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

When the heart is injured, fibroblasts transition from a quiescent structural role into contractile and synthetic myofibroblasts. This is crucial for the initial healing response, for example scar formation to prevent ventricular wall rupture after myocardial infarction (MI), but excessive fibrosis is maladaptive, impairs cardiac function and contributes to heart failure progression. While cytosolic calcium ( i Ca 2+ ) elevation has been shown to be necessary for myofibroblast transdifferentiation, other Ca 2+ domains, such as mitochondrial calcium ( m Ca 2+ ), have not been explored. Recent studies have reported that the Mcu gene encodes the channel‐forming portion of the mitochondrial calcium uniporter complex (MCU) and is required for acute m Ca 2+ uptake. To examine the role of m Ca 2+ uptake in cardiac fibrosis, we generated a conditional, fibroblast‐restricted Mcu knockout mouse by crossing our Mcu flox mice with a transgenic mouse expressing a tamoxifen‐inducible cre recombinase under the control of the collagen 1a2 promoter ( Mcu fl/fl x Col1a2‐CreERT; fibro MCU‐cKO). fibro MCU‐cKO mice and controls were subjected to myocardial infarction by permanent ligation of the left coronary artery and cardiac function was examined weekly by echocardiography. Ablation of fibroblast Mcu significantly potentiated left ventricular dysfunction ( Figure 1) over the course of study. Further, fibro MCU‐cKO mice displayed increased fibrosis, evaluated by quantifying Mason's trichrome staining and qPCR examination of the fibrotic gene program. To further examine the cellular mechanisms responsible for the observed increase in fibrosis we isolated mouse embryonic fibroblasts (MEFs) from Mcu fl/fl mice and deleted Mcu using adenovirus encoding cre (Ad‐Cre). We first confirmed loss of acute m Ca 2+ uptake by challenging Mcu −/− MEFs with numerous pro‐fibrotic ligands (AngII, TGF‐β, Endothelin‐1) and monitored i Ca 2+ and m Ca 2+ transients using genetically encoded and spatially restricted Ca 2+ sensors. Loss of Mcu ablated m Ca 2+ uptake and enhanced i Ca 2+ transient amplitude, suggesting mitochondria may serve as a buffer to pro‐fibrotic i Ca 2+ signaling. Functionally, Mcu −/− MEFs displayed decreased cell migration and proliferation and increased contractile function in response to TGF‐β stimulation as examined using a collagen gel retraction assay, which was paralleled by increased expression of smooth muscle α‐actin (α‐SMA) ( Figure 1). Mcu −/− MEFs also exhibited a more negative mitochondrial membrane potential and enhanced oxidative phosphorylation capacity in comparison to control cells, results which suggest m Ca 2+ signaling may elicit substrate utilization and energetic changes to support or drive myofibroblast transdifferentiation. In summary, these findings suggest that m Ca 2+ signaling may be a novel regulatory mechanism in myofibroblast transdifferentiation and cardiac fibrosis. Support or Funding Information 5R01HL123966‐02 MECHANISMS OF MITOCHONDRIAL CALCIUM EXCHANGE IN HEART FAILURE 1A) fibro MCU cKO and control mice were analyzed by echocardiography and percentage of fractional shortening (%FS) was acquired before and every week thereafter myocardial infarction for 4 weeks. B) Mcu fl/fl MEFs were treated with Ad‐Cre to knockout MCU or Ad‐βgal as a control and then subjected to treatment with TGF‐β or AngII. Immunofluorescence was performed by co‐staining MEFs with α‐SMA antibody and DAPI. Mean pixel intensity of α‐SMA was calculated.