Significance: Here, we present unequivocal evidence of physical association of AMPK holoenzymes with mitochondrial reticulum (mitoAMPK) across multiple mouse tissues with evidence of conservation in human skeletal muscle and heart. We demonstrate that mitoAMPK is activated heterogeneously across the mitochondrial reticulum by mitochondrial energetic stress. Finally, we present evidence that suggests activation of mitoAMPK in skeletal muscle is required for mitophagy. We propose that mitoAMPK responds to mitochondrial microenvironment cues to maintain energetic homeostasis through mitochondrial quality control.

Coronary artery disease (CAD) is characterized by atherosclerotic plaque formation in the arterial wall. CAD progression involves complex interactions and phenotypic plasticity among vascular and immune cell lineages. Single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures, but human cell phenotypes remain controversial. Here, we perform an integrated meta-analysis of 22 scRNA-seq libraries to generate a comprehensive map of human atherosclerosis with 118,578 cells. Besides characterizing granular cell-type diversity and communication, we leverage this atlas to provide insights into smooth muscle cell (SMC) modulation. We integrate genome-wide association study data and uncover a critical role for modulated SMC phenotypes in CAD, myocardial infarction, and coronary calcification. Finally, we identify fibromyocyte/fibrochondrogenic SMC markers (LTBP1 and CRTAC1) as proxies of atherosclerosis progression and validate these through omics and spatial imaging analyses. Altogether, we create a unified atlas of human atherosclerosis informing cell state-specific mechanistic and translational studies of cardiovascular diseases.

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. Methods and Results: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3 . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD. By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques. The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.