Coronary Sinus Neuropeptide Y Levels and Adverse Outcomes in Patients With Stable Chronic Heart Failure

Key Points Question Is the adrenergic cotransmitter neuropeptide Y (NPY) associated with outcomes in patients with stable heart failure (HF)? Findings In a cohort of patients with stable HF undergoing cardiac resynchronization therapy device implantation, coronary sinus blood was sampled for NPY levels. A threshold level of NPY was identified, which was associated with death, heart transplant, and ventricular assist device placement; molecular studies on human sympathetic neurons indicated increased release of NPY in HF patients. Meaning Using NPY, hyperadrenergic activation associated with adverse outcomes may be identifiable in patients with stable HF.

of death.

Stellate ganglia immunohistochemistry
The study was approved by the UCLA institutional review board and written informed consent by the patient or appropriate designee obtained. Stellate ganglia from controls (organ donors at the time of heart/lung organ procurement, n=4) with structurally normal hearts, and CHF patients undergoing cardiac sympathetic denervation (the resection of the lower half of both SG, and second through fourth paravertebral ganglia, n=13) were collected, fixed in formalin, and paraffin embedded. Sections 5μm thick were taken for immunostaining with antiserum to NPY (ab112473; 1:2000 dilution). Diaminobenzidine reaction (Life Technologies,) was used for detection. Slides were scanned (Scanscope, Aperios Systems) for digital analysis (Tissue Studio, Definiens Inc.).

Stellate ganglia quantitative polymerase chain reaction
Stellate ganglia samples were collected from patients with CHF undergoing cardiac sympathetic denervation (n=4 from 2 patients), or from organ donors (n=6 from 3 patients) with structurally normal hearts at the time of organ procurement. Two technical repeats were performed per patient (one form each stellate ganglia). Total RNA from human tissue was extracted using an RNeasy Protect mini kit (Qiagen). For reverse transcription, first-strand cDNA was synthesized from 1 μg of total RNA with the iScript™ cDNA Synthesis kit (Biorad). Quantitative polymerase chain reaction was conducted in a total of 20 µl containing 10 µl of Taqman Universal PCR Master mix (Applied Biosystems), 4 µl of cDNA (10ng/µl), 1 µl of 20X specific primers for Taqman Gene Expression Assays (Hs00173470_m1 for human NPY, Quantitative real-time RT-PCR was performed in a 96-well clear optical reaction plate (Applied Biosystems), and thermal cycling conditions were: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Results were analyzed with the ABI Prism 7000 Sequence Detection System software (Applied Biosystems). Gene expression was normalized to GAPDH.

Statistical analysis Bivariate relations of NPY with clinical variables
Continuous variables are reported as mean ± standard deviation (SD) or median with interquartile range if not normally distributed, and nominal variables as frequency or percentages. The p values for mean NPY comparisons across categorical or two group (binary) predictors were computed using the t-test since NPY was normally distributed. NPY immunoreactivity in control and cardiomyopathy groups was compared using the Mann-Whitney test for non-normally distributed data. The association between continuous predictors such as age and NPY was assessed using the Spearman correlation (rs), and by simple linear regression.
Linearity of relationship between a continuous potential predictor versus NPY was assessed by comparing the linear model to a restricted cubic spline fit for the same predictor via a likelihood ratio test.

NPY versus MACE
The relationship between CS NPY and time to MACE was assessed by considering NPY as a continuous predictor using restricted cubic splines (RCS) in a Cox proportional hazard regression and by searching for a critical NPY threshold that best separated low from high MACE hazard.
This NPY threshold was estimated using a tree structured survival analysis using MACE hazard as the outcome and carrying out binary recursive partitioning. The hazard ratio (HR) and its 95% confidence interval limits (CI) and corresponding p value are reported. Once the critical NPY threshold was determined, the effect of NPY was assessed both ignoring and controlling for the critical covariates of age, reduced glomerular filtration rate and LVEF using a Cox proportional hazard model.

Clinical characteristics predictive of NPY levels
The distribution of NPY levels in the cohort is shown in efigure 1 (mean 85.1±31pg/ml). We examined whether relevant clinical characteristics were related to NPY levels. As illustrated in efigure 2, higher CS NPY levels were seen as estimate glomerular filtration rate (eGFR) decreases (rs= -0.36, p=0.0002), and blood urea nitrogen (rs=0.30, p=0.0018) and serum creatinine levels increase (rs=0.22, p=0.023). Interestingly, CS NPY was negatively correlated with LV internal dimensions in diastole (rs= -0.35, p=0.0004) and systole (rs= -0.30, p=0.0033), and with left atrial diameter (rs = -0.23, p=0.022). There was no association between LVEF and NPY levels (rs = 0.06, p=0.54). Baseline six-minute walk test (6MWT) distance, an objective measure of functional status of patients, and NT-ProBNP levels, a biomarker for HF symptoms and prognosis correlated with NPY levels (rs= -0.32, p=0.012, and rs = 0.33, p=0.008, respectively). One-sentence Summary: The adrenergic co-transmitter neuropeptide Y is associated with adverse outcomes in stable heart failure patients.