Probenecid Improves Cardiac Function in Subjects with a Fontan Circulation and Augments Cardiomyocyte Calcium Homeostasis

Abstract

Subjects with functionally univentricular circulation who have completed staged single ventricle palliation, with the final stage culminating in the Fontan procedure, are often living into adulthood. However, high morbidity and mortality remain prevalent in these patients, as diastolic and systolic dysfunction of the single systemic ventricle are linked to Fontan circulatory failure. We presently investigated the effects of probenecid in post-Fontan patients. Used for decades for the treatment of gout, probenecid has been shown in recent years to positively influence cardiac function via effects on the Transient Receptor Potential Vanilloid 2 (TRPV2) channel in cardiomyocytes. Indeed, we observed that probenecid improved cardiac function and exercise performance in patients with a functionally univentricular circulation. This was consistent with our findings from a retrospective cohort of patients with single ventricle physiology where TRPV2 expression was increased. Experiments in isolated cardiomyocytes associated these positive actions to augmentation of diastolic calcium homeostasis.

Keywords: Congenital heart disease; Fontan procedure; Probenecid; TRPV2; Univentricular circulation.

Figure 1.

CONSORT diagram showing enrollment, recruitment and subjects that completed the study.

Figure 2:

Effect of probenecid on systolic function. A, Change in global longitudinal strain (%) in systole and diastole. B, Relationship between change in global longitudinal strain and baseline global longitudinal strain after probenecid and placebo. C, Representative figures. (a compared with no change; b compared with placebo).

Figure 3.

Effect of probenecid on exercise parameters. A. Change in Max VO₂ (maximum rate of oxygen consumption); Max work (maximum work); Max VE/VCO₂ slope (slope of the linear relationship between ventilation and carbon dioxide output). B. Representative graphs, with red square marking Max VO₂. (a compared with no change; b compared with placebo).

Figure 4.

Change in TRPV2 expression in samples from bi-ventricular non-failing controls (BVNF), single ventricle non-failing samples (SVNF), and single ventricle failing samples (SVHF). A, relative mRNA expression in all three groups. B, relative protein expression in all three groups and C; representative western blots.

Figure 5.

Effects of probenecid on cardiomyocyte Ca²⁺ homeostasis. A, Representative recordings of fluo-4 Ca²⁺ transients across a range of stimulation frequencies. B and C, Mean measurements of Ca²⁺ transient magnitude and decay kinetics. D, Change in resting Ca²⁺ levels, presented as diastolic fluorescence normalized to 0.5 Hz.

Figure 6.

Probenecid augments SERCA function in cardiomyocytes. A, Representative recordings for caffeine-elicited Ca²⁺ transients following 1 Hz pacing. B and C, Mean measurements of caffeine decay kinetics and release magnitude. D. SERCA activity was estimated by comparing the decay of caffeine and 1 Hz transients. E. Oxalate-supported monitoring of Ca²⁺ uptake into SR homogenates was assessed upon addition of ATP, Ca²⁺ leak was measured by application of thapsigargin (TG), and releasable SR content was assessed by application of CMC. F, G, and H, Mean measurements showed no effect of probenecid, supporting that SERCA stimulation in intact cardiomyocytes is indirect.