Endolysosomal two‐pore channels regulate autophagy in cardiomyocytes

Abstract

Key pointsr Two-pore channels (TPCs) were identified as a novel family of endolysosome-targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na + channels able to control endolysosomal fusion, a key process in autophagic flux.r Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure.r We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy.r We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts.Abstract Autophagy participates in physiological and pathological remodelling of the heart. The endolysosomal two-pore channels (TPCs), TPC1 and TPC2, have been implicated in the regulation of autophagy. The present study aimed to investigate the role of TPC1 and TPC2 in basal and induced cardiac autophagic activity. In cultured cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 transcripts and protein levels that paralleled the increase in autophagy identified by increased LC3-II and decreased p62 levels. Small interfering RNA depletion of TPC2 alone or together with TPC1 increased both LC3II and p62 levels under basal conditions and in response to serum starvation, suggesting that, under conditions of severe energy depletion (serum plus glucose starvation), changes in the autophagic flux (as assessed by use of bafilomycin A1) occurred either when TPC1 or TPC2 were downregulated. The knockdown of TPCs diminished cardiomyocyte viability under starvation and simulated ischaemia. Electron micrographs of hearts from TPC1/2 double knockout mice showed that cardiomyocytes contained large numbers of immature lysosomes with diameters significantly smaller than those of wild-type mice. In cardiac tissues from humans and rats, TPC1 and TPC2 transcripts and protein levels were higher in females than in males. Furthermore, transcript levels of TPCs correlated negatively with p62 levels in heart tissues. TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes (i.e. there is a negative effect on cell viability under stress conditions in their absence) and they are differentially expressed in male and female human and murine hearts, where they correlate with markers of autophagy.

Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions

“…60 As well, mRNA expression of TPC1 was detected by RT-PCR in heart, kidney, spleen, liver, lung and adipose tissue in mice, 20 in heart tissue in rats, 40 and in human myocardium, where TPC1 has increased expression in patients with dilated cardiomyopathy 67 and in women compared to men, 40 a gender difference also confirmed in rats. Protein levels of TPC1 were observed by western blot in human and rat heart tissue.…”

“…Protein levels of TPC1 were observed by western blot in human and rat heart tissue. 40,67 Regarding TPC2, RNA gel blot analysis showed the same tissue distribution as TPC1 in mice, although with lower expression, 60 and, in human, TPC2 was detectable in lung, placenta, small intestine, liver, kidney, spleen, thymus, colon, skeletal muscle, heart and brain. 4 RT-PCR showed TPC2 mRNA expression in pancreas in mice, 21 in liver and heart in rats, 40,68 and in the human heart.…”

“…40,67 Regarding TPC2, RNA gel blot analysis showed the same tissue distribution as TPC1 in mice, although with lower expression, 60 and, in human, TPC2 was detectable in lung, placenta, small intestine, liver, kidney, spleen, thymus, colon, skeletal muscle, heart and brain. 4 RT-PCR showed TPC2 mRNA expression in pancreas in mice, 21 in liver and heart in rats, 40,68 and in the human heart. 40,67 Protein expression of TPC2 was detectable by protein gel blot in liver in mice, 44 and in human and rat hearts, 40,67 and by immunofluorescence in human pancreatic b-cells.…”

“…4 RT-PCR showed TPC2 mRNA expression in pancreas in mice, 21 in liver and heart in rats, 40,68 and in the human heart. 40,67 Protein expression of TPC2 was detectable by protein gel blot in liver in mice, 44 and in human and rat hearts, 40,67 and by immunofluorescence in human pancreatic b-cells. 21 …”

“…Today it is known that TPCs have a role not only in ion signaling but also in intracellular vesicle trafficking, [18][19][20] participating in a wide range of pathophysiological processes; that is why in the last years TPCs have received an increased attention from the main journals in the field, and they have been related to the regulation of many biological functions at different levels, including pancreatic b-cell function, 21,22 thermogenesis, 23 nutrient sensing, 6 endolysosomal transport and functions, 19,20 exocytosis, 24,25 cytokinesis, 26 fertilization and embryogenesis, 27 cell differentiation, [28][29][30][31] angiogenesis, 32 endothelium activation mediated by histamine, 33 smooth muscle contraction, 34 autophagy, [35][36][37][38][39][40] skin pigmentation, 41 or even to the Ebola virus infection mechanism. 42 As well, recent studies have suggested their possible implication in the pathogenesis of Alzheimer disease, 36 46 Thus, TPCs have become attractive therapeutic targets, although it is necessary to go deeper into their main functions and mechanisms of action not only to clinically relevant compounds could be designed but also to understand the pathophysiology of an increased wide range of diseases related to TPCs function/dysfunction.…”