Purpose: Cardiomyocyte apoptosis may have a detrimental impact on cardiac structure and function in patients with severe aortic stenosis (AS), as shown for other cardiopathies. On the other hand, microRNAs may regulate this process.
Methods: Endomyocardial biopsies were obtained from 28 patients with AS during valve replacement. Left ventricular (LV) morphology and function were assessed by echocardiography. Cardiomyocyte apoptotic index (CMAI) was quantified by TUNEL. Bax, Bcl-2 and BNIP3 were determined by western blot. MicroRNA profile was studied in 10 patients (5 from each subgroup) by TLDA, validating those of interest by real time RT-PCR in the whole population. microRNAs potential target genes were identified with the Microcosm, miRanda and PicTar databases.
Results: Cardiomyocyte apoptosis was increased (P<0.01) in AS patients compared with control subjects. By cluster analysis of CMAI, 2 subgroups of patients were identified: subgroup 1 (CMAI≤0.08%; n=16) and subgroup 2 (CMAI>0.08%; n=12). The higher activation of the apoptotic process in subgroup 2 patients was verified by a higher expression (P<0.05) of the Bax/Bcl-2 and BNIP3/Bcl-2 ratios. Subgroup 2 patients showed a lower cardiomyocyte density (P<0.001) and LV ejection fraction (P<0.05), and a higher prevalence of clinical heart failure (P<0.05) and NT-proBNP levels (P<0.05) than patients from subgroup 1. Of interest, CMAI was inversely associated (P<0.05) with cardiomyocyte density and LV ejection fraction and directly associated (P<0.05) with NT-proBNP in all patients.
The TLDA analysis showed 70 microRNAs altered (P<0.05) more than 4-fold in patients from subgroup 2 compared with subgroup 1, with 64 down-regulated and 6 up-regulated. Focusing on those with the highest fold-change, the repression in patients from subgroup 2 of miR-10b and miR-338-3p (P<0.05), which present pro-apoptotic predicted target genes (e.g. caspase 9 and Fas ligand), was validated in the whole AS population. Similarly, the up-regulation (P<0.05) of miR-198, with anti-apoptotic target genes like Bcl-x, was validated. These microRNAs were associated (P<0.05) with the CMAI. Additionally, miR-10b inhibition in HL-1 adult cardiomyocytes increased (P<0.05) the susceptibility of these cells to suffer apoptosis both in baseline conditions and in the presence of staurosporine.
Conclusion: Cardiomyocyte apoptosis is increased in AS patients, mainly in a subgroup of patients, and is associated with diminished cardiomyocyte density and the development of heart failure. MiR-10b down-regulation may be involved in this process, emerging as a potential therapeutic target.