#46 Myocardin preserves mitochondrial function and prevents cell death during cardiac development.
Wajihah Mughal, University of Manitoba; Matthew Martens, University of Manitoba; Jared Field, Children’s Hospital Research Institute of Manitoba; Donald Chapman, University of Manitoba; Yan Hai, University of Manitoba; Richard Keijzer, University of Manitoba; Joseph Gordon, University of Manitoba
Ten newborns a day are affected by congenital heart defects, contributing to infant mortality. As the underlying cause of cardiac abnormalities remain unknown, we hypothesize that Myocardin, a cardiac protein that modulates genes, can maintain cell health by regulating mitochondrial function to prevent cardiac cell death during cardiac development.
A rat cardiac tissue culture model (H9C2) was genetically manipulated or exposed to drugs (n=3) and compared to control (empty vector/scrambled or drug vehicle). The primary outcome assessed cell viability while the secondary outcome assessed mitochondrial function by measuring permeability transition pore closure, membrane potential, oxidative stress and calcium localization by fluorescent imaging. Gene and protein expression was determined by qPCR, protein immunoblot and immunofluorescence, respectively. An empty vector (ds-Red) was used to control for transfection efficiency, cells were counter-stained with nuclear Hoechst/DAPI and fluorescent signal was normalized to area.
Tissue culture experiments illustrate that loss of Myocardin function reduces cell viability by 25% (95%CI, 17.74 to 31.76; p<0.0001), reduces mitochondrial function and reduces expression of miR-133a in comparison to control. Loss of Myocardin also increases expression of a mitochondrial death protein Nix, which is reversed by miR 133a inhibition. An in vivo Myocardin knockout mouse embryo model indicates an increase in cardiac Nix signal in comparison to wild type. A series of in vitro gain of function studies demonstrate expression of Myocardin or miR 133a independently restore cell viability (p<0.01), mitochondrial function (p<0.01) and reduces Nix protein levels. To assess the calcium crosstalk between the endoplasmic reticulum (ER) and mitochondria, Nix localized to the ER (Nix-ER) increases mitochondrial calcium uptake in comparison to control.
Our data supports the hypothesis that Myocardin activation of miR 133a prevents Nix-induced cell death to restore cardiac survival and the therapeutic potential of miR-133a molecules in the management of congenital heart defects.