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After haemodynamic values had again returned to baseline, an endomyocardial biopsy was performed to exclude myocarditis (according to the Dallas criteria13) or specific heart muscle disease. Several (at least three) endomyocardial biopsy specimens were obtained from the septal wall of the right ventricle. Biopsy samples for mRNA analysis were frozen immediately in liquid nitrogen and stored at ?80��C until use. A diagnosis of MA was made if the pressure difference between the strong and weak beats was ��?4?mmHg Cobimetinib continuously for the LV pressure data analysed, as described previously.9 The occurrence of MA was determined independently by two experienced cardiologists (AH and HI). Quantitative reverse transcription�Cpolymerase chain reaction (RT-PCR) analysis of the mRNA expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2a, the ryanodine RyR2 receptor, phospholamban, click here calsequestrin and the Na+�CCa2+ exchanger was performed using a Prism 7700 Sequence Detector (Perkin-Elmer, Foster City, CA, USA), as described previously.14 The amount of each mRNA was normalized against the corresponding amount of GAPDH mRNA. All patients were followed up prospectively for the occurrence of primary events, defined as cardiac death (death from worsening heart failure or sudden death), unscheduled readmission to hospital for worsening heart failure or receipt of an implantable cardioverter defibrillator (ICD) because of life-threatening arrhythmia. Data are presented as the mean?��?SD. ALG1 Baseline characteristics and haemodynamic variables were compared among groups by one-way factorial analysis of variance (anova); if a significant difference was detected, inter-group comparisons were performed using Scheff����s multiple-comparison test. Measurements from the strong and weak beats of MA were also compared with paired Student��s t-test. Interobserver variability in the identification of MA was assessed with Cohen��s kappa (��) coefficient. Cox��s proportional hazard regression analysis was used to identify independent predictors of cardiac events. We also performed a stepwise forward selection procedure. Cumulative cardiac event estimates were calculated by the Kaplan�CMeier method; differences between the survival curves were assessed by the log rank test. All analyses were performed with spss 12.0 software (SPSS, Chicago, IL, USA) and P?