Mechanisms underlying low flow-low gradient aortic stenosis
by Manar El Kenani
Date of Examination:2020-10-23
Date of issue:2020-11-09
Advisor:Prof. Dr. Dörthe Katschinski
Referee:Prof. Dr. Wolfram-Hubertus Zimmermann
Referee:PD Dr. Sven Thoms
Persistent Address:
http://dx.doi.org/10.53846/goediss-8300
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Abstract
English
Aortic stenosis (AS) remains the most common valvular disease in the elderly with an increasing prevalence, owing to the predominance of degenerative etiologies; hence, represents a major public health burden. The majority of patients with severe AS exhibit normal flow across the aortic valve and high transvalvular mean gradient (ΔPm), those patients are identified as (NFHGs-AS). Approximately 30% of patients with AS show discordant echocardiographic parameters with aortic valve area (AVA) <1 cm2, consistent with severe AS, and the ΔPm <40 mmHg, consistent with mild/moderate AS in association with low forward flow. This entity is the so-called low flow low gradient severe AS (LFLGs-AS). Populations with LFLGs-AS are characterised by either an impaired ejection fraction (EF) (classical LFLGs-AS) or a normal EF (paradoxical LFLGs-AS). Historically, degenerative AS was thought to be a solely valvular disease, involving passive accumulation of calcium (Ca2+) on the surface of the aortic valve leaflet. However, this concept has been evolved. AS is a disease of the valve and the myocardium. Although, AS typically leads to left ventricle (LV) hypertrophy in order to maintain normal wall stress and cardiac output, the LV response varies extensively in terms of geometry and function between the AS subtypes. Therefore, it is increasingly being acknowledged that severe AS phenotypes, in particular the paradoxical LFLGs-AS, follow different antecedent pathological pathways rather than being an end-stage high-gradient disease. To date, these pathways remain elusive. The overall goal of this study is to identify the functional and molecular alterations underlying the LFLGs-AS with particular focus on the paradoxical LFLGs-AS. First, we have performed proteomic comparison of the LV myocardium from 6 NFHGs-AS versus 5 paradoxical LFLGs-AS patients, using the state-of-the-art mass spectrometry with special focus on the extracellular matrix (ECM) proteins. Proteomic analysis revealed differentially abundant proteins involved in Ca2+ dependent cardiac contraction and relaxation. Additionally, the Raf kinase inhibitor protein (RKIP), was upregulated in paradoxical LFLGs-AS myocardial protein extracts. However, no major changes were identified on the level of ECM proteins between the investigated groups. Second aim of this study is to generate an experimental mouse model that recapitulates the clinical features of the paradoxical LFLGs-AS disease in human. Besides the severity of AS, the degree of hypertrophy in paradoxical LFLGs-AS patients is highly influenced by genetic variation. From this perspective, we have subjected wild type mice from two different backgrounds (C57Bl/6J and FVB/N) to short- and long-term pressure overload (PO) induced by transverse aortic constriction (TAC) surgery. One week of PO resulted in a significant LV hypertrophy in both strains, which was more pronounced in the C57Bl/6J mice. Cardiac function assessment using echocardiography revealed impaired systolic and diastolic functions in C57Bl/6J-TAC mice. Conversely, FVB/N-TAC mice showed mild diastolic dysfunction with well-preserved systolic function. Moreover, C57Bl/6J mice exhibited significant fibrosis and myocyte apoptosis, indicating pathological myocardial remodelling. These adverse changes were less prominent in FVB/N mice. On the molecular levels, both TAC groups showed re-expression of the fetal gene program. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII), favoring maladaptive remodelling and arrhythmias, was activated in both TAC groups. In FVB/N-TAC mice, additional signalling kinases were also activated, including the protein kinase B (Akt) and the protein kinase A (PKA). Echocardiography of C57Bl/6J mice at 12 weeks (i.e. long-term PO) of TAC operation showed promoted eccentric hypertrophy, marked decrease in the EF and the reverse longitudinal strain rate. Dramatic myocardial impairment was confirmed by pressure-volume (PV) loop analysis, which demonstrated significant alterations in systolic (EF, dP/dtmax, stroke work, stroke volume), as well as diastolic parameters (Tau, EDPVR, dP/dtmin). Decreased stroke volume index (SVI) indicates a low flow state and consequently a low gradient in C57Bl/6J mice. These criteria mirror the classical LFLGs-AS human disease. In contrast, echocardiographic and hemodynamic measurements of FVB/N-TAC mice at 12 weeks of TAC illustrated severe concentric remodelling, preserved LV EF, but with diastolic dysfunction and stiffness as indicated by increased Tau, EDPVR and decreased dP/dtmin. Consistent with compromised diastolic function, the left atrium size and weight were significantly increased. Despite the normal EF, the LV failed to preserve a normal stroke volume, probably due to the small LV cavity and impaired LV filling. Similar to the C57Bl/6J mice, the SVI was also decreased in the FVB/N mice after TAC, highlighting a low ventricular outflow. Furthermore, 30-40% of FVB/N-TAC mice develop advanced form of the phenotype in the form of severe atrial enlargement, associated with high mortality, promoted LV hypertrophy and fibrosis. Our results showed that the major clinical characteristics of paradoxical LFLGs-AS patients were captured by the FVB/N-TAC mice. Additionally, FVB/N mice showed increase in the RKIP protein expression; this further links our mouse model to the proteomics results of the paradoxical LFLGs-AS. Our study highlighted that the developed phenotype in C57Bl/6J-TAC mice is a typical progression from severe PO hypertrophy to decompensation and heart failure. In contrast, FVB/N mice response to PO is more likely to be distinct phenotype of adverse concentric remodelling and diastolic dysfunction that develop progressively upon long standing PO. This study provides the first insight into the cardiac proteomic profile of paradoxical LFLGs-AS, as well as an experimental mouse model that exhibits characteristics of the paradoxical LFLGs-AS disease. These findings may contribute to a better understanding of the underlying disease pathogenesis as well as the development of more selective therapeutic approaches for the paradoxical LFLGs-AS.
Keywords: low flow low gradient severe aortic stenosis; Cardiac remodelling; Pressure overload; Mouse models; Raf kinase inhibitor protein
