Loss of the Coronary Artery Disease Risk Gene LMOD1 in Vascular Smooth Muscle Cells Triggers Rapid-Onset Coronary Atherosclerosis

Abstract

Background: Atherosclerosis is the primary underlying cause of coronary artery disease. LMOD1 is a coronary artery disease risk gene whose role in coronary artery pathophysiology is unknown. Whole-body loss of Lmod1 causes a lethal neonatal visceral myopathy in mice, necessitating unique approaches for the study of vascular smooth muscle cell (VSMC) phenotypes.

Methods: Control mice (Lmod1^WT carrying a Cre recombinase allele) and VSMC-restricted Lmod1 knockout mice (Lmod1^SMKO) were subjected to various atherogenic regimens. Atherosclerosis and LMOD1 (leiomodin 1) expression in mouse and human coronary arteries were assessed by histopathology and confocal immunofluorescence microscopy. Coronary arteries from Lmod1^WT and Lmod1^SMKO mice were analyzed with assorted stains and antibodies, immunogold lineage tracing, and spatial metabolomics. Aortic smooth muscle cells from Lmod1^WT and Lmod1^SMKO mice were subjected to lipid loading with or without lentivirus carrying wild-type or actin nucleation-deficient Lmod1 (Lmod1^ND. Mice harboring an intronic deletion of Lmod1 or Lmod1^ND were engineered using clustered regularly interspaced short palindromic repeats.

Results: A lethal neonatal visceral myopathy occurred in Lmod1^SMKO mice using Myh11-CreER^T2, prohibiting further investigation. In contrast, Lmod1^SMKO mice generated with Itga8-CreER^T2 survived and were therefore used in all subsequent studies. Under atherogenic conditions, Lmod1^SMKO mice displayed little vascular disease in several organs but developed diffuse, occlusive coronary atherosclerosis with fibrous caps. No such disease was observed in Lmod1^WT mice. Time-course studies documented lipid insudation and VSMC migration into the intima of coronary arteries of Lmod1^SMKO mice as early as 8 days after the regimen. Immunogold lineage tracing revealed that 46% of coronary plaque cells were of VSMC origin. Spatial metabolomics uncovered multiple lipid species within coronary atheromata of Lmod1^SMKO mice. In vitro studies demonstrated elevated lipid accumulation in Lmod1^SMKO VSMCs, which was rescued by viral-mediated Lmod1^WT or Lmod1^ND expression. An intronic deletion of Lmod1, comprising a conserved orthologous sequence where the single nucleotide variant associated with coronary artery disease exists, showed attenuated LMOD1 expression. Heterozygous Lmod1^SMKO mice, with a comparable reduction in LMOD1, displayed no coronary artery disease. Similarly, VSMC-restricted expression of Lmod1^ND resulted in negligible coronary atherosclerosis.

Conclusions: Under atherogenic conditions, Lmod1^SMKO mice present with rapid-onset coronary atherosclerosis. LMOD1 safeguards coronary homeostasis, apparently in an actin nucleation-independent manner.

Keywords: atherosclerosis; coronary vessels; microscopy, electron; smooth muscle.