# Modeling the Effects of Calcium Overload on Mitochondrial Ultrastructual Remodeling
## 2 Materials and Methods  
Ethical Approval. This work conformed to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and was approved by Michigan State University's Institutional Animal Care and Use Committee. Mitochondria Isolation and Protein Quantification. Cardiac mitochondria were isolated from guinea pig hearts using differential centrifugation, as described previously [8,14,22]. Briefly, Hartley albino guinea pigs weighing 350-450 g (4-6 weeks) were injected with heparin (500 units/mL) in the intraperitoneal cavity. The animals were anesthetized with 4-5% isoflurane and decapitated by a guillotine. After decapitation, a thoracotomy was performed, and the heart was perfused with a cold cardioplegia solution and homogenizedusing a handheld electronic homogenizer at 18,000 rpm for 20 s. The mitochondrial protein content was quantified using the BIO-RAD bovine serum albumin (BSA) standard set kit and the bicinboninic acid (BCA) assay. The mitochondrial suspension was diluted to a working concentration of 40 mg/mL and kept on ice for the duration of the experiment (2-3 h). The substrate stock solutions were neutralized to a pH of 7.0. Mitochondrial Quality Control. The mitochondrial quality was determined using an Oxygraph 2k (Oroboros Instruments Corp., Innsbruck, Austria). The O2k chambers were loaded with 2 mL of a respiratory buffer containing 130 mM KCl, 5 mM K2 HPO4, 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 1 mM MgCl2, 1 mM ethylene glycol-bis(\(\beta\)-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 0.1 % (w/v) BSA at a pH of 7.1 and 37 \({}^{\circ}\)C. All subsequent experiments were done using this buffer and temperature. At 0 min, 5 mM sodium pyruvate and 1 mM L-malate were added, followed by 0.1 mg/mL of mitochondria. Here, we defined the leak state as the rate of oxygen consumption by mitochondria only in the presence of substrates. At 5 min, a 500 \(\upmu\)M bolus of adenosine diphosphate (ADP) was added to induce maximal ADP-stimulated respiration. Quality was assessed by computing the respiratory control ratio (maximal ADP-stimulated rate divided by the leak rate). Only mitochondria with a respiratory control ratio (RCR) value greater than or equal to 16 were used in the experiments. Calcium Effects on Respiration and Oxidative Phosphorylation. The calcium effects on mitochondrial leaking and ADP-stimulated respiration were determined by quantifying changes in the proton leak and ADP-stimulated respiration rates after a calcium challenge. At 0 min, 5 mM sodium pyruvate, 1 mM L-malate, and 0.1 mg/mL of mitochondria were injected into each 2 mL chamber containing the respiratory buffer. At 5 min, a calcium bolus of 50 \(\upmu\)M of calcium chloride was injected. At 10 min, 500 \(\upmu\)M of ADP was added induce the maximal ADP-stimulated respiration. Cryo-Electron Microscopy (Cryo-EM) Sample Vitrification and Imaging. Isolated mitochondria were suspended at a concentration of 0.1 mg/mL in a 2 mL respiration buffer with 5 mM sodium pyruvate and 1 mM L-malate. At the collection times indicated, 5 \(\upmu\)L samples were pipetted from the mitochondrial suspension and deposited on Quantifoil R2/2 holey carbon grids that were pretreated for 1 min using a Pelco EasiGlo glow discharge unit. Prior to the mitochondria addition, 5-10 \(\upmu\)L of 10 nm nanogold markers were applied to the grids, which were then air dried. These beads provided markers for fiducial alignment of the tilt series. The grids were blotted to thin the water layer and subsequently plunged into liquid ethane at room temperature using a Vitrobot Mark IV with automated temperature regulation, blotting, and humidity control (Fisher Scientific). The Vitrobot chamber was set at 4 \({}^{\circ}\)C with 100% humidity, and the grids were blotted with a blot force of 1. The grids were then transferred and stored in liquid nitrogen until imaging. Tilt series were performed using an FEI Talos Arctic at 200 keV in low-dose conditions, operated using Thermofisher's Tomography software.