Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA020

Poster Communications

ATP release from cardiomyocytes involves cystic fibrosis transmembrane conductance regulator (CFTR), pannexin1 (Panx1), Ca signaling and mitochondria

H. J. Ballard1, S. Y. Wang1

1. School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.

Cardiac interstitial ATP increases with work, and ATP breakdown products contribute to blood flow regulation. We investigated ATP release in cardiomyocytes isolated by collagenase digestion from adult SD rat hearts. Cells were treated with lactic acid (LA, 10mM) which mimics increased work; ATP released into the bathing medium was measured with the luciferase assay and expressed as mean±SEM % of release from control cells. All changes were P<0.05 in ANOVA. LA treatment increased extracellular ATP to 215±19% (n=79), which was blocked by inhibiting LA entry to cells with α-cyanohydroxycinnamic acid. Total internal reflection microscopy (TIRF; n=4) showed that LA addition increased Ca in the zone <150nm below the cell membrane; the intracellular Ca chelator, BAPTA, or the Na/Ca exchanger inhibitor, SN6, blocked both Ca elevation and ATP release (47±3% with BAPTA or 12±2% with SN6 (n=16)), suggesting that Ca signaling in the near-membrane area is involved in ATP release. LA increased intracellular cAMP from 0.40±0.03 to 0.58±0.03nM (n=14); the LA-induced increase in ATP release was blocked by inhibition of CFTR with CFTRinh172 (75±3% (n=24) without or 70±11% (n=16) with LA) or by CFTR siRNA (57±7% without or 61±3% with LA (n=12)), suggesting that CFTR activation is a necessary step in the signaling pathway. In HCO3-free medium, extracellular ATP decreased to 39±4% (n=10) in the presence of LA, suggesting that CFTR-mediated or CFTR-regulated HCO3 entry to the cell is required for ATP release. Cardiomyocytes were homogenised and mitochondria isolated by differential centrifugation. Increasing HCO3 in the mitochondrial incubation medium from 0 to 40 mM doubled cytochrome C expression (n=6) and increased mitochondrial ATP release from 32±8 to 237±59% (n=16), which was reduced to 0.5±0.04% (n=8) by DIDS, suggesting that increased ATP release from mitochondria in the near-membrane area may drive the increase in cellular ATP release. The LA-induced increase in ATP release from cardiomyocytes was blocked by inhibition of pannexins with Fast Green-FCF (64±4% (n=19) without or 56±7% (n=16) with LA) or by Panx1 siRNA (54±3% without or 51±4% with LA (n=12)) suggesting that Panx1 may be the ATP release channel. Immunofluorescent staining (n=25) showed that CFTR and Panx1 did not colocalise in cardiomyocytes, which rules out direct interaction between them. In a live-cell caspase assay, intracellular caspase activity increased within 10s of adding LA to the medium, and medium ATP in the presence of LA was reduced from 174±6% (n=56) to 94±6% (n=32) by caspase 3 inhibitor, suggesting that Panx1 may be activated by caspase cleavage during LA treatment. These data suggest that LA stimulates cardiomyocytes to release ATP via Panx1 through a pathway that involves entry of Ca and HCO3, CFTR activation and mitochondrial signaling.

Where applicable, experiments conform with Society ethical requirements