The ethics committee of the Medical Association of Rheinhessen approved this study. The experiments were carried out in the experimental laboratory of the Department of Cardiothoracic and Vascular Surgery of the University Hospital of Mainz.
All the patients were informed and gave written consent for the use of intraoperative resected tissue in further research. All the experiments were carried out according to the Declaration of Helsinki.
Eighteen female and 18 male patients with pure severe mitral valve insufficiency who were scheduled for elective mitral valve surgery were included in the study. The exclusion criteria were aged <18 years, emergency surgery, atrial fibrillation, primary pulmonary hypertension, other valve lesions, coronary artery disease, an Ejection Fraction of <50 %, and endocarditis.
Skinned fiber preparation
Right auricle tissue was taken from the 36 patients prior to implementation of extracorporal circulation and prepared for skinned fiber experiments. The process of skinned fiber preparation has been described in detail elsewhere [
8‐
10]. Briefly, the fibers were collected in the operating theater after resecting the right auricle. Using the no-touch-technique, the auricular tissue was transferred to an ice-cooled vial (4 °C) filled with an oxygenated cardioplegic solution (1000 ml of Krebs-Henseleit solution in mmol/L of 118.07 NaCl, 11.1 C
6H
12O
6 + H
2O, 4.7 KCK, 25 NaHCO
3, 1.2 KH
2PO
4, 1.2 MgSO
4 + 7 H
2O, and 1.8 CaCl
2 + 2H
2O). From this solution, 100 ml were removed and 30 mmol/L of an ATP-sensitive potassium canal inhibitor, C
4H
2NO
2 (2,3-butanedione monoxime), were added. The auricular was then placed in a dish filled with 20 ml of a preparation solution (4 °C) (amounts in mM: C
3H
4N
2 68.08, NaN
3 65.01, C
14H
24N
2O
10 380.4, C
4H
10O
2S
2.154.3, MgCl
2 × 6 H
2O 203.3, C
10H
14N
5O
13 P
3 Na
2 605.2). Subsequently, the muscle bundles were resected from the auricle and placed in a test tube containing 9900 μl of the preparation solution, including 1 % Triton-X-100 (100 μl), for 24 h at 4 °C on a shaking device. The fibers were skinned after being rolled for 24 h on a special rotation device to remove membrane-dependent properties [
9]. Following this procedure. the fiber bundles were prepared for the experimental setup.
The experimental setup consisted of a specially designed “gradient measurement device,” in which the concentration inside the perfusion chamber was automatically increased stepwise during the experiment (Scientific Instruments, Heidelberg, Germany). The device consists of two pumps, which transports and withdraws a specific amount of the solution in and out of a perfusion chamber. In this perfusion chamber, the skinned fiber was fixed between two forceps and immersed in the solution. As the calcium-containing solution was transported to the perfusion chamber, the concentration was increased in a stepwise manner. When the skinned fiber started to contract, the resulting force-development curve was recorded by the attached computer system. Of course, to achieve a special calcium concentration, which is calculated by the attached computer system, several parameters (the concentration of EGTA, free calcium concentration, tubing volume, volume of the perfusion chamber etc.) have to be entered in the computer system before starting the experiments. Thus, the concentration of calcium can be calculated precisely (Gradient Program, Scientific Instruments, Heidelberg).
For the experimental set-up, the fibers were cut in strips of 2–2.5 mm × 0.3 mm and fixed between two forceps in a cuvette, which served as a perfusion chamber. To achieve steady state conditions, the cuvette was perfused with a relaxation solution (contents in mM: C3H4N2 68.08, C4H8N3O5PNa2 + 4 H2O 327.2, NaN3 65.01, C14H24N2O10 380.4, MgCl2 203.3, C4H10O2S2.154.2, and C10H14N5O13 P3 Na2 605.2) and 400 U/ml of creatine kinase (Boehringer, Mannheim, FRG). To induce contraction, solution calcium and CaCl2 (147.02 mM) were added. The free calcium concentration was obtained by mixing the relaxation and contraction solution in appropriate proportions. The desired calcium concentrations were calculated by a computer program (Scientific Instruments, Heidelberg, Germany), following the equation of Fabiato and Fabiato and given as pCa (−log of free [Ca]+) until steady state conditions were observed. Afterwards, the fibers were carefully prestretched to 20 mg to achieve optimal overlapping of contractile elements (according to a cardiomyocyte sarcomere length of 2 μm). After achieving a steady state, the fibers were exposed to the contraction solution. A computer program calculated the desired calcium concentrations (i.e., pCa 6.5 until 4.0) (Scientific Instruments Heidelberg, Germany).
The fibers were exposed to a continuous increase in the calcium concentration, starting at pCa 6.5 (lowest calcium concentration) and ending at pCa 4.0 (highest calcium concentration). The rising calcium concentration and responding force development were simultaneously recorded and sampled on the attached computer. Three fibers from each patient were exposed to cycles of increasing calcium concentrations. Thus, the final analysis consisted of 54 data sets for female fibers and 54 data sets for male fibers.
Statistical analysis
The statistical analysis was performed using SPSS software 13.0 for PC (Analytical Software, Chicago, IL, USA). Values are expressed as the mean ± S.D, and numbers of patients are expressed as percentages. The Shapiro–Wilk test was used to verify normal statistical distributions. The Welch test was used to evaluate statistically significant differences between males and females in RAP, wedge, LVESV, height, weight, and BMI. The Wilcoxon rank-sum test was used to verify statistically significant differences in the force values of pCa 6.5–4.0, TAPSE, age, and EF. A Chi square test was used to evaluate differences in the prevalence of the dilatation of the mitral valve ring, LA and LV dilatation, and grade of TR and MR. Statistical significance was determined using an alpha level of < 0.05.