Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria

Abstract

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Figure 1. InsP₃R Deficient Cells Have Elevated Autophagy in Nutrient Rich Conditions

(A) Western blot of LC3 or tubulin in WT and DT40-KO cells (top) and quantification of LC3-II/(LC3-I + LC3-II) (bottom) expressed as fold increase over WT levels (n = 8). (B) TEM of DT40-KO cells showing autophagosomes (AV, arrows), and summary of cell profiles containing AV. 400 profiles counted in 4 experiments. Bars, 50 nm. (C) Confocal images of GFP-LC3 in WT and KO DT40 cells (left and middle panels). Arrows show LC3 puncta (900 cells from 3 experiments with 15 random fields/experiment). *** p < 0.001, ** p < 0.01; Bar, 10 μm. See also Figure S1.

Figure 2. InsP₃R Dependence of Autophagy Suppression in Nutrient Rich Conditions

(A) XeB (2 μM for 1 h) induces autophagy in WT DT40 cells to levels observed in DT40-KO cells (n = 6). (B) TEM of WT (left) and KO (right) cells showing AV (arrows and insets) after 1 h incubation with 2 μM XeB. Bars, 500 nm. Bottom panel summary of 100 cells per experiment counted from 15 fields (n = 3). (C) Current recordings in isolated nuclei from DT40-KO cells expressing D2550A-InsP₃R-3. Upper: typical gating, arrow represents closed channel current level. Bottom: currents during voltage ramps in control solution (black) and in presence of 10 mM luminal Ca²⁺ (red). Lack of normal shift in reversal potential indicates absence of Ca²⁺ permeability. (D) Expression of InsP₃R-3, but not Ca²⁺-impermeable D2550A-InsP₃R-3, suppresses autophagy in KO cells (n = 3). (E) TEM of AV (arrows) in cells stably-transfected with D2550A-InsP₃R-3. Bar = 500 nm. (F) RyR-2 expression fails to suppress autophagy in KO cells (n = 3). *** p < 0.001; ** p < 0.01; NS not significant. See also Figures S2, S3 and S4.

Figure 3. Lack of InsP₃R Ca²⁺ Release Activity Actives Pro-Survival Mechanisms

(A and B) Glucose (A) and glutamine (B) consumption measured daily in DT40 cells. (C) Cytochalasin B (Cyt B)-sensitive deoxyglucose uptake by WT and KO cells (n = 3). (D) Effects of autophagy inhibition by 3MA in nutrient-rich media on viability (PI-negative cells) of WT and KO cells after 24 hr (n = 3). (E and F) Survival of WT and KO cells in starvation conditions (Hank’s) in absence (E) and presence (F) of 3MA (n = 3). (G) Death (PI-positive cells) of Atg5-deficient primary hepatocytes induced by XeB (n = 3). (H) Recovery of WT and KO cells after 24 hr starvation. Same number of cells seeded in complete medium (n = 3). Error bars shown when bigger than points. *** p < 0.001, ** p < 0.01.

Figure 4. Pro-Survival Autophagy Induced By Lack of InsP₃R Ca²⁺ Signaling is Associated with Activation of AMPK but not mTOR

(A) DT40-KO have higher levels of P-AMPK than DT40-WT cells. P-AMPK/AMPK expressed as fold increase over WT levels (n = 8). (B) XeB (2 μM for 1 h) increases P-AMPK in WT cells to levels observed in KO cells (n = 6). (C) InsP₃R-3 but not Ca²⁺-impermeant D2550A-InsP₃R-3 reduces high P-AMPK in KO cells to control (WT) levels (n = 3). (D) Expression of RyR-2 fails to reduce P-AMPK in KO cells (n = 3). (E) mTOR phosphorylation is similar in KO and WT cells and is unaffected by XeB (2 μM for 2 h). P-mTOR/mTOR levels expressed as fold increase over WT levels (n = 5). (F and G) Phosphorylation of mTOR substrates 4E-BP1 and p70^S6K in KO and WT cells treated or not with 2 μM XeB for 2h. P-4E-BP1/4E-BP1 or P-p70^S6K/p70^S6K levels expressed as fold increase over WT levels (n = 5). (H and I) AMPK inhibition by compound C (CC; 2.5 μM for 1 hr) represses constitutive autophagy and elevated P-AMPK observed in KO cells (n = 5). (J) XeB (2 μM, 1 hr) increases AMP/ATP ratio in hepatocytes. AMP/ATP ratio expressed as average fold increase (mean ± S.E, n = 3) over untreated conditions. *p < 0.05, ** p < 0.01,*** p < 0.001, NS not significant. See also Figure S3.

Figure 5. Pro-Survival Autophagy Induced By Lack of InsP₃R Ca²⁺ Signaling is Mediated by AMPK

(A) α1-AMPK expression in HEK-293 cells stably expressing DOX-inducible WT or dominant negative (DN) α1-AMPK in presence or absence of DOX. Cells transfected with empty vector (EV) used as control. AMPK/tubulin expressed as fold increase over basal levels (DOX untreated) (n= 6). (B and C) XeB (2 μM for 1 h) failed to trigger either autophagy or AMPK phosphorylation in cells expressing DN-AMPK. Over-expression of WT α1-AMPK increased autophagy (n = 6). (D) α1-AMPK levels in HEK cells transiently transfected with α1-AMPK or irrelevant (NT) siRNA (n = 3). (E and F) XeB fails to induce autophagy or P-AMPK in cells transiently transfected with α1-AMPK siRNA (n = 3). (G) HEK-293 cells stably expressing inducible α1-AMPK shRNA were exposed to DOX for 72 hr (n = 3). (H and I) XeB fails to induce autophagy and P-AMPK in cells expressing inducible α1-AMPK shRNA (n = 3). (J – M) Treatment of DT40-KO (J and K) and HEK-293 (L and M) cells with methyl-pyruvate (MP; 5 μM for 1 hr) reduced autophagy and AMPK hyper-phosphorylation (n = 3). *p < 0.05, ** p < 0.01, *** p < 0.001, NS, not significant. See also Figures S3 and S4.

Figure 6. Lack of InsP₃R Ca²⁺ Signaling and Inhibition of Mitochondrial Ca²⁺ Uptake Are in the Same Pathway that Regulates Bioenergetics and Autophagy

(A) Oxygen consumption rates (OCR) in WT (red) and KO (black) DT40 cells exposed sequentially to (a) oligomycin, (b) FCCP and (c) rotenone plus myzothiazol. (B) Basal and maximal OCR in WT and KO DT40 cells (n =3). (C and D) Basal and maximal OCR in DT40 and HEK-293 cells treated or not with XeB (2 μM for 1 hr) (n = 3). (E – F) The Ca²⁺ ionophore ionomycin (100 nM) reversed inhibitory effects of XeB (E) and Ru360 (F) on OCR. (G – J) Effects of inhibition of mitochondrial Ca²⁺ uptake by Ru360 (10 μM for 1 h) in absence or presence of XeB on autophagy and P-AMPK in DT40 (G and H) and HEK-293 (I and J) cells (n = 3). (K and L) Effects of methyl-pyruvate (MP, 5 μM) on autophagy and P-AMPK evoked by 10 μM Ru360 in HEK-293 cells (n = 3). (M and N) Effects of mitochondrial Ca²⁺ uptake inhibition by Ru360 on basal and maximum OCR in DT40 (M) and HEK-293 (N) cells (n = 3). (O) Lactate generation in WT and KO cells after 24h in normal media (n = 3) *** p < 0.001, NS not significant. See also Figure S4.

Figure 7. Normal Growth Medium Supports Constitutive InsP₃R Ca²⁺ Release

(A and B) DT40-KO cells and XeB-treated HEK-293 cells have higher levels of phospho-PDH (n = 3). (C – E) DCA treatment normalizes PDH hyper-phosphorylation (C; 2 mM for 1h), enhances OCR (D; added acutely at “a”) and suppress autophagy (E; 2 mM for 1h) in KO cells to WT levels. (n = 3). (F) Effect of transient transfection (48 h) with specific or irrelevant (NT) siRNA on InsP₃R-1 expression in SH-SY5Y cells. InsP₃R-1/tubulin (n = 3) expressed relative to basal levels. (G – J) Effects of InsP₃R-1 knockdown (G and H) or XeB (10 μM, 1 h) (I and J) on autophagy and P-AMPK in SH-SY5Y cells (n=3). (K) Effects of InsP₃R-1 knockdown on basal and maximum OCR in SH-SY5Y cells (n=3). (L) Representative fluorescence recordings of Ca²⁺ release events in unstimulated SH-SY5Y cells in either Hank’s solution or normal culture media. (M) Quantification of Ca²⁺ release events in SH-SY5Y cells exposed either to Hank’s or normal media (n=3 experiments). (N) Effects of XeB (10 μM for 30 min) on spontaneous Ca²⁺ release events recorded in SH-SY5Y cells in normal media (n=3 experiments). **p < 0.01 ***p < 0.001. See also Figure S5 and Video S1.