Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Abstract

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture. This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples. Protocol Reagent Preparation1. The relaxation and preservation solution (RP Solution) is prepared as previously described with minor modifications 1 . Briefly, the RP Solution consists of 2.77mM CaK2EGTA, 7.23mM K2EGTA, 20mM imidazole, 0.5mM dithiothreitol, 20mM taurine, 50mM K-MES, 6.56 MgCl2, 5.7mM ATP, 14.3mM phosphocreatine, pH 7.1, adjusted at room temperature (RT). Filter the solution through a 0.45-μm filter to sterilize. Divide into 15 mL portions (Falcon polypropylene tubes) and store at -20°C See discussion for recipes. 2. The Mitochondrial Respirometry Solution (MiR05) is prepared as previously described 2 and contains 0.5mM EGTA, 3mM MgCl2. 6H2O, 20mM taurine, 10mM KH2PO4, 20mM HEPES, 1g/L BSA, 60mM potassium-lactobionate, 110mM sucrose, pH 7.1, adjusted at 30°C. Filter the solution through a 0.45-μm filter to sterilize. Divide into 50 mL portions (Falcon polypropylene tubes) and store at -20°C. See discussion for recipes. The oxygen solubility factor for MiR05 at 30°C and 37°C is 0.92 3 .

Sulforaphane protects the heart from doxorubicin-induced toxicity

“…50 mg of myocardial tissue were freshly isolated from the left ventricles of control and treated mice and dissected in relaxing solution on ice to prepare fiber bundles as described previously [49, 50]. Myocardial fibers were permeabilized by gentle agitation in relaxing solution supplemented with 50 mg/ml saponin for 20 min at 4°C.…”

Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model

“…High-resolution respirometry was performed on freshly isolated tissue samples from the left ventricle as described elsewhere (13, 14). Briefly, myocardial tissue samples were permeabilized by gentle agitation in relaxing solution supplemented with 50 mg/ml saponin for 30 min at 4°C, followed by washing in ice-cold respiration media for 10 min.…”

Diminishing impairments in glucose uptake, mitochondrial content, and ADP-stimulated oxygen flux by mesenchymal stem cell therapy in the infarcted heart

“…Saponin-permeabilized, cardiac fibers from the peri-infarct area were prepared as described (16). High-resolution respirometry (Oroboros Instruments, Innsbruck, Austria) was performed in duplicate at 37°C in MiR05 (0.5 mM EGTA, 3 mM MgCl 2·6H2O, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 1 g/l BSA, 60 mM potassiumlactobionate, 110 mM sucrose, pH 7.1, adjusted at 30°C).…”