Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals Is Controlled by the Stoichiometry of MICU1/2 and MCU

SUMMARY Mitochondrial Ca2+ uptake through the Ca2+ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca2+-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca2+] threshold for Ca2+ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca2+ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca2+ tuning of oxidative metabolism.


SUPPLEMENTAL EXPERIMENTAL PROCEDURES Chemicals
Standard chemicals were purchased from Fisher Scientific or Sigma-Aldrich. Thapsigargin and CGP-37157 were from Enzo Life Sciences (Plymouth Meeting, PA). Fluorescent Ca 2+ -indicator dyes, and Pluronic F-127 were from Molecular Probes or Teflabs. Chelex 100 Sodium form was purchased from BioRad.
AAV and DNA constructs AAV9-Luciferase was from Penn Vector Core (Philadelphia, PA) and AAV9-MICU1 was custom prepared by Vector Biolabs (Philadelphia, PA). FLAG tagged-MCU was described in (Patron et al., 2014).

Cell culture and transfection
HeLa cells were grown in DMEM, 10% FBS and 1% penicillin/streptomycin and were transiently transfected with siRNA or plasmid DNA as described earlier (Csordas et al., 2013). MICU1-KO HEKs were kindly provided by Dr. Vamsi Mootha and grown as previously described . Transfection in HEKs cells was performed using Lipofectamine 3000 (Thermo Fisher).

Heart, skeletal muscle and liver mitochondria isolation
Mice were euthanized by cervical dislocation. Heart was rapidly excised and washed in cold isolation buffer containing 225mM Mannitol, 75mM Sucrose, 20mM Hepes and 0.1mM EGTA, at pH 7.4. Heart tissue was finely minced and incubated for 1min in the isolation buffer with 0.5% BSA and 3U/g of tissue of proteases (Sigma). Then, the cardiac tissue was homogenized with a glass Teflon potter homogenizer and the homogenate centrifuged at 500g/5min/4°C to remove nuclei and cellular debris. The supernatant was next centrifuged at 9,000g/10min/4°C and the mitochondrial pellet was resuspended in 225mM Mannitol, 75mM Sucrose, and 10mM Hepes at pH 7.4 and kept on ice. After liver harvesting, liver mitochondria were isolated by differential centrifugations. Briefly, liver was minced in a cold isolation buffer containing 250mM Sucrose, 10mM Tris/HCl, 0.1mM EGTA and 0.5% BSA, at pH 7.4 and homogenized in a glass Teflon potter. Cellular debris were removed by two consecutive centrifuges at 370g/10min/4°C. Supernatant was then centrifuged at 7,500g/10min/4°C and mitochondrial pellet washed once in isolation buffer without BSA before a new centrifugation at 7,500g/10min/4°C. Finally, liver mitochondria were resuspended in 250mM Sucrose and 10mM Tris/HCl at pH 7.4 and kept on ice. Skeletal muscles were carefully dissected, minced and homogenized in a glass Teflon potter in the homogenization buffer containing 140mM KCl, 20mM Hepes, 5mM MgCl 2 , 2mM EGTA, 1mM ATP(Di-K) at pH 7.0 with 1% BSA and 2U/g of tissue of proteases (Sigma). After centrifugation at 500g/10min/4°C, the collected supernatant was further centrifuged at 9,000g/8min/4°C. Pellet was resuspended in the isolation medium (140mM KCl, 20mM Hepes, 5mM MgCl 2 , 1mM EGTA at pH 7.0) and incubated on ice for 5min for myofibrillar re-polymerization. The two previous centrifuge steps were repeated and the final mitochondrial pellet resuspended in the isolation medium. Protein content was determined by the Lowry method (BioRad).

Live cell calcium imaging
RCaMP and ratiometric pericam were described in (Akerboom et al., 2013;Nagai et al., 2001). For imaging experiments, the cells were pre-incubated in a serum-free extracellular medium (ECM, 120 mM NaCl, 5 mM NaHCO 3 , 10 mM Na-HEPES, 4.7 mM KCl, 1. Fura2/furaFF and TMRM fluorescence were recorded simultaneously using 340-380 nm excitation and 500 nm emission, and 545 nm excitation and 580 nm emission, respectively. Complete dissipation of ΔΨ m was elicited by addition of 2 µM FCCP. Calibration of the fura signal was carried out at the end of each measurement, adding 1 mM CaCl 2 , followed by 10 mM EGTA/Tris, pH 8.5.

RNA isolation and qPCR
Total RNA were extracted from cells using TRIzol reagent (Ambion) and then treated with RQ1 DNase (Promega). Mice liver and heart RNA were isolated using the animal tissue RNA purification kit (Norgen) according to the manufacturer's instructions. Quantification was done on the nanophotometer Pearl (Implen).Then cDNA was synthesized using SuperScript III (Invitrogen) and used for qPCR reactions using SYBR Green (Invitrogen) on an ABI Prism 7000 sequence detection system (Life Technologies). Data were analyzed using the comparative 2-ΔΔCt method. Ct of the gene of interest was normalized to that of β-actin for heart and liver comparison. Primers used are listed below: