Introduction:
Aortic valve stenosis (AS), the leading form of cardiac valve pathologies, has a prevalence of 9.8% in Western elderly populations 80 years or above. While untreated AS is associated with high mortality, different hemodynamic subtypes can range from normal left-ventricular function to severe heart failure. However, the molecular mechanisms underlying four current echocardiographically classified AS phenotypes are unknown. Here, we used direct proteomic analyses of left-ventricular biopsies to identify unique biomarkers leading to subtype-specific AS mechanisms.
Methods:
Left-ventricular endomyocardial biopsies were obtained from 38 patients during transcatheter aortic valve replacement (median age [interquartile range], 77.0 [7] years at intervention; 26% female). Based on echocardiographic criteria, samples were divided into: 1) normal ejection fraction (EF)/high-gradient; 2) low EF/high-gradient; 3) low EF/low-gradient; and 4) paradoxical low-flow/low-gradient AS. Left-ventricular tissue from non-failing (NF) donor hearts served as control. Samples for proteome expression profiling were lysed and digested using pressure cycling technology followed by data-independent acquisition mass spectrometry (DIA-MS). For superresolution imaging informed by proteomic results, left-ventricular biopsies were directly fixed and embedded in paraffin for immunofluorescence antibody and wheat germ agglutinin labeling for triple-color STimulated Emission Depletion (STED) microscopy. Proteomic and subcellular cardiomyocyte data were correlated with echocardiographic parameters by univariate regression modeling.
Results:
In DIA-MS, 2.273 proteins were reproducibly detected and quantified throughout each individual left-ventricular biopsy. Unbiased proteomic analyses segregated unique proteotypes and identified three AS-specific subtypes. 160 proteins were differentially abundant between AS subtypes and NF, including connexin-43 and the cardiac Ryanodine Receptor (RyR2) SR Ca2+ release channel. In additional histomorphologic analyses, distinct proteotypes were associated with major AS-subtype specific myocardial hypertrophy differences (cardiomyocyte cross section area in µm² (mean ± SEM): NF) 342 ± 65, 1) 1231 ± 230, 2) 975 ± 166, 3) 801 ± 54, 4) 455 ± 78; p < 0.01). STED imaging further showed major functionally relevant protein changes in cardiomyocyte microdomains. Specifically, STED imaging revealed that connexin-43 plaques were diminished at the intercalated disc across all AS subtypes. In contrast, subcellular RyR2 cluster fragmentation and disruption of the functionally important association with transverse tubules occurred only in patients with left-ventricular systolic dysfunction.
Discussion:
Direct proteomic profiling and STED imaging of small left-ventricular biopsies enabled to identify novel molecular biomarkers and subcellular mechanisms of maladaptive remodeling in hemodynamic AS subtypes. Taken together, our data provide a proteomic and nanopathological basis for diagnostic subclassification of distinct forms of compensated versus decompensated AS subtypes, as well as new rationales for prognostic and mechanistic subtype analysis.