Abstract
When the heart is injured, quiescent fibroblasts differentiate into contractile, synthetic myofibroblasts. Initially fibrosis is reparative, but when chronic it becomes maladaptive and contributes to HF. Intracellular Ca 2+ ( i Ca 2+ ) signaling is reported to be necessary for myofibroblast transdifferentiation yet the role of mitochondrial Ca 2+ ( m Ca 2+ ) exchange has not been explored. The Mcu gene encodes the channel-forming subunit of the m Ca 2+ uniporter channel (MCUc) and is required for acute m Ca 2+ uptake. To examine the contribution of m Ca 2+ in cardiac fibrosis, we generated conditional, fibroblast-specific knockout mice by crossbreeding Mcu fl/fl mice with Col1a2-CreERT mice (Col1a2- Mcu -/- ), permitting tamoxifen-inducible gene deletion in adult mice. Col1a2- Mcu -/- mice and controls were subjected to ligation of the left coronary artery and cardiac function was examined by echocardiography. Loss of fibroblast Mcu worsened LV function and increased fibrosis, as evaluated by Mason’s trichrome staining and qPCR analysis of fibrotic gene expression. To examine the cellular mechanisms responsible for the increased fibrosis we isolated mouse embryonic fibroblasts (MEFs) from Mcu fl/fl mice and deleted Mcu with Cre-adenovirus. When challenged with pro-fibrotic ligands (TGF-β and AngII), Mcu -/- MEFs exhibited decreased m Ca 2+ uptake and enhanced i Ca 2+ transient amplitude. Loss of Mcu promoted myofibroblast transdifferentiation: increased α-SMA expression and contractile function (gel retraction) and decreased migration and proliferation. Mcu -/- MEFs were more glycolytic with increased phosphorylation (inactivation) of pyruvate dehydrogenase. Genetic activation of glycolysis with a Pfk2 mutant in WT MEFs promoted myofibroblast differentiation. Conversely, genetic inhibition of glycolytic flux ablated the increased transdifferentiation observed in Mcu -/- MEFs. Further, TGF-β and AngII altered the expression of regulatory MCUc components in WT MEFs. Our results suggest that alterations in m Ca 2+ uptake and bioenergetic pathways are necessary for myofibroblast transdifferentiation. Thus, energetic signaling represents a novel therapeutic target to impede HF progression and other progressive fibrotic diseases.
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