Primate Model Carrying LMNA Mutation Develops Dilated Cardiomyopathy

Abstract

To solve the clinical transformation dilemma of lamin A/C (LMNA)-mutated dilated cardiomyopathy (LMD), we developed an LMNA-mutated primate model based on the similarity between the phenotype of primates and humans. We screened out patients with LMD and compared the clinical data of LMD with TTN-mutated and mutation-free dilated cardiomyopathy to obtain the unique phenotype. After establishment of the LMNA c.357-2A>G primate model, primates were continuously observed for 48 months, and echocardiographic, electrophysiological, histologic, and transcriptional data were recorded. The LMD primate model was found to highly simulate the phenotype of clinical LMD. In addition, the LMD primate model shared a similar natural history with humans.

Keywords: CRISPR-mediated adenine base editing; dilated cardiomyopathy; lamin A/ C; primate model.

Graphical abstract

Figure 1

Mapping of LMNA Variants and Histology Characteristics of Heart Tissue (A) Diagrams representing the structure of lamin A/C (LMNA) and the distribution of LMNA variants. (B) Histologic characteristics. (B-i) Hematoxylin-eosin staining revealed nuclear malformation of cardiomyocytes in the group with LMNA-mutated dilated cardiomyopathy (LMD). (B-ii and B-iii) Histologic analysis suggested increased cell vacuolation and intercellular space in the LMD group. (B-iv) Heart tissue of end-stage LMD was characterized by fibrosis. (B-v) Heart tissue of LMD was characterized by myocyte disarray. DCM = dilated cardiomyopathy.

Figure 2

Construction of LMNA c.357-2A>G Primate Model and Identification at the Molecular Level (A) The schematic showed the process of generating DCM monkeys (upper) and general outcomes of LMNA c.357-2A>G mutations mediated by ABEmax in monkeys (below). (B) Heat map of the editing efficiency in mutated type (MT)-monkey fibroblast. (C) Reverse transcription–polymerase chain reaction to amplify LMNA gene Ex1 to Ex3 of MT and wild-type (WT) monkey’s fibroblast (upper) and percentage of messenger RNA (mRNA) alternative splicing–based TA-cloned sequencing (below). (D) Comparison of protein levels between the MT and WT monkeys via Western blot analysis. LMNA was normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Compared with WT monkeys (n = 4), the MT monkeys (n = 3) exhibited lower LMNA protein expression using a 2-sided Student’s t-test (P = 0.047). ∗P < 0.05. ABE = adenine base editing; other abbreviations as in Figure 1.

Figure 3

Echocardiographic and Weight Characteristics in Primates The continuous change curve of echocardiographic and weight data between the WT group (192015, 192017, 192028, and 192030) and the MT group (192007, 192009, 192011, and 192018) (left). The month 42 echocardiographic and weight data between the WT group (192015, 192017, 192028, and 192030) and the symptomatic MT group (192007, 192009, and 192011) (right). (A) Left ventricular ejection fractions (LVEF). (B) Fraction shortening (FS). (C) Aortic peak systolic velocity (APSV). (D) Left ventricular end-diastolic diameter (LVEDD). (E) Left ventricular end-systolic diameter (LVESD). (F) Left ventricular posterior wall thickness (LVPW). (G) Left atrial diameter (LAD). (H) Weight. Values are presented as mean ± SD. Unpaired 2-sided Student’s t-test was performed when comparing data from 2 groups. ∗P < 0.05. ns = not significant; other abbreviations as in Figure 2.

Figure 4

ECG and Histologic Characteristics in Primates (1) Timeline of symptom onset in MT group primates. (B) Kaplan-Meier curves for the monkeys in the MT and WT groups. (C) Twelve-lead electrocardiogram (ECG) characteristics of WT (normal ECG phenotype) and MT (atrial fibrillation) monkeys. (D) Single-lead ECG characteristics of WT (normal ECG phenotype) and MT (192011, atrial flutter; 192007, atrial fibrillation; 192009, supraventricular premature contraction) individuals. (E) Histologic characteristics of LMNA c.357-2A>G primates (192007 and 192011). (Ei) Nuclear malformation of cardiomyocytes in LMNA c.357-2A>G primates. (Eii) Increased intercellular space in LMNA c.357-2A>G primates. (Eiii) Fibrosis of heart tissue in LMNA c.357-2A>G primates. (Eiv) Heart tissue of LMNA c.357-2A>G primates was characterized by myocyte disarray. HF = heart failure; other abbreviations as in Figures 1 and 2.

Figure 5

RNA-Sequencing Analysis Revealed Transcriptional Alteration in LMNA c.357-2A>G Primates (A) Heatmap showing the up-regulated and down-regulated differentially expressed genes (DEGs) in the left ventricular tissue of LMNA c.357-2A>G primates. (B) Volcano plot of DEGs. (C) Pearson correlation heatmap of RNA-sequencing showing distinct clustering of monkey samples from the WT and MT groups. (D) Gene ontology enrichment analysis of the down-regulated genes (n = 714) in the MT group. (E) Gene ontology enrichment analysis of the up-regulated genes (n = 807) in the MT group. (F) Gene set enrichment analysis (GSEA) plot showing the biological term of laminin complex (Gene Ontology [GO]: 0043256). (G) GSEA plot showing the biological term of sequestering of metal ion (GO: 0051238). (H) GSEA plot showing the biological term of extracellular matrix assembly (GO: 0085029). The Benjamini-Hochberg false discovery rate procedure was used for multiple testing. Abbreviations as in Figure 2.