This study examined 22 patients with severe symptomatic MR who had undergone valve operations for heart failure. Exclusion criteria were previous myocardial infarction, febrile disorder, infectious or inflammatory disease, autoimmune disease, malignancy, chronic renal failure (serum creatinine >2.5 mg/dL), acute or chronic viral hepatitis or use of immunosuppressive drugs. Twelve patients had persistent AF [mean (± SD) duration, 47.8 ± 70.2 months; duration range, 1 to 240 months] before surgery (MR AF patients). The sample included ten males and two females with a mean (± SD) age of 67 ± 7 years old (age range, 58 to 81 years old). Ten patients with no history and no records of electrocardiograms of AF before surgery had symptomatic severe MR (MR sinus patients). The sample included two males and eight females with a mean (± SD) age of 56 ± 10 years old (age range, 33 to 68 years old). Informed consent was obtained from all study subjects. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Review Committee for Human Research at our institution. Normal adult left atrial tissue samples were purchased from BioChain Institute, Inc, USA, Novus, USA, and G-bioscience, USA for histochemical and immunochemical studies. These normal atrial tissues were used as the normal controls.
Transthoracic echocardiographic examinations were performed on all patients using a 2.5 MHz transducer attached to a commercially available echo Doppler machine (Sonos 7500; Hewlett-Packard; Palo Alto, CA) on the day before valve surgery. Echocardiographic measurements were performed according to the recommendations of the American Society of Echocardiography.
Measurements of left atrial pressure were performed within one month before surgery.
Atrial tissue was sampled from the left atrial appendage. After excision, atrial tissues were immediately frozen in liquid nitrogen or embedded in optimal cutting temperature compound, and stored at −80 Celsius to be held for later immunofluorescence staining and immunoblotting.
Frozen tissue sections (5 μm) were fixed for 10 min with 4 % paraformaldehyde and then exposed for 50 min in 5 % BSA (bovine serum albumin), followed by incubation with the corresponding antibodies in the double staining procedures. Primary antibodies included antibodies against ROCK1, ROCK2 (1:50 dilution; Santa Cruz, CA, USA), phalloidin-FITC F-actin (counterstaining for myocyte identification; 1:1000 dilution; Sigma, MO, USA), and cleaved caspase-3 (1:200 dilution; Cell signaling, MA, USA) at 4 °C overnight. For confocal microscope, the secondary detection systems were Alex Fluor 488 (green)/594 (red) goat-anti-mouse IgG (AnaSpec, CA, USA) conjugated with ROCK2, and Alex Fluor 594 (red) goat-anti-rabbit IgG (AnaSpec, CA, USA) conjugated with F-actin and caspase-3 that were diluted 1:500 for 30 min at 37 °C. Nuclei were stained with Hoechst 33258 (1:1000 dilution; Sigma, MO, USA). All images of each specimen were captured and examined at high magnification (600×) using an Olympus FV10I-Oil confocal microscope (Tokyo, Japan).
For immunostaining quantification, atrial samples were analyzed with at least 50 randomly chosen cells per each sample. Cell area, myolytic area, integrated intensities of each antibody (calculated after correction of background noise), percentage of co-localization, and Pearson’s coefficient in each myocyte were obtained and calculated by Olympus Fluoview software (Tokyo, Japan). The expression levels of ROCK2, ROCK1 and cleaved caspase 3 were presented as integrated intensities. Atrial myocytes were scored by morphometry as mildly myolytic if <10 % of the sarcomere content was absent, and moderately-to severely myolytic if >10 % of the sarcomere was absent. For co-localization analysis, the average intensity of each antibody was used as co-localization threshold.
Three normal adult left atrial tissue samples were purchased from BioChain Institute, Inc, USA (76 female, 70 female, and 24 male).
Tissues extracts were prepared by PRO-PREP™ protein extraction solution (Intron biotechnology, Gyeonggi-do, Korea). Homogenates were centrifuged at 14000 rpm for 30 min at 4 °C to yield supernatants. The concentrations of sample proteins and 3 normal human left atrial proteins [purchased from Novus, USA (77 male), Biochain, USA (62 female), and G-bioscience, USA (24 male)] were determined by the Bradford method (Bio-Rad) according to the supplier’s instructions. Protein extracts were size-fractionated using SDS-PAGE electrophoresis at 7 °C overnight and electro-transferred onto PVDF membranes for 3 h on ice. Membranes were blocked in Tris-buffered saline, with 0.1 % Tween-20 (TBST) and 5 % BSA at room temperature for 2 h. Primary antibodies included phosphorylation level of myosin-binding subunit of myosin light chain phosphatase (pMBS) (1:1000 dilution; Cyclex, Nagano, Japan), total MBS (tMBS) (1:1000 dilution; Cell signaling, MA, USA), ROCK1 (1:500 dilution; Santa Cruz, CA, USA), ROCK2 (1:500 dilution; Santa Cruz, CA, USA), cleaved caspase-3 (1:1000 dilution; Cell signaling, MA, USA) and GAPDH (1:5000 dilution; Millipore, MA, USA) and were used to react with the blots at 4 °C overnight in 5 % BSA. The blots were washed three times in TBST and incubated at room temperature for 1 h with horseradish peroxidase-labeled secondary antibody at dilutions of 1:5000 in TBST containing 5 % BSA. Following three washings, blots were incubated with Immobilon Western chemiluminescent HRP substrate (Millipore, MA, USA). Densitometry analysis was conducted using Quantity One 1-D Analysis Software (Bio-red, Berkeley, California).
Data were presented as means ± SD or SEM. Categorical variables between the MR AF patients, MR sinus patients, and the normal control subjects were compared using chi-square test or Fisher exact test as appropriate. Moreover, continuous variables in the three groups were compared using a Kruskal-Wallis test. Continuous variables between the two groups of study patients were compared with a Mann–Whitney U test. Co-localization analysis between the expression site of ROCKs and the expression site of cleaved caspase-3 in the left atrial myocytes was performed and presented as Pearson’s correlation coefficient. The correlation between the expression levels of ROCKs in the left atrial myocytes and left atrial dimension was performed with the Spearman’s correlation. Finally, statistical analyses were performed using the statistical software program (SPSS version 19.0; SPSS Inc.; Chicago, Illinois, U.S.A.). All p values were two-sided, and the level of statistical significance was set at 0.05.