MITOCHONDRIAL CALCIUM EXCHANGE LINKS METABOLISM WITH THE EPIGENOME TO CONTROL CELLULAR DIFFERENTIATION

Abstract

Fibroblast to myofibroblast differentiation is essential for the initial healing response, but excessive myofibroblast activation leads to pathological fibrosis. Upon injury, quiescent fibroblasts differentiate into contractile, synthetic myofibroblasts. Initially fibrosis is reparative, but when chronic it contributes to organ dysfunction and failure. Cytosolic calcium (cCa2+) signaling is necessary for myofibroblast differentiation yet the role of mitochondrial calcium (mCa2+) has not been explored. cCa2+ signaling is rapidly integrated into the mitochondrial matrix via the mitochondrial calcium uniporter channel (mtCU), a mechanism theorized to integrate cellular energetic demand with metabolism and respiration. This is intriguing, as it is now appreciated that metabolic reprogramming is required for numerous cellular differentiation programs. The Mcu gene encodes the channel-forming subunit of the mtCU and is required for acute mCa2+ uptake. To examine the contribution of mCa2+ signaling to myofibroblast differentiation, we isolated mouse embryonic fibroblasts (MEFs) from Mcufl/fl mice and deleted Mcu with adenovirus-expressing Cre recombinase. Mcu-/- MEFs exhibited decreased mCa2+ uptake and enhanced cCa2+ transient amplitude when treated with ATP (purinergic, IP3-mediated Ca2+ release). Loss of Mcu promoted myofibroblast differentiation: increased alpha-smooth muscle actin (α-SMA) expression and contractile function (gel retraction), increased myofibroblast gene expression, and decreased proliferation. Further, we found that treatment of wild-type fibroblasts with fibrotic agonists – transforming growth factor beta (TGFβ) and Angiotensin II (AngII) – increased expression of the mtCU gatekeeper, MICU1, to modulate mtCU activity and down-regulate mCa2+ uptake. This suggests that fibrotic agonists signal to acutely inhibit mCa2+ uptake to initiate myofibroblast differentiation. Next, we evaluated the relationship between mCa2+ uptake, metabolism, and myofibroblast differentiation. Fibrotic stimuli increased glycolysis and loss of MCU augmented this phenotype. In addition, genetic activation of glycolysis promoted myofibroblast differentiation, while genetic inhibition of glycolysis ablated the increased differentiation observed in Mcu-/- MEFs. We hypothesize that loss of mCa2+ uptake promoted aerobic glycolysis by reducing the activity of key Ca2+-dependent enzymes such as pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogase (αKGDH). Metabolomic analysis revealed a multitude of changes induced by both TGFβ and the loss of MCU, including increased levels of pyruvate, consistent with inactive PDH. In addition, metabolite quantification showed TGFβ increased alpha-ketoglutarate (αKG) levels ~2-fold and this increase was augmented by loss of Mcu. This is interesting because αKG promotes histone and DNA demethylation by modulating αKG-dependent dioxygenases. Indeed we observed that TGFβ and loss of MCU induced demethylation of histone lysine residues. Further, using ChIP-qPCR we found that TGFβ decreased H3K27me2 marks at the periostin and platelet-derived growth factor receptor alpha loci, which are early and robust indicators fibroblast activation. Finally, to examine the contribution of mCa2+ in cardiac fibrosis, we generated conditional, fibroblast-specific knockout mice by crossbreeding Mcufl/fl mice with Col1a2-CreERT mice (Mcufl/fl x Col1a2-Cre), permitting tamoxifen-inducible gene deletion in adult mice. Loss of Mcu (Mcufl/fl x Col1a2-Cre) increased myofibroblast differentiation and exacerbated fibrosis following myocardial infarction or chronic AngII infusion. In summary, our data linked changes in mCa2+ uptake with metabolic alterations necessary for chromatin modifications and activation of the myofibroblast gene program. While mCa2+ signaling is most well known and studied for its role in cell death, we have demonstrated a previously unrecognized role for modulation of mCa2+ uptake as a key regulator of myofibroblast differentiation.