Date Published: March 12, 2019
Publisher: Public Library of Science
Author(s): Martin Steinmetz, Charlotte Schmitter, Tobias Radecke, Anja Stundl, Georg Nickenig, Christian Schaefer, Nadjib Schahab, Mariuca Vasa-Nicotera, Jan-Malte Sinning, Marc W. Merx.
Transcatheter aortic valve replacement (TAVR) is a therapeutic option for patients with aortic valve stenosis at increased surgical risk. Telomeres are an established marker for cellular senescence and have served to evaluate cardiovascular diseases including severe aortic valve stenosis. In our study, we hypothesized that telomere length may be a predictor for outcome and associated with comorbidities in patients with TAVR.
We analyzed leucocyte telomere length from 155 patients who underwent TAVR and correlated the results with 1-year mortality and severe comorbidities. The cohort was subdivided into 3 groups according to telomere length. Although a trend for a positive correlation of telomere length with a lower EuroSCORE could be found, telomere length was not associated with survival, aortic valve opening area or cardiovascular comorbidities (peripheral, coronary or cerebrovascular disease). Interestingly, long telomeres were significantly correlated to a reduced left ventricular ejection fraction (LVEF).
In elderly patients with severe aortic valve stenosis, leucocyte telomere length did not predict post-procedural survival. The correlation between long telomere length and reduced LVEF in these patients deserves further attention.
Calcific aortic valve disease (CAVD) has a high prevalence of about 2% among the elderly ≥ 65 years, and is the most common heart valve pathology. Chronic inflammatory processes rather than passive degeneration are essential for the morphological changes in the aortic cusps leading to calcification . To date, the only therapeutic option is aortic valve replacement either through cardiac surgery or transcatheter aortic valve replacement (TAVR). Whether biomarkers help to predict the outcomes of patients undergoing TAVR is an ongoing debate.
Samples of 155 patients undergoing TAVR were enrolled after written informed consent in the study that was approved by the local medical ethics committee (Ethikkommission an der Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn). DNA samples were obtained from whole blood samples. DNA was extracted with the QIAamp DNA Blood Mini Kit (Qiagen). The isolated DNA was measured with a PEQLAB Nano Drop 2000c spectrophotometer. Telomere length was analyzed with a PCR-based technique that compares telomere repeat sequence copy number to single-copy gene (36B4) copy number in a given sample as published elsewhere. Duplicate DNA samples were amplified in parallel PCR reactions of 0,0768 ng genomic DNA, Takyon Rox SYBR Mastermix blue dTTP and either telomere primers (forward: 5’-GGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’; reverse: 5’- GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’); or 36B4 primers (forward: 5’-CAGCAAGTGGGAAGGTGTAATCC-3’; reverse: 5’-CCCATTCTATCATCAACGGGTACAA-3’). All PCRs were run on a ThermoFisher 7500 Fast Real-Time PCR System (initially 50° C for 2 min and 95°C for 10 min, then 15 s at 95°C, 30 s at 58°C and 30 s at 72° C for 40 cycles). The linear range of the assay was determined by using serially diluted DNA (1,92 ng, 0,384 ng, 0,0768 ng, 0,01536 ng). Both PCR reactions exhibited good linearity across this input range (r2 > 0.95). Test samples were checked to be within this range, outliers were diluted as necessary and re-run. 6 study samples (diluted four times), a calibrator sample, and one no-template control sample (all in triplets) were processed per run. The calibrator sample was used to enhance inter-assay comparability.
Baseline characteristics of patients before TAVR that were grouped according to their telomere length, i.e. from shortest (1st tertile) to longest telomere length (3rd tertile). Baseline characteristics are listed in Table 1. 30-day and 1-year survival were not significantly different between the 3 groups with short, intermediate and long telomere length (30-days: 1st tertile 98.1%, 2nd tertile 98.1%, 3rd tertile 98.0%; 1-year: 1st tertile 90.4%, 2nd tertile 76.9%, and 3rd tertile 92.2%; all p>0.05) (Fig 1A).
The results of our retrospective analysis suggest, that telomere length in peripheral blood is not useful as a biomarker to predict outcomes in elderly patients with various co-morbidities undergoing TAVR. We have used an experimental setup using quantitative PCR with a reference gene, and creating a very good inter-assay comparability by adding a calibrator sample. The advantage of this approach is the very easy and rapid analysis with standard quantitative real time PCR. Peripheral, carotid or coronary artery disease in combination with severe aortic valve disease was not associated with further telomere shortening. A single cardiovascular co-morbidity obviously does not significantly promote or inhibit telomere shortening in our cohort. Nevertheless, telomere length might reflect global disease burden. The logistic EuroSCORE comprises variables for organ (dys-)function, active inflammation, or chronic diseases and hence reflects overall disease burden with a cardiovascular pronunciation. We found a non-significant tendency that it was negatively correlated to telomere length, suggesting that a higher degree of frailty may be linked to shorter telomeres in an adequately powered study. The contribution of a single risk factor or disease probably cannot be extrapolated in patients with complex co-morbidities, because—depending on the specific disease—cellular activation and turnover may have even opposite effects on telomere length and senescence. In a recent study by Kurz and co-workers, calcific aortic valve disease but not coronary artery disease was linked to shorter telomeres. On the contrary, in patients with premature myocardial infarction, telomere length was inversely correlated with disease burden. In ApoE/TERC-deficient mice with significantly disturbed telomerase activity and shorter telomere, atherogenesis progress more slowly when compared to ApoE-deficient controls, probably a consequence of immuno-incompetence. Thus, it is finally not clear whether short telomeres lead to or result from pronounced atherogenesis.