Right Ventricle Has Normal Myofilament Function But Shows Perturbations in the Expression of Extracellular Matrix Genes in Patients With Tetralogy of Fallot Undergoing Pulmonary Valve Replacement

Abstract

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

Keywords: extracellular matrix; myofibril; pulmonary valve replacement; small leucine rich proteoglycan; tetralogy of Fallot.

Figure 1

Passive stiffness and Ca²⁺ activated force in “permeabilized” cardiac myocytes from patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR) are similar to those from right ventricle (RV) donors. A, A typical force trace showing activation of a single myocyte by an increase in solution Ca²⁺ and the measurement of Ca²⁺‐activated force and crossbridge kinetics (k _tr). For (B, C, D, and F), each small symbol shows the result from 1 myocyte, and each large symbol shows the mean derived from replicate myocytes from each donor or patient. The horizontal bars show the overall mean±SD, calculated using each mean value derived from replicate myocytes (large symbols) as a single datum for the statistical analysis. B, Passive force, measured as the difference between passive forces at sarcomere lengths 2.0 and 2.3 µm. There were 19 RV donor myocytes (1–6 myocytes from each of 7 donors used in this panel) and 16 rToF‐PVR myocytes (1–4 myocytes from each of 6 patients with rToF undergoing PVR used in this panel). C, Maximum Ca²⁺‐activated force, corrected for myocyte cross‐sectional area. There were 23 donor myocytes (1–6 myocytes from each of 7 donors) and 19 rToF‐PVR myocytes (1–4 myocytes from each of 7 patients). D, Rate of redevelopment of force (k _tr) after a release/restretch protocol at maximum Ca²⁺. There were 21 donor myocytes (1–6 myocytes from each of 7 donors) and 19 rToF‐PVR myocytes (1–4 myocytes from each of 7 patients). E, Force–Ca²⁺ relationship. All forces are expressed relative to the maximum force at 30 µmol/L Ca²⁺. In this panel, the symbols show overall mean±SD for each [Ca²⁺]. There were 1 to 6 myocytes from each of 5 donors and 1 to 4 myocytes from each of 7 patients. F, Ca²⁺ sensitivity, expressed as the pCa50 value (−log₁₀ of [Ca²⁺] required for 50% activation of force), and serves as a summary variable for the same data as in (E). Statistical analysis showed no significant differences (P>0.05, unpaired t tests) between the overall mean data for RV donor and patients with rToF undergoing PVR for any of the measured parameters.

Figure 2

The collagen matrix in myocardium of patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR) is similar to that in right ventricle (RV) donor myocardium. A, quantitative polymerase chain reaction analysis of mRNA expression for COL1A2 (collagen I), COL3A1 (collagen III), and LOX (lysyl oxidase) indicated no differences between rToF‐PVR and RV donor myocardium. Each data point shows the result from 1 donor or rToF‐PVR patient. B, Picrosirius red staining of collagen fibers in RV donor and rToF‐PVR heart tissue sections with quantitation of the dark‐red‐stained collagen fibers as a percentage of total tissue area. C, The same sections were viewed under polarized light to assess the presence of irreversibly linked collagen as a percentage of total tissue area. No statistical differences were observed between groups for all assays described. RV donor, n=7; rToF‐PVR, n=5. Values are expressed as mean±SD. Scale=30 μm.

Figure 3

Analysis of the transcriptome identifies gene‐expression changes in myocardium of patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR). A, Principal component analysis of the gene‐expression profile of each tissue sample in relation to all others in the first 3 principal components revealed clear functional grouping of samples according to right ventricle (RV) donor (black) and rToF‐PVR (magenta). Each data point shows the result from 1 RV donor or rToF‐PVR patient. B, Hierarchical clustering also showed rToF‐PVR samples were functionally grouped according to gene‐expression profile. Selected thresholds were a false‐discovery rate (q) of 0.47 and P=0.004. C, Volcano analysis revealed that the most profound differences occurred in downregulated genes. The transcripts ASPN, LUM, and OGN were upregulated in rToF‐PVR samples. Statistical measures applied to the data set before analysis were a P value of 0.004 and a false‐discovery rate of 0.47. RV donor, n=6; rToF‐PVR, n=7.

Figure 4

Gene ontology (GO) analysis implicated tissue remodeling and inflammation pathways as dysregulated in myocardium of patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR). Holistic analysis of the differentially expressed genes in rToF‐PVR myocardium compared with right ventricle donor against the GO‐annotated database of affected biological processes ordered according to P value.

Figure 5

Schematic representation of the top‐scoring, statistically significant pathway map indicated downregulation of genes involved in immune response in myocardium of patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR) . The top‐scoring map (map with the lowest P value) based on enrichment distribution sorted by "statistically significant maps" was the Immune response_Lectin induced complement pathway. This shows negative regulation (blue thermometer) of most genes involved in this pathway, implying suppression of immune pathways in rToF‐PVR myocardium. Only 1 gene displayed upregulated expression (red thermometer). Many genes in the second and third top‐scoring maps overlapped with this map. The material in this figure is reproduced under a licence from Clarivate Analytics. This material may not be copied or redistributed in whole or in part without the written consent of Clarivate Analytics.

Figure 6

Gene set enrichment analysis showing the expression patterns of the extracellular matrix (ECM) proteoglycan gene set in patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR) compared with right ventricle (RV) donor myocardium. A, Gene set enrichment analysis of extracellular matrix proteoglycans displaying some enrichment of genes at the leading edge. B, Corresponding heat map of genes and expression profiles for 6 RV donor samples and 7 rToF‐PVR samples.

Figure 7

Validation of small leucine‐rich proteoglycan (SLRP) gene and protein expression shows asporin to be upregulated in several myocardial samples from patients with repair of tetralogy of Fallot undergoing pulmonary valve replacement (rToF‐PVR). A, The mRNA validation of gene expression was performed by quantitative polymerase chain reaction analysis for the genes encoding asporin (ASPN), lumican (LUM), and osteoglycin (OGN); n=7 per group. Each data point shows the result from 1 right ventricle (RV) donor or rToF‐PVR patient. Values are expressed as mean±SD, indicated P values were returned after performing the Mann–Whitney U test. The P values were unchanged if the single rToF‐PVR outlier was removed and the data were analyzed using the Student unpaired t test. B, Protein expression analysis for these genes was performed by western blotting using antibodies probing for asporin, lumican, and osteoglycin. Ponceau S shows equal loading of the gel lanes. Densitometry was performed for quantitation of protein abundance relative to ponceau S; RV donor, n=7; rToF‐PVR, n=5.

Figure 8

Immunostaining of sections for asporin shows localization in extracellular domains and the intercalated disc in human right ventricle. Asporin expression in right ventricle myocardium of both cohorts appeared in the extracellular domains in between the lateral membranes of cardiomyocytes suggestive of extraxcellular matrix localization (red arrows) or as regularly spaced bands indicative of intercalated disc staining (white arrows). Antibody to the sarcomere protein, myomesin, was used as a cardiomyocyte marker, and DAPI was used to stain nuclear DNA. Scale bar=10 μm. PVR indicates pulmonary valve replacement; rToF, repair of tetralogy of Fallot; RV, right ventricle