Rationale: Cilp1 (cartilage intermediate layer protein 1) is a secreted extracellular matrix protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in postmyocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of patients with heart failure. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with NCilp1 (N-terminal half of the protein) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared with wild-type littermates. Conversely, NCilp1-transgenic mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 (mammalian Target of Rapamycin Complex 1) signaling were downregulated in KO hearts compared with wild-type hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and extracellular matrix gene expression analysis indicated that while MI caused significant stiffness in wild-type hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

BACKGROUND: Aortic aneurysm/dissection (AAD) is a life-threatening disease lacking effective pharmacological treatment. Protein ubiquitination plays a pivotal role in cardiovascular diseases. However, the possible contribution of the E3 ubiquitin ligase March2 (membrane-associated RING [really interesting new gene] finger protein 2) to the cause of AAD remains elusive. METHODS: Integrated single-cell RNA sequencing analysis was conducted in human AAD tissues. Based on the screening results, we generated a mouse line of smooth muscle cell–specific March2 knockout. β-Aminopropionitrile monofumarate was used to establish AAD. Cleavage under targets and tagmentation and cleavage under targets and tagmentation–quantitative polymerase chain reaction were performed to identify possible target genes for histone H3K18 lactylation. RESULTS: March2 expression was downregulated in aorta from patients with AAD or β-aminopropionitrile monofumarate–induced AAD mice. β-Aminopropionitrile monofumarate–induced AAD was significantly accentuated in March2 global (March2 ^–/– ) and vascular smooth muscle cell–specific deletion (March2 ^fl/fl ; Tagln ^Cre ) mice, whereas the AAD pathology was rescued by rAAV9-SM22α (smooth muscle 22α)-March2 (recombinant adeno-associated virus serotype 9 expressing Flag-tagged March2 under SM22α promoter). March2 interacted with PKM2 (pyruvate kinase M2) to promote K33-linked polyubiquitination. Deficiency of March2 lessened PKM2 dimer-to-tetramer conversion in AAD and overtly exacerbated AAD-induced histone H3K18 lactylation in vascular smooth muscle cells by fostering glucose metabolism reprogramming, thereby promoting p53-driven apoptotic transcriptional response—a hallmark of AAD pathogenesis. TEPP-46 (tetraethyl pyrophosphate), a PKM2-specific activator, pronouncedly alleviated March2 deficiency–deteriorated AAD pathology. CONCLUSIONS: Our findings demonstrated that March2 is a novel endogenous defender that prevents AAD by inhibiting vascular smooth muscle cell apoptosis, suggesting that March2 represents a potential therapeutic target for AAD.

Data-independent acquisition (DIA) is an emerging technology for large-scale proteomic studies. DIA data analysis methods are evolving rapidly, and deep learning has cut a conspicuous figure in thi...