CARD19 interacts with MICOS complex proteins and protects against mitochondrial dysfunction in macrophages

Abstract

Abstract CARD19 is a mitochondrial protein of unknown function; gene expression databases indicate that CARD19 is highly expressed in myeloid cells. We have observed that Card19 −/− mice injected with LPS display elevated levels of some pro-inflammatory cytokines relative to Card19 +/+ mice. Because CARD19 is a mitochondrial protein and mitochondrial dysfunction is frequently associated with immune dysregulation and chronic inflammation, we sought to identify the function of CARD19 in macrophages. We demonstrated via super resolution microscopy that endogenous CARD19 colocalizes with mitochondrial markers in bone marrow derived macrophages (BMDMs) and has a punctate distribution. Through immunoprecipitation and mass spectrometry analyses, we determined that CARD19 interacts with MIC19 and other components of the mitochondrial contact site and cristae organizing system (MICOS) complex. We further demonstrated that CARD19 colocalizes with MIC19 in mitochondrial sub-domains via super resolution microscopy. We then measured outcomes of mitochondrial stress in BMDMs. Consistent with previous reports of MICOS deficiencies, we found that Card19 −/− BMDMs have a modestly decreased oxygen consumption rate (OCR) measured by a Seahorse extracellular flux analyzer relative to Card19 +/+ BMDMs. Additionally, Card19 −/− BMDMs display elevated levels of mitochondrial reaction oxygen species (mROS) and a population of mitochondria with a decreased mitochondrial membrane potential, as measured by flow cytometry. Based on these data, we propose that CARD19 may be a previously unknown regulator of MICOS function which potentially links this complex to other mitochondrial and/or non-mitochondrial CARD proteins in macrophages.