Introduction
Cardiac pressure overload causes left ventricular remodeling with pathological hypertrophy by inducing a chronic burden on the left ventricle (LV). To identify potential therapy targets and better understand the pathophysiology of the disease, a pig model was established in which luminal narrowing resembling aortic stenosis is achieved by stent placement in the descending aorta using a catheter-based technique.
Methods
In a catheter-based intervention a stent is placed in the descending aorta that gradually reduces the aortic lumen to about 40%. By guiding a coronary catheter through the deposited stent hemodynamic measurements proximal and distal to the stent were taken. LV angiographies were performed at baseline and after eight weeks. Systematic heart sampling, including the aorta proximal and distal to the stent was performed. Sirius red staining of LV was conducted to achieve comprehensive insights into progression of fibrotic remodeling. A qPCR panel comprising 13 target genes was thoroughly optimized to achieve ideal amplification parameters, including optimal slope, R² and reaction efficiency, ensuring high accuracy and reproducibility. Subsequently, the expression of hypertrophy-associated genes, particularly those involved in fibrotic remodeling such as COL1A2 and COL3A1, as well as extracellular matrix regulators, was quantified. Snap frozen tissue samples with high collagen levels from the septal region of stented and control animals were pulverized and pooled (stented vs. control) for snRNA sequencing.
Results
Differences in systolic blood pressure of up to 25 mmHg proximal and distal to the stent were observed. Comparison of distal to proximal aortic wall thickness showed a mean increase of 55.32%. Sirius red staining revealed significantly increased collagen content in the left ventricle of stented pigs compared to control animals (stented: 17.16 % of LV [n=6] vs. control: 7.42% of LV [n=5], p=0.0003). Analysis of hypertrophy associated gene expression revealed no significant differences between stented pigs and controls. Single nuclei RNA sequencing of septal, high in collagen, myocardium showed unaltered major cardiomyocyte markers. Although fibroblast abundance was unchanged, gene expression profiles indicated a potential shift from collagen VI dominance in control animals to increased collagen IV expression in stented animals.
Conclusion
High differences in systolic blood pressure and subsequently thickened aortic walls, as well as the significantly increased collagen levels of stented pigs, which confirm enhanced myocardial fibrosis, support the validity of this animal model for pressure overload-induced cardiac hypertrophy. The observed shift in fibroblast collagen expression from type VI to type IV suggests the initiation of extracellular matrix remodeling. Ongoing in-depth investigations of the sequencing datasets intend to refine cluster identities and validate key marker genes via qPCR and histological assays. The resulting insights are anticipated to reveal a broad set of candidate targets for AAV-based gene therapy designed to alleviate cardiac fibrosis after pressure overload.
