Functional Characterization of Cardiac Mitochondrial Subpopulations in Naïve Pigs

Abstract

Cardiac mitochondria exist as two different subpopulations: A) subsarcolemmal mitochondria (SSM), which are present just below the sarcolemmal membrane, and B) interfibrillar mitochondria (IFM), which are present in between the contractile myofibrils. SSM supply ATP for the transport of protein, while IFM supply ATP for active muscle contraction. These two individual subpopulations are demonstrated to be functionally different in several disease models and respond differently to therapeutic compounds. Our interest in these cardiac mitochondrial subpopulations relate to their potential role in novel therapies of cardiopulmonary resuscitation (CPR) after prolonged untreated cardiac arrest. First, our goal was to identify and characterize the function of the two cardiac mitochondrial subpopulations in naïve pigs. Pigs are a commonly used large animal model for CPR, therefore hearts from 39–45 kg Yorkshire pigs (n=8) were used for this study. Hearts were harvested from animals under anesthesia and ~3g of left ventricular tissue was used for isolation of SSM and IFM using differential centrifugation. Mitochondrial protein yield was measured through Bradford assay. Mitochondrial respiration to determine the amount of oxygen consumed in the process of producing ATP was measured using a Clarke‐ electrode oxymeter. Respiratory Control Index was measured a ratio of state 3 and state 4 respiration using Complex‐1 (Pyruvate+Malate) and Complex‐2 (Rotenone + Succinate) substrates. Mitochondrial tolerance to Calcium stress was assessed by measuring the intensity of Calcium Green, an extra‐mitochondrial Calcium sensitive dye upon continuous addition of calcium to isolated mitochondria present in a Ca2+‐ free buffer. The rate of ATP produced by the mitochondria is measured by luciferin‐luciferase mediated bioluminescence. Mitochondrial size and complexity were measured by flow cytometry, using Mitotracker Green™, a mitochondrial membrane permeable dye. Preliminary results indicate that Complex‐1 and Complex‐2 mediated ATP synthesis were both significantly higher in the IFM when compared to SSM (p=0.0058 for Complex‐1 and p=0.0258 for Complex‐2). In addition, Complex‐2 mediated Calcium retention appeared to be higher in IFM when compared to the SSM. No differences were observed in either the SSM or IFM on their ability to produce ATP as evidenced by the Respiratory Control Index. Future studies include further characterization of mitochondrial function by measuring the enzyme activities of electron transport chain and Krebs's cycle proteins as well as closer examination of the phospholipid bilayer composition of SSM and IFM via electron microscopy. Furthermore, this study's characterization of naïve animal's mitochondria subpopulations will serve as a control for our future studies in identifying the role of SSM and IFM in cardiac arrest followed by CPR.Support or Funding InformationNational Institutes of Health: 5R01HL123227‐03 to Demetris Yannopoulos