Endolysosomal two-pore channels regulate autophagy in cardiomyocytes

Abstract

Key points: 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. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure. 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. 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.

Figure 1. TPCs levels increase under starvation in cardiomyocytes

RT‐PCR (A) and a western blot (B and C) show increases of TPC1 and TPC2 after 12/24 h of serum starvation or serum plus glucose starvation in neonatal rat cardiomyocytes (n = 6). A western blot (D and E) confirmed the increase of TPCs after serum starvation or after serum plus glucose starvation in HL‐1 cells (n = 7). RT‐PCR (F) confirmed increases of TPCs transcripts after 24 h of serum starvation in human cardiomyocytes (n = 4). Bar graphs show the mean ± SEM. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01.

Figure 2. Effect of starvation on pAMPK, AMPK, mTOR, pmTOR, LC3 and p62 in cardiomyocytes

A, western blot corresponding to neonatal rat cardiomyocytes lysates immunostained with pAMPK, AMPK, pmTOR and mTOR (n = 5). B, representative western blot of HL‐1 lysates immunostained with pAMPK, AMPK, mTOR and pmTOR (n = 5). C, western blot corresponding to neonatal rat cardiomyocytes lysates immunostained with LC3 and p62. D, western blot corresponding to HL‐1 lysates immunostained with LC3 and p62 (n = 5). Bar graphs show the mean ± SEM. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01.

Figure 3. Levels of expression of TPCs after knockdown

Confirmation of knockdown of TPC1 and TPC2 by using RT‐PCR (A) and western blot (B) in neonatal rat cardiomyocytes (n = 6). Bar graphs show the mean ± SEM. Statistical significance (Mann–Whitney U test): **P ≤ 0.01; ***P ≤ 0.001.

Figure 4. TPC2 and TPC1/2 downregulation increases LC3II in neonatal rat cardiomyocytes

A and B, immunocytochemistry/confocal analysis showed that downregulation of TPC2 and TPC1/2 (but not of TPC1) increases basal GFP‐LC3 (n = 4). C, immunoblotting confirmed these results for LC3II/GAPDH ratio (n = 5). Bar graphs show the mean ± SEM. Statistical significance (Mann–Whitney U test): *P ≤ 0.05 vs. basal control; **P ≤ 0.01 vs. basal control; ***P ≤ 0.001 vs. basal control; ^# P ≤ 0.05 vs. non‐starved siTPC1.

Figure 5. TPC downregulation affects autophagic flux in neonatal rat cardiomyocytes

A, downregulation of TPC2 decreases autophagic flux (assessed by comparison of LC3II/GAPDH ratios between bafilomycin A1‐treated and bafilomycin A1‐non treated groups in cells in which TPC1 or TPC2 was silenced) in neonatal rat cardiomyocytes both under basal and serum starvation (24 h) conditions, whereas downregulation of TPC1 shows a tendency in the same direction (n=6). Under severe energy deprivation (serum plus glucose starvation for 16 h) conditions, either TPC1 or TPC2 downregulation causes alterations in autophagic flux (n = 7). B, immunocytochemistry/confocal analysis confirmed the above results for basal conditions (n = 4). Bar graphs show means ± SEM. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01; ^# P ≤ 0.05 vs. basal control; ^## P ≤ 0.01 vs. basal control. n.s., not significant.

Figure 6. TPC downregulation affects autophagy progression in neonatal rat cardiomyocytes

Downregulation of TPC2 and TPC1/2 induces accumulation of p62 under basal and starvation conditions (A to C; n = 5). Downregulation of TPC1 induces accumulation of p62 only after starvation (B and C; n = 5). Bar graphs show the mean ± SEM. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01.

Figure 7. Electron micrographs of cardiac tissue from TPC1/2 KO and wild‐type mice

A, electron micrographs show increase in lysosome number and decrease in lysosome diameters in TPC1/2 KO vs. wild‐type mice. B, distribution of number vs. diameter of lysosomes (left) and statistical analysis of lysosome number (right) in TPC1/2 KO vs. wild‐type mice (n = 63 cardiomyocytes). Statistical significance (Mann–Whitney U test): *P ≤ 0.05.

Figure 8. The knockdown of TPCs decreases the viability of cardiomyocytes under ischaemia and starvation

A, statistical analysis of MTT assays (n = 8) showing that, when cardiomyocytes were cultured under conditions of simulated ischaemia for 6 h, the silencing of TPCs significantly decreased cell viability vs. controls (cardiomyocytes subjected to simulated ischaemia and transfected with a siRNA negative control with no homology to known gene sequences). B, statistical analysis of MTT assays (n = 8) confirming that, in cardiomyocytes subjected to serum deprivation for 24 h, knockdown of TPC2, with or without TPC1, also decreased cell viability vs. controls. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01.

Figure 9. Sex‐dependence of cardiac TPCs levels and autophagy markers in cardiac surgery patients

TPC1 and TPC2 transcripts are increased in women vs. men (A: women: n = 100; men: n = 199) hearts. Western blot confirmed increases of TPCs protein levels (B: women: n = 11; men: n = 11) in women hearts. C, human cardiac protein levels of LC3I (women: n = 14; men: n = 16), LC3II (women: n = 14; men: n = 16) and p62 (women: n = 10, men: n = 9), but not the LC3II/LC3I ratio (women: n = 14, men: n = 16), are lower in women than in men. D, negative correlation between p62 and TPCs transcripts in human hearts (n = 15). E, absence of a significant influence of sex in the cardiac protein levels of the Rab GTP‐ase Rab7 (women: n = 8; men: n = 8) and of the lysosomal associated membrane protein LAMP‐1 (women: n = 8; men: n = 8). Bar graphs show the mean ± SEM or median with interquartile range. The correlation coefficients were measured as the Spearman ρ. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. ρ, Spearman's rho.

Figure 10. Sex‐dependence of cardiac TPCs levels and autophagy markers in rats

TPC1 and TPC2 transcripts are increased in females vs. males in rat (A: female: n = 9; male n = 9) hearts. Western blot confirmed increases of TPCs protein levels in rat (B: female: n = 7; male: 7) hearts. C, rat cardiac protein levels of LC3I, LC3II and p62, but not the LC3II/LC3I ratio, are lower in females (n = 7) than in males (n = 7). D, negative correlation between p62 and TPCs transcripts in rat hearts (n = 23). E, absence of a significant influence of sex in the cardiac protein levels of the Rab GTP‐ase Rab7 and of the lysosomal associated membrane protein LAMP‐1 (female: n = 10; male: n = 10). Bar graphs show the mean ± SEM or median with interquartile range. The correlation coefficients were measured as Spearman ρ. Statistical significance (Mann–Whitney U test): *P ≤ 0.05; **P ≤ 0.01; ρ, Spearman's rho.