Increased Vasopressin 1A Receptor Expression in Failing Human Heart

To the Editor

Plasma levels of the neurohormone arginine vasopressin (AVP) are increased in patients with heart failure (HF) and there is a direct relationship between a rise in AVP levels and increased morbidity and mortality. (1) AVP activates a family of distinct G protein-coupled receptors: V_1A receptors (V_1A-R) in the heart, vasculature and liver and V₂ receptors (V₂-R) in the kidney. Activation of V₂-Rs causes reabsorption of free water, with excessive activation leading to hyponatremia. V₂ antagonists increase serum levels of sodium, but do not have a salutary effect on clinical outcome in patients with HF and they increase serum levels of AVP. (2) Recent studies demonstrate that over-expression of the V_1A-R in a mouse model diminished cardiac function independent of an effect on the vasculature. (3) However, little is known about the regulation of the V_1A-R in the failing heart. The present study was therefore undertaken to evaluate the expression of the V_1A-R in failing and non-failing human heart.

Human left ventricular myocardium was obtained from 25 subjects with end-stage HF undergoing heart transplant (19 male, 6 female, mean age 51.2 ± 2.7) or from 8 organ donors (1 male, 7 female, mean age 59.3 ± 3.2 years) whose hearts were unsuitable for donation owing to blood type, age or size incompatibility. Nine of the transplant recipients had HF secondary to ischemic cardiomyopathy. All had severe left ventricular dysfunction: the mean left ventricular ejection fraction (LVEF) was 11.9 ± 0.8%. At the time of surgery, three of the transplant recipients were receiving dobutamine alone, 11 were receiving milrinone alone and eight were receiving both. The non-failing hearts demonstrated normal left ventricular function by echocardiography (LVEF 60.4 ± 2.2%). Tissue aliquots were removed from the LV free wall, rapidly frozen in liquid nitrogen and stored at -70°C. (4) The Temple University Institutional Review Board approved the study and consent was obtained for all subjects.

Reverse-transcribed cDNA from myocardial mRNA (1 μg) was used to determine the expression of human V_1A-R (AVPR1A) using ribosomal protein S18 (RPS18) for normalization of gene expression data. RNA was extracted with RNeasy Fibrous Tissue Midi Kit (Qiagen). The primers for AVPRIA were F- 5’-CTTGAAGGAGATGGCCACTAAA-3’ and R- 5’-GTGATCGTGACGGCTTACAT-3’. The primers for RPS18 were F- 5’- CTTTGCCATCACTGCCATTAAG-3’ and R - 5’-ATCACACGTTCCACCTCATC-3’. The delta-delta CT method (Applied biosystems) was used to calculate relative quantitation (RQ) values.

Membrane fractions for radioligand binding assays were prepared as previously described. (5) The protein concentration was determined by the method of Lowry. V_1A-R levels were measured by saturation of radioligand ¹²⁵I-p-AVP (PerkinElmer, NEX310010UC) binding. (K_d 35 pM for human recombinant V _1A –R). Membrane preparations (~40 μg protein) were incubated with ¹²⁵I^-p-AVP (4 to 300 pmol/L) in buffer (mmol/L: Tris 50, EDTA 5, 0.1% BSA) either alone or with 5 μ mol/L of the V_1A selective blocker SR49059. Incubation was at 25°C for 2 hours in a volume of 100 μL and steady-state kinetics were achieved in specific binding. The reaction was terminated with ice-cold incubation buffer and rapid vacuum filtration through glass fiber filters. Each filter was washed 3X with 7 ml of ice-cold 10 mM Tris-HCl plus 0.1% BSA. Radioactivity was determined in a Gamma counter. All assays were performed in duplicate. Receptor density was normalized to membrane protein. The dissociation constant (K_d) and the maximal number of binding sites (B_max) for ¹²⁵I-p-AVP were determined by Scatchard analysis of saturation binding isotherms with Graphpad Prism. Non-specific binding was 30% and was subtracted from total binding. When using the radio-labeled antagonist specific for V_1A-R (K_d 30pmol for human recombinant V_1A-R) all Scatchard plots were linear and the binding curves fit a one binding site model.

As seen in Figure 1, the density of V_1A-R was significantly increased in failing heart when compared with non-failing controls without any change in the affinity of the ligand for the receptor. That the change in density was due to an increase in receptor expression was supported by the fact that there was a comparable increase in the levels of the mRNA encoding V_1A-R. (Figure 1) This represents the first report demonstrating changes in V_1A-R expression in HF. The majority of the patients were receiving inotropic therapy and therefore our findings may not be generalizable to patients with less severe disease. Interestingly, our findings are in contrast with the decrease in the expression of β₁-adrenergic and angiotensin type 1 receptors that characterize the failing human heart. (4) Further studies will be required to better understand the molecular mechanisms responsible for this difference as well as to determine whether these changes in receptor density contribute to diminished cardiac function observed in patients with end stage HF and elevated the levels of AVP.

Figure 1. V1AR expression is increased in human heart failure.

Saturation radioligand binding analysis with [¹²⁵I]-p-AVP indicates a significant increase in V1AR plasma membrane expression in failing hearts over non-failing hearts (B_max, B), while V1AR affinity for ligand was not different between failing and non-failing hearts (K_d, C), unpaired t-test with welch’s correction for unequal variance. C) Realtime PCR reveals a significant increase in AVPR1A expression in failing human hearts versus non-failing human hearts. AVPR1A expression is normalized to RPS18 and data are presented as RQ with RQmin and RQmax as error bars, unpaired t-test with welch’s correction for unequal variance.