Genetic Ablation of Fibroblast Mitochondrial Calcium Uptake Increases Myofibroblast Transdifferentiation and Exacerbates Fibrosis in Myocardial Infarction

Abstract

When the heart is injured, fibroblasts transition from a structural role into contractile, synthetic myofibroblasts. This is crucial for scar formation after myocardial infarction to prevent ventricular rupture, but excessive fibrosis is maladaptive and leads to heart failure. While intracellular calcium (iCa2+) elevation has been shown to be necessary for myofibroblast transdifferentiation, the mitochondrial calcium (mCa2+) domain has not been explored. Mcu encodes the channel-forming portion of the mitochondrial calcium uniporter complex and is required for acute mCa2+ uptake. We generated a conditional, fibroblast-restricted Mcu knockout mouse by crossing Mcufl/fl mice with a mouse expressing a tamoxifen-inducible Cre recombinase under control of the collagen1a2 promoter (fibroblast Mcu-cKO). Fibroblast Mcu-cKO mice and controls were subjected to ligation of the left coronary artery and cardiac function was examined weekly by echocardiography. Deletion of fibroblast Mcu worsened left ventricular dysfunction and increased fibrosis. To examine cellular mechanisms responsible we isolated mouse embryonic fibroblasts (MEFs) from Mcufl/fl mice and deleted Mcu using Cre-adenovirus. When challenged with pro-fibrotic ligands, Mcu-/- MEFs exhibited decreased mCa2+ uptake and enhanced iCa2+ transient amplitude. Mcu-/- MEFs displayed enhanced myofibroblast transdifferentiation as evidenced by decreased migration and proliferation as well as increased α-SMA expression and gel contraction. Metabolically, Mcu-/- MEFs display a shift towards more glycolysis with less oxidative phosphorylation, correlating with increased phosphorylation (inactivation) of the pyruvate dehydrogenase subunit E3. These results suggest that alterations in mitochondrial buffering of pro-fibrotic iCa2+ signaling and changes in energetic pathways may be mechanisms driving myofibroblast transdifferentiation and fibrosis.