Mitral regurgitation (MR) is the second most prevalent valvular heart disease after aortic valve stenosis [
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Committee for Human Research of Chang Gung Memorial Hospital (102-2219C). Written informed consent was obtained from each study patient.
This study enrolled 18 severe nonischemic MR patients with HF in sinus rhythm (age: 57 ± 11 years), 12 patients with severe degenerative aortic valve disease and HF in sinus rhythm (age: 60 ± 12 years; aortic stenosis in 5, aortic regurgitation in 5, combined aortic stenoregurgitation in 2), and 16 control subjects without valve disease and HF. Exclusion criteria included previous myocardial infarction, febrile disorder, infectious or inflammatory disease, autoimmune disease, malignancy, acute and/or chronic viral hepatitis, and current use of immunosuppressive drugs.
Eleven left atrial tissue samples from normal adults were purchased for use as normal controls. Of these, six tissue samples were used for gene studies (24-year-old Caucasian male, 27-year-old Caucasian male, 30-year-old Asian male, 60-year-old Caucasian female, 76-year-old Caucasian female, and 77-year-old Caucasian male; BioChain, Newark, CA, USA). Five tissue samples were used for measuring tissue angiotensin II and angiotensin 1~7 concentrations. Of these, one sample (35-year-old Caucasian female) was obtained from G-Biosciences (St Louis, MO, USA), and four samples (49-year-old African American male, 60-year-old Caucasian female, 62-year-old Caucasian female, and 77-year-old Caucasian male) were obtained from BioChain (Newark, CA, USA).
During surgery, small specimens of atrial tissue were collected from the left atrial free wall of patients with MR and aortic valve disease. Excised atrial tissues were immediately frozen in liquid nitrogen and stored at −80°C for subsequent analyses.
Blood samples collected from MR patients, aortic valve disease patients, and control subjects without valve disease and HF were stored in tubes containing EDTA. The blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C, aliquoted into Eppendorf tubes, and stored at −80°C.
Plasma angiotensin II concentration was measured with an enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, USA) according to the manufacturer instructions. The EIA kit includes angiotensin standard, antiangiotensin II IgG tracer, glutaraldehyde, borane trimethylamine, Ellman reagent, assay, and wash buffers. The standard curve range was 0.98–125 pg/mL. An aliquot of plasma was assayed, and all samples were tested in duplicate. The EIA plate was read at 405 nm with an auto plate reader (
Plasma angiotensin 1~7 concentrations were measured with a human angiotensin 1~7 ELISA kit (MyBioSource, San Diego, USA) according to the manufacturer instructions.
To determine angiotensin II and angiotensin 1~7 concentrations, human left atrial tissues were dissected and lysed by sonication with RIPA buffer (Cell Signaling, MA, USA) and supplemented with 1% protease inhibitors. The lysates were incubated on ice for 30 minutes and then cleared with centrifugation. The supernatants were used to measure angiotensin II and angiotensin 1~7 concentrations in the left atrium with a human angiotensin II EIA kit (Cayman Chemical, Ann Arbor, USA) and a human angiotensin 1~7 ELISA kit (MyBioSource, San Diego, USA) according to the manufacturer instructions.
The RNAs were extracted from the left atrial myocardial tissue using a RiboPure™ kit (Ambion, NY, USA) according to the manufacturer protocol and then reverse transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed using TaqMan chemistry on a 7500 Fast Real-Time PCR System (Applied Biosystems). TaqMan primers and probe mixtures were also purchased from Applied Biosystems. Table
TaqMan real-time PCR assay identification.
Gene name | Assay identification |
---|---|
Hs00174179_m1 | |
Hs01085333_m1 | |
Hs01096939_m1 | |
Hs00169126_m1 | |
Hs00989750_m1 | |
Hs01586213_m1 | |
Hs00264902_m1 | |
Hs00162760_m1 | |
Hs00252959_m1 | |
Hs00174265_m1 | |
Hs0113415_g1 | |
Hs00893646_m1 | |
Hs00153510_m1 | |
Hs00156558_m1 | |
Hs00267157_s1 | |
Hs00150019_m1 | |
Hs00982555_m1 | |
Hs03929097_g1 |
Data were presented as mean ± SD (baseline characteristics) or SEM (plasma and tissue angiotensin II and angiotensin 1~7 concentrations and gene expressions). Categorical variables (excluding New York Heart Association functional class) between MR patients and aortic valve disease patients were compared using Fisher exact tests. Categorical variables among MR patients, aortic valve disease patients, and control subjects were compared using chi-square test. Chi-square test was also used to compare New York Heart Association functional class between MR patients and aortic valve disease patients. Continuous variables among three groups were analyzed by Kruskal-Wallis test, and continuous variables between two groups were analyzed by Mann-Whitney test. Covariates were adjusted according to analysis of covariance results. Statistical analysis was performed using commercial statistical software (SPSS for Windows, version 22). A
Table
Baseline clinical characteristics of the study subjects.
MR ( |
AVD ( |
NC ( |
||
---|---|---|---|---|
Age (years) | 57 ± 11 | 60 ± 12 | 49 ± 12 | 0.053 |
Male (%) | 5 (27.8%) | 9 (75.0%) | 10 (62.5%) | 0.024 |
Smoking (%) | 2 (5.6%) | 1 (8.3%) | 2 (12.5%) | 0.772 |
Body mass index (kg/m2) | 23.7 ± 2.6 | 25.3 ± 3.4 | 23.6 ± 3.4 | 0.168 |
Hypertension (%) | 8 (44.4%) | 7 (58.3%) | 0 (0.0%) | 0.002 |
Diabetes mellitus (%) | 3 (16.7%) | 1 (8.3%) | 0 (0.0%) | 0.227 |
Hyperlipidemia (%) | 6 (33.3%) | 3 (25.0%) | NA | 0.704a |
NYHA | 0.324a | |||
Functional class I (%) | 2 (11.1%) | 3 (25.0%) | ||
Functional class II (%) | 7 (38.9%) | 5 (41.7%) | ||
Functional class III (%) | 9 (50.0%) | 3 (25.0%) | ||
Functional class IV (%) | 0 (0.0%) | 1 (8.3%) | ||
Aortic valve disease (%) | 0 (0.0%) | 12 (100.0%) | ||
Tricuspid regurgitation (%) | 7 (38.9%) | 1 (8.3%) | 0.099a | |
3 (16.7%) | 2 (16.7%) | 1.000a | ||
Calcium channel blockers (%) | 5 (27.8%) | 5 (41.7%) | 0.461a | |
Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers (%) | 14 (77.8%) | 4 (33.3%) | 0.024a | |
Systolic blood pressure (mmHg) | 127.3 ± 21.7 | 131.5 ± 17.0 | 124.3 ± 12.3 | 0.672 |
Diastolic blood pressure (mmHg) | 77.8 ± 11.2 | 71.6 ± 7.7 | 75.6 ± 8.7 | 0.364 |
Heart rate (beats/min) | 78.2 ± 12.6 | 71.1 ± 10.6 | 78.9 ± 9.1 | 0.162 |
Creatinine (mg/dL) | 0.9 ± 0.6 | 0.9 ± 0.2 | 0.8 ± 0.2 | 0.089 |
eGFR (mL/min/1.73 m2) | 86.2 ± 22.3 | 79.0 ± 17.3 | 92.4 ± 17.1 | 0.093 |
White blood cell count (103/ |
6.1 ± 1.7 | 5.8 ± 1.5 | 6.0 ± 1.4 | 0.775 |
Left atrial diameter (mm) | 43.7 ± 5.1 | 38.8 ± 4.7 | NA | 0.019a |
Left atrial maximal volume (mL) | 86.4 ± 38.8 | 58.8 ± 38.0 | NA | 0.083a |
Left atrial ejection fraction (%) | 51.6 ± 14.4 | 44.7 ± 19.5 | NA | 0.376a |
Left ventricular end-diastolic diameter (mm) | 56.3 ± 6.4 | 58.3 ± 11.9 | NA | 0.384a |
Left ventricular ejection fraction (%) | 67.7 ± 11.0 | 62.2 ± 12.9 | NA | 0.396a |
Data are presented as mean ± SD or number (percentage); AVD: aortic valve disease; MR: mitral regurgitation; NC: control subjects without valve disease and heart failure; NYHA: New York Heart Association; a
Plasma angiotensin II concentrations did not significantly differ between MR patients and aortic valve disease patients (35.18 ± 9.03 vs. 30.13 ± 8.45 pg/mL,
Plasma angiotensin II (Ang II) (a) and angiotensin 1~7 (Ang 1~7) (b) concentrations in mitral regurgitation (MR) patients with heart failure, in aortic valve disease (AVD) patients with heart failure, and in control subjects without valve disease or heart failure (NC).
The MR patients had significantly higher plasma angiotensin 1~7 concentrations compared to aortic valve disease patients (2.42 ± 0.68 vs. 0.96 ± 0.16 ng/mL,
To determine the effects of MR and HF on the gene expression profiles of the renin-angiotensin system, gene expression profiles of the renin-angiotensin system in the left atrium were compared in left atrial tissues from MR patients with HF (
Table
Comparison of mRNA levels through quantitative PCR in the left atria among MR patients with heart failure, aortic valve disease patients with heart failure, and normal controls.
Gene name | MR ( |
AVD ( |
NC ( |
|||
---|---|---|---|---|---|---|
MR vs. NC | AVD vs. NC | MR vs. AVD | ||||
12.22 ± 0.48 | 12.37 ± 0.31 | 9.85 ± 0.66 | 0.023 | 0.005 | 0.657 | |
11.61 ± 0.69 | 12.83 ± 0.34 | 7.57 ± 0.32 | 0.003 | 0.002 | 0.149 | |
13.27 ± 0.36 | 14.05 ± 0.47 | 12.04 ± 0.29 | 0.013 | 0.010 | 0.126 | |
8.86 ± 0.24 | 9.52 ± 0.31 | 8.14 ± 0.10 | 0.007 | 0.002 | 0.068 | |
5.18 ± 0.28 | 4.91 ± 0.25 | 4.09 ± 0.22 | 0.007 | 0.053 | 0.594 | |
6.24 ± 0.25 | 5.13 ± 0.19 | 4.44 ± 0.31 | 0.003 | 0.071 | 0.006 | |
9.47 ± 0.42 | 8.83 ± 0.24 | 7.89 ± 0.08 | 0.002 | 0.007 | 0.424 | |
10.60 ± 0.29 | 10.00 ± 0.18 | 8.72 ± 0.30 | 0.005 | 0.020 | 0.214 | |
10.52 ± 0.55 | 10.66 ± 0.73 | 7.03 ± 0.52 | 0.001 | 0.005 | 0.923 | |
11.71 ± 0.48 | 11.73 ± 0.47 | 9.94 ± 0.59 | 0.042 | 0.042 | 0.949 | |
7.91 ± 0.55 | 6.17 ± 0.27 | 5.18 ± 0.40 | 0.005 | 0.039 | 0.013 | |
11.08 ± 0.54 | 10.66 ± 0.23 | 7.88 ± 0.66 | 0.010 | 0.002 | 0.441 | |
13.88 ± 0.46 | 13.43 ± 0.38 | 11.50 ± 0.94 | 0.063 | 0.053 | 0.298 | |
11.30 ± 0.77 | 10.59 ± 0.41 | 7.64 ± 0.71 | 0.007 | 0.014 | 0.441 | |
6.35 ± 0.90 | 12.19 ± 1.44 | 16.39 ± 0.69 | 0.001 | 0.028 | 0.016 | |
4.89 ± 0.14 | 6.08 ± 0.31 | 0.007 | ||||
4.56 ± 0.28 | Unmeasurable |
Data are presented as mean ± SEM; quantitative RT-PCR values are presented in △Cq units; MR: mitral regurgitation; AVD: aortic valve disease; NC: purchased samples from normal subjects.
Compared to normal controls, the aortic valve disease patients had significantly downregulated expressions of
Figure
Quantitative determination of mRNA of (a) cathepsin A (
The MR patients with and without treatment with renin-angiotensin system blockers (
The MR patients (
This study showed that gene expression patterns of the renin-angiotensin system in the left atrium in MR patients with HF differed from those in aortic valve disease patients with HF and normal controls. Notably, for three genes in the renin-angiotensin system (
To date, the gene expression profiles of the renin-angiotensin system in the atrial myocardium of MR patients have never been examined. A previous study using an atrial fibrillation pig model showed that atrial myocytes express all components of the renin-angiotensin system and undergo structural changes in response to rapid atrial pacing [
Cathepsins, which are lysosomal proteases known to degrade unwanted intracellular or endocytosed proteins, reportedly play functional roles in the pathogenesis of heart disease by contributing to matrix turnover, chamber dilation, and structural remodeling [
Leucyl/cystinyl aminopeptidase (
The
Taken together, the data obtained in this study indicate that expressions of
This study has several limitations. First, although significant differences were observed among the groups in this study, the number of subjects was relatively small. Therefore, studies with larger sample sizes are warranted. Second, most of the MR patients enrolled in the study had received renin-angiotensin system blockers, which may have modified the expressions of some genes. Notably, however, expressions of the 13 genes of the renin-angiotensin system did not significantly differ between MR patients treated with and without renin-angiotensin system blockers. Finally, this study did not specifically investigate the functional and regulatory roles of
Expressions of genes in the renin-angiotensin system, especially
The data used to support the findings of this study are included within the article.
The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
The authors have no conflicts of interest to declare.
Wen-Hao Liu and Yen-Nan Fang contributed equally in this work.
This work was supported by a grant from the National Science Council of Taiwan (grant NSC 102-2314-B-182A-107-MY2). The authors gratefully acknowledge the Chang Gung Medical Foundation Kaohsiung Chang Gung Memorial Hospital Biobank and Tissue Bank Core Laboratory (CLRPG8B0033, CLRPG8E0161, and CLRPG8F1702) for its excellent technical support.