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Literature Review | Latest Research: Exploring the Pathogenesis of Cardiac Fibrosis Through Gene-Edited Pigs!

Introduction:

Mutations in the MYBPC3 gene, which encodes cardiac myosin-binding protein C (cMyBP-C), are the most common cause of hypertrophic cardiomyopathy (HCM). Myocardial fibrosis (MF) plays a critical role in the pathogenesis and progression of HCM. However, the mechanisms by which MYBPC3 mutations induce MF remain unclear. In this study, the researchers employed cytosine base‑editing technology to generate a porcine model carrying the R495Q mutation and observed early‑onset myocardial fibrosis shortly after birth in these mutant pigs.

 

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Hypertrophic cardiomyopathy (HCM) is a heterogeneous disorder that affects individuals of different sexes, ethnicities, and races. HCM is primarily caused by autosomal dominant mutations in sarcomeric genes, with an estimated prevalence of approximately 1 in 500 to 1 in 200 in the general population. Clinically, it is characterized by left ventricular (LV) hypertrophy, excessive myocardial contractility, reduced ventricular compliance, arrhythmogenic remodeling, and fibrosis.

Over the past two decades, substantial evidence has demonstrated that MYBPC3 is the most frequently mutated gene in HCM, accounting for approximately 40%–50% of all HCM mutations.

Cardiac myosin-binding protein C (cMyBP-C), a flexible rod‑shaped protein, is a critical component of the cardiac sarcomere. In HCM patients with MYBPC3 gene mutations, 90% harbor heterozygous frameshift, nonsense, or splice‑site mutations that give rise to premature stop codons (PTCs) and truncated cMyBP‑C proteins.

Cardiac fibrosis is a hallmark of most myocardial pathologies, yet therapeutic options remain limited. Diseases associated with cardiac fibrosis carry a high mortality rate, and their incidence increases with age. Myocardial fibrosis (MF) can be characterized by the proliferation of cardiac fibroblasts (CFs) and excessive collagen deposition in myocardial tissue; it serves as a key marker and determinant in the pathogenesis of hypertrophic cardiomyopathy (HCM).

The growth factor TGF-β is the most extensively studied mediator of fibroblast activation, with TGF-β1 playing a pivotal role in pathological fibrosis. In addition to Smad‑dependent signaling, TGF-β1 can also trigger noncanonical pathways involving the activation of transforming growth factor‑β‑activated kinase 1 (TAK1), which is thought to contribute to maladaptive cardiac remodeling. Constitutively active TAK1 overexpression in the myocardium can induce cardiac hypertrophy and heart failure.

In this study, the researchers established an HCM pig model carrying the R495Q mutation and observed severe myocardial fibrosis shortly after birth in these mutant piglets. They found that MYBPC3 and several other cardiac‑related genes are expressed in fibroblasts, and that CRISPR‑mediated genetic disruption of MYBPC3 in fibroblasts impairs the NF‑κB/TGF‑β1/HIF‑1α–driven aerobic glycolysis signaling cascade, thereby promoting the activation of MYBPC3‑expressing cells into myofibroblasts.

 

Experimental Results      

The MYBPC3 R495Q mutant pig model exhibits premature myocardial fibrosis.

First, the researchers established MYBPC3 R495Q–mutant porcine fetal fibroblasts (PFFs) using CBE, single-cell cloning, and Sanger sequencing (Supplementary Figure S1a). Electroporation with CBE and a MYBPC3‑targeting sgRNA resulted in the conversion of the CCG codon to CTG in exon 16 of the porcine MYBPC3 gene (Supplementary Figure S1b). Using heterozygous R495Q PFFs, mutant pigs were generated via somatic cell nuclear transfer (SCNT). In total, three surrogate sows carried pregnancies to term, and six piglets were delivered without complications.

All piglets were genotyped by sequencing, and three neonates carried mutations at the target site. Sequencing analysis of the six highest‑ranked off‑target sites revealed no editing (Supplementary Figure S1d). Piglets carrying the MYBPC3 R495Q mutation began to die shortly after birth; all three died within 10 days, whereas all wild‑type piglets survived.

Quantitative RT‑PCR (qRT‑PCR) analysis revealed that, compared with WT piglets, the expression of MYBPC3 in the myocardium of R495Q‑mutant pigs was significantly reduced (Figure 1A). Western blotting demonstrated a marked decrease in cMyBP‑C protein levels in the hearts of R495Q‑mutant pigs (Figures 1B and 1C). Serological assays indicated that, in the mutant pigs, highly sensitive markers of myocardial injury and fibrosis—cardiac troponin T (cTnT) and type I procollagen carboxy-terminal propeptide (PICP)—were significantly elevated (Figures 1D and 1E).

Figure 1

 

Fibrosis-related genes, such as type I collagen (COL1A1), alpha-smooth muscle actin (a-SMA), and the fibrotic cytokine transforming growth factor‑β1 (TGF‑β1), exhibited significantly elevated transcript and protein expression levels in the hearts of mutant pigs (Fig. 1A–C). Furthermore, researchers observed inflammatory infiltrates in cardiac tissue sections (Supplementary Fig. S1e) and markedly increased pro-inflammatory cytokine levels in the serum samples of mutant pigs (Supplementary Fig. S1f–i).

Lactate concentrations were significantly elevated in the serum of mutant pigs (Figure 1F) and in cardiac muscle tissue (Supplementary Figure S1j). Furthermore, Masson’s trichrome staining of paraffin‑embedded sections revealed severe myocardial fibrosis in the mutant pigs (Figures 1G and 1H). Taken together, these findings indicate that the MYBPC3 R495Q mutation leads to pronounced myocardial fibrosis and inflammation.

 

MYBPC3 is expressed in cardiac fibroblasts.

The early-onset MF phenotype in the mutant pigs suggests that MYBPC3 deficiency may play a potential role in regulating fibroblast transformation.

The researchers first examined the expression of MYBPC3 in primary porcine cardiac fibroblasts (PCFs). Immunofluorescence staining revealed that both MYBPC3 and myosin light chain 3 (MYL3) were readily detectable in wild-type PCFs, which were positive for the fibroblast marker vimentin (VIM) (Figure 2A).

To investigate whether the expression of MYBPC3 and MYL3 in cardiac fibroblasts is species-specific, the researchers examined their expression in primary human cardiac fibroblasts (HCFs) and mouse cardiac fibroblasts (MCFs), finding that both MYBPC3 and MYL3 are expressed in these cells (Figure 2A).

Figure 2

 

Furthermore, the researchers found that MYBPC3 and MYL3 are also expressed in NIH3T3 cells, a commonly used mouse embryonic fibroblast cell line (Figure 2A). Western blot analysis confirmed the expression of the cardiac proteins MYBPC3, MYL3, and TNNT2 in NIH3T3 fibroblasts as well as in primary mouse, porcine, and human cardiac fibroblasts.

 

Deletion of MYBPC3 promotes fibroblast differentiation.

To investigate the role of MYBPC3 in fibroblasts, researchers used the CRISPR/Cas9 system to knock out the MYBPC3 gene in NIH3T3 mouse fibroblasts (MYBPC3‑ko). A guide RNA (gRNA) targeting exon 2 of the murine Mybpc3 gene was co-transfected into NIH3T3 cells together with a plasmid expressing Cas9. PCR–sequencing confirmed efficient indel formation at the gRNA target site, while no editing was observed at the five predicted off-target sites. In Mybpc3‑ko fibroblasts, both Mybpc3 mRNA and protein expression were abolished.

Next, the researchers investigated whether disruption of Mybpc3 could induce fibroblast activation. As shown in Figures 3A–C, Mybpc3 cleavage significantly increased the transcriptional and protein expression of the myofibroblast markers COL1A1 and α‑SMA, suggesting that Mybpc3 disruption can promote fibroblast activation.

Figure 3

 

 

Deletion of MYBPC3 enhances aerobic glycolysis in fibroblasts.

Researchers observed that the glucose concentration in the culture medium and cell lysates of MYBPC3‑KO fibroblasts decreased, while lactate levels increased and rose more rapidly than in wild-type fibroblasts (Fig. 4A–D). ATP production was also elevated in MYBPC3‑KO fibroblasts (Fig. 4E). Furthermore, compared with wild-type controls, the transcriptional and translational levels of key glycolytic enzymes—GCK, PFKM, and LDHA—were significantly upregulated in MYBPC3‑KO fibroblasts (Fig. 4F–H).

Inhibition of glycolysis using the glucose analog 2-deoxy-d-glucose (2-DG) significantly reduced the expression of glycolytic genes and fibrosis‑related marker genes (COL1A1 and α‑SMA), but had no effect on TGF‑β1 expression (Fig. 4F–H).

Figure 4

These results indicate that the loss of the Mybpc3 gene leads to metabolic reprogramming in fibroblasts.

 

Mybpc3 deficiency can be mediated through the NF-κB signaling pathway.

The researchers examined the NF-κB signaling pathway in wild-type and MYBPC3‑KO fibroblasts. In MYBPC3‑KO fibroblasts, phosphorylated p65 serves as a marker of NF‑κB activity, whereas the TGF‑β1 inhibitor SB‑431542 does not affect the activation of the NF‑κB pathway (Fig. 6A & B). NF‑κB plays a critical role in regulating the expression of pro‑inflammatory cytokines.

Figure 6

 

The researchers found that the levels of CCL2, IL‑1β, IL‑6, and TNF‑α were significantly elevated in both the culture medium and cell lysates of MYBPC3‑KO fibroblasts, whereas the NF‑κB signaling inhibitor PDTC markedly suppressed these cytokines in MYBPC3‑KO fibroblasts (Fig. 6C–F). PDTC also significantly reduced the expression of TGF‑β1 and HIF‑1α (Fig. 6G), indicating downstream regulation by NF‑κB. These findings suggest that Mybpc3 deficiency in fibroblasts activates an NF‑κB‑mediated signaling cascade, thereby driving their activation.

 

Discussion

In this study, we generated a porcine HCM model carrying a MYBPC3 mutation using CRISPR/Cas9 genome-editing technology. This resulted in the development of an early‑onset HCM phenotype in the MYBPC3‑mutant pigs. Furthermore, MYBPC3 and other myofibrillar proteins were found to be expressed in primary cardiac fibroblasts as well as in NIH3T3 fibroblasts.

The researchers’ findings reveal a novel intrinsic fibroblast mechanism that, via TGF‑β1, HIF‑1α, and aerobic glycolysis, links MYBPC3 deficiency to the activation of an NF‑κB‑mediated signaling cascade, thereby promoting the premature development of myocardial fibrosis in HCM (Figure 7).

Figure 7

 

In this study, the researchers demonstrated that mutations in the MYBPC3 gene activate the NF-κB signaling pathway. NF-κB is a key regulator of TGF-β1 levels, and in hepatic macrophages, a crosstalk mechanism may exist between NF-κB and the TGF-β/Smad signaling axis.

Studies have shown that mutations in MYBPC3 enhance HIF‑1α expression and aerobic glycolysis. The researchers demonstrated that TGF‑β1 can upregulate HIF‑1α expression in MYBPC3‑KO fibroblasts, an effect that can be reversed by SB‑431542. Moreover, both the shift from oxidative phosphorylation to aerobic glycolysis (the Warburg effect) and increased lactate production—phenomena previously observed in R495Q‑mutant pigs—were also evident in MYBPC3‑KO fibroblasts, and this metabolic reprogramming could be reversed by the aerobic glycolysis inhibitor 2‑DG.

In summary, the deficiency of MYBPC3 in cardiac fibroblasts can promote their transdifferentiation into myofibroblasts via the NF-κB/TGF‑β1/HIF‑1α/aerobic glycolysis signaling cascade, leading to premature myocardial fibrosis. These findings underscore the importance of targeting cardiac fibroblasts as an additional therapeutic target in the future development of treatments for HCM patients harboring MYBPC3 mutations.

 

References:

DOI: https://doi.org/10.21203/rs.3.rs-1724020/v1

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