Monday, May 19, 2025
5/19/2025
Mitochondria in cardiomyocyte-fibroblast transcellular cross-talk - Abstract: Heart is a complex organ characterized by homeostatic crosstalk between cardiomyocytes and fibroblasts. This homeostasis is altered upon cardiac injury, wherein cardiomyocyte-derived factors are known to mediate fibrobroblast to myofibroblast differentiation as a repair mechanism. Contrary to the classical factors, we found intact mitochondria to be the conduits of trans-cellular signaling between cardiomyocytes and fibroblasts. Since phosphoinositide 3-kinase γ knock out (PI3Kγ KO) mice have a pro-fibrotic phenotype with cardiac fibrosis at baseline with age, proteomics was performed on conditioned media/secretome of isolated adult cardiomyocytes from PI3Kγ KO and wild type (WT). Secretome from PI3Kγ KO cardiomyocytes was significantly enriched in mitochondrial proteins, despite no differences in cardiomyocyte apoptosis between PI3Kγ KO and WT. Interestingly, increased uptake of MitoTracker was observed in the PI3Kγ KO cardiomyocyte secretome compared to WT. MitoTracker staining was observed only in the mitochondrial enriched buoyant fraction following sucrose-density gradient fractionation showing intact mitochondria in the cardiomyocyte secretome. Transmission electron microscopy showed budding mitochondria from cardiomyocytes not only in PI3Kγ KO, but also in WT. Finding of lower amounts of mitochondria in the WT secretome compared to PI3Kγ KO indicates a) PI3Kγ negatively regulates mitochondrial release from cardiomyocytes, and b) intact mitochondria is a part of an yet to be determined cardiomyocyte-fibroblast trans-cellular homeostatic cross-talk whose dysregulation may underlie cardiac fibrosis. Acute myocardial ischemia (MI) leads to cardiomyocyte injury/death that could release mitochondria which would be taken up by subjacent fibroblasts leading to accelerated myofibroblast differentiation and scar formation to maintain structural integrity. Consistently, incubation of C57Bl/6 WT primary cardiac fibroblasts with sucrose-gradient purified labeled mitochondria from the cardiomyocyte secretome showed mitochondrial uptake and myofibroblast differentiation. Importantly, only intact mitochondrial fraction of the cardiomyocyte secretome showed myofibroblast differentiation compared to non-mitochondrial fractions or broken/fragmented mitochondria. Based on these exciting preliminary data, we hypothesize that intact mitochondria are trans-cellular signaling conduits between cardiomyocytes and fibroblasts accelerating myofibroblast differentiation. To test whether this trans-cellular cross-talk occurs between cardiomyocytes and fibroblasts, we plan to employ a repertoire of ex vivo tools and reagents, and unique genetically engineered mouse models to address the hypothesis through these studies: Aim 1: To test whether cardiomyocyte-specific expression of inactive PI3Kγ determines release of intact mitochondria through regulation of SNARE protein Vti1A, Aim 2: To evaluate whether fibroblast uptake of cardiomyocyte-mitochondria is necessary and sufficient to mediate myofibroblast differentiation/fibrosis, and Aim 3: To determine whether uptake of cardiomyocyte mitochondria by cardiac fibroblasts activates TLR4-STAT3 pathway to mediate myofibroblast differentiation.
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