Study of the role of the new endolysosomal receptors two pore channels (TPCN1 and TPCN2) in human, mouse and rat heart

Abstract

Purpose: Mammalian two-pore channel proteins (TPCN1, TPCN2) are recently identified as ion channels in intracellular endosomes and lysosomes whose role at cardiac level is completely unknown. Our aim was to study the cardiac effect of knocking out TPCN1 in mice as well as studying the regulation of expression of cardiac TPCN1 and TPCN2 by cardiovascular diseases and metabolic conditions. Methods: We carried out a differential proteomic analysis of murine cardiac ventricle between controls (n=3) and knockout (n=3) mice for TPCN1 (TPCN1 KO). Two dimensional gel electrophoresis was performed. Detection was made with SYPRO Ruby and identification by MALDI-TOF/TOF. RT-qPCR was used to quantify mRNA expression and inmunoblotting to confirm protein expression of TPCN1 and TPCN2 in left-ventricular myocardium from patients with heart failure (HF, n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy, and non-diseased donors (CTL, n = 6), as well as in three/four week-old male Sprague-Dawley rats fed with standard or high-fat diet and in male adult Zucker diabetic fatty (ZDF) and lean (ZL) rats. Results: In murine cardiac tissue, we observed 27 differential spots (fold-change>1.8 and p<0.05), all elevated in TPCN1 KO versus wt mice. The most relevant protein change observed was FABP3 (Fatty acid binding protein 3) and it was validated by western blot (p=0.0313). Cardiac transcript expression of TPCN1 (p=0.018) and TPCN2 (p=0.001) increased in heart failure patients relative to controls. Increases remained significant for TPCN2 in all groups (ICM, p=0.008; DCM, p=0.001) but for TPCN1 only in DCM (p=0.015). In male Sprague-Dawley rats, we observed an increase in TPCN1 (p=0.012) and TPCN2 (p=0.0041) cardiac mRNA levels in high fat (n=13) versus control (n=13) rats. On the contrary, in Zucker rats, we observed a decreased of TPCN1 (p=0.0001) and TPCN2 (p=0.0005) mRNA levels in ZL (n=10) versus ZDF (n=10). Conclusion: In TPCN1 KO mice heart, there is an increase in FABP3 which suggest that cardiac fatty acid transport is altered by the lack of TPCN1. Both receptors seem to be key regulators in the rat heart metabolism, since their gene expression levels are regulated by diet and obesity. TPCN1 expression is clearly increased in HF as a whole also tended towards a cardiomyophaty difference. TPCN2 up regulation could also be involved in a general mechanism underlying heart failure, at least with ICM and DCM etiology. The current data provide the first evidence that the endolysosomal system might be involved in the regulation of cardiac metabolism.