a mild reduction in renal mass is not deleterious to the heart, yet exploration of the impact of unilateral nephrectomy (Nx) on myocardial gene responses is undefined. We hypothesized that even when ventricular function and structure are grossly preserved after Nx, such a reduction in renal mass and function would result in significant alterations in the cardiac genome. Wistar rats, Sham(S)(n=10), Nx(n=9). 4 weeks after Nx we assessed proteinuria, UNaV, UcGMP and BNP. Cardiac function was assessed by echo. GFR was determined by inulin clearance and renal blood flow (RBF) by PAH. Blood was obtained for BNP, aldosterone and cGMP. Kidneys and hearts were harvested. Gene microarrays were conducted of left ventricle (LV) myocardium (Affymetrix GeneChip® Rat Genome 230 2.0). GFR strongly tended to decrease and RBF was decreased in Nx (S:8±1, Nx:5±1 ml/min, p<0.005). Glomerular volume was increased in Nx (S:1±0.04, Nx:1.6±0.1 u 3 ×10 6 , p<0.001) while UNaV and volume were not different. There was no proteinuria in Nx. EF, LV end systolic and diastolic diameters were normal. Blood pressure (BP) was unchanged. There was no activation of the systemic RAAS or BNP. Importantly, 278 cardiac genes changed their expression after Nx (1.5 fold, P<0.05). 11% were increased and 89% decreased. Pathways involved included cell adhesion, growth and proliferation. We conclude that Nx, independently of alterations in circulating RAAS or BNP, BP or myocardial structure and function, results in widespread changes in myocardial gene expression. Further long‐term studies are needed to define the impact of these early gene responses to myocardial structure and function.