Morphological, functional, and molecular endpoints of myocardial hypertrophy: a cross-analysis of aortic valve stenosis and hypertrophic cardiomyopathy

Jan Federspiel (Homburg (Saar))1, H.-J. Schäfers (Homburg (Saar))2, J. Dudek (Würzburg)3, A. Nickel (Würzburg)3, J. Van der Velden (Amsterdam)4, P. Schmidt (Homburg (Saar))1, C. Maack (Würzburg)3, V. Sequeira (Würzburg)3

1Universität des Saarlandes Institut für Rechtsmedizin Homburg (Saar), Deutschland; 2Universitätsklinikum des Saarlandes Klinik für Thorax- und Herz-Gefäßchirurgie Homburg (Saar), Deutschland; 3Universitätsklinikum Würzburg Deutsches Zentrum für Herzinsuffizienz Würzburg, Deutschland; 4Amsterdam University Medical Centers Department of Physiology - Amsterdam Cardiovascular Sciences Amsterdam, Niederlande


Background: Basal interventricular septum (IVS) hypertrophy is a frequent finding in both, patients with aortic valve stenosis (AVS) and patients with hypertrophic cardiomyopathy (HCM). In HCM, well-characterized structural and molecular changes, such as increased myofilament Ca2+-sensitivity, disordered myosin state and metabolic changes, are paving the way for advancements in treatment. Specifically, for obstructive HCM patients, myosin inhibitors improve symptoms and induce reverse remodelling. Pathomechanisms in AVS, however, warrant more in depth research.

Purpose: The present study analysed whether AVS patients undergo morphological and molecular alterations similar to those occurring in HCM.

Methods: Septal myectomy samples from patients with HCM and AVS were collected (2 cohorts from 2 hospitals). Nonfailing (NF) left ventricular (LV) myocardium obtained at autopsy served as control. Due to limited availability, control tissue could not be included in all analyses. Cohort 1 (Myectomy n=26; NF control n=6): Histomorphologic and -morphometric assessments using standard stains based on published grading schemes were performed (no control). Analyses of protein kinase A (PKA, kit Enzo®) and creatine kinase (CK) activity (kit abcam®; both colorimetric assays), and 4-hydroxynonenal (4-HNE) content (SDS-PAGE; oxidative stress index) were performed. Clinical characteristics were obtained from patient charts and transthoracic echocardiography. Cohort 2 (Myectomy n=58; NF control n=12): Functional assessments of myofilament Ca2+-activation using demembranated cardiomyocyte force measurements at increasing Ca2+-concentrations (reported as half maximal effective concentration, EC50), with and without exogenous PKA administration. Analysis by 1D-gel electrophoresis and staining (ProQ-Diamond, SYPRO Ruby) evaluated myofilamental protein phosphorylation levels.

Results: The patients in both cohorts were comparable in terms of age (overall median 53 years; p=0.081) and sex distribution (overall 64% males; p=0.606). Cohort 1: AVS and HCM were comparable regarding LV ejection fraction (AVS median 65%, HCM median 70%; p=0.095) and end-diastolic IVS thickness (AVS median 20mm; HCM median 23mm; p=0.135). HCM, AVS, and controls had comparable levels of 4-HNE, PKA and CK activities. Structurally, AVS and HCM were comparable in terms of fibrosis, vacuolization, and cardiomyocyte alterations. Cohort 2: Myofilament Ca2+-sensitivity was elevated in both HCM (EC50=2.64±0.14µM) and AVS (EC50=2.28±0.13 µM) samples compared to controls (EC50=3.08±0.08 µM; p<0.05, respectively), with no differences between HCM and AVS. PKA treatment normalized myofilament Ca2+-sensitivity to control levels (EC50=3.35±0.14), in both HCM (EC50=3.09±0.13 µM) and AVS (EC50=3.53±0.35 µM) samples, and led to control-like phosphorylation of myofilament PKA-targets in HCM and AVS myocardium.

Conclusion: Despite different up-stream triggers for myocardial hypertrophy, being it afterload increases or endogenous in nature, analogous structural and molecular alterations in HCM and AVS were observed. This suggests that divergent pathomechanisms converge into shared downstream events, which are associated with an increased myofilamental Ca2+-sensitivity due to decreased myocardial PKA activity. Such unified downstream events provide a rationale for targeting sarcomeric (de)phosphorylation in patients with AVS (i.e., post-AV replacement) to foster reverse remodelling and improve function.

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