Study population
This was an prospective observational study, which began in January 1998 and ended in December 2019. A total of 86 consecutive newly diagnosed PPCM patients were enrolled at the HF clinic of Beijing Chaoyang Hospital. We obtained the demographic data and related information by in-person interview using the structured questionnaire. The inclusion criteria were as follows: (1) age, between 18 and 40 years old, (2) cardiac function, New York Heart Association functional classes (NYHA) II-IV, (3) symptoms of HF, happened in the last month of pregnancy or during the first 5 months postpartum, (4) no other identifiable causes for HF, and (5) LVEF < 45% by transthoracic echocardiography. Exclusion criteria were as follows: (1) clinical conditions with increased levels of autoantibody, such as rheumatoid arthritis, HIV, and evidence for sepsis, (2) moderate-severe anemia, (3) metabolic disorders such as thyroid disease, or (4) moderate-severe hepatic or renal dysfunction. The study protocol complied with the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Chaoyang Hospital, Beijing, China. All the PPCM patients provided written informed consent before study entry.
Serum anti-M2-R detection
About 2 mL of blood was withdrawn from the antecubital vein of each patient when enrolled into this study and after five years treatment. Serum samples were separated by centrifugation at 3,000 rpm for 10 min and stored at –20 °C. Peptide corresponding to the sequence of the second extracellular loop of human M2-muscarinic receptor (amino acid sequence number 169-193: V-R-T-V-E-D-G-E-C-Y-I-Q-F- F-S-N-A-A-V-T-F-G-T-A-I), was synthesized by Genomed (Genomed Synthesis, Inc., San Francisco, CA, USA) with the solid-phase method of Merrifield. The purity of the peptide was 98%, on the basis of HPLC analysis on a Vydac C-18 column. Levels of serum anti-M2-R was measured with SA-ELISA and positive was defined as a ratio of (sample A - blank A)/(negative control A - blank A) ≥ 2.1 [7]. Titers of autoantibodies was the highest when this ratio ≥ 2.1 with serum diluted from 1:20 to 1:160. The coefficient of variation of intra-assay and inter-assay were less than 5%.
Beginning of the standard pharmacological regimen
All the patients received digoxin additional to standard therapy regimens (perindopril or losartan, metoprolol, furosemide, and spironolactone). Perindopril was taken at an initial dose of 2 mg/day, and then uptitrated according to the blood pressure. If perindopril wasn't tolerated, losartan was used instead. Metoprolol was taken at an initial dose of 12.5 mg/day that was up-titrated over a 2–4-week period by doubling the twice-daily amount to the maximum tolerated dose or a target of 100 mg/day [2]. The maximum tolerated heart rate and blood pressure were 60–75 bpm and 120/65 ± 10/5 mmHg, respectively. The initial dosage of furosemide was 10-20 mg/day, which allowed to increase if a patient displayed signs or symptoms of HF progression. The dosage of spironolactone was 10-20 mg/day. Digoxin was taken at an initial dose of 0.125 mg/day and then adjusted according to the serum digoxin concentration (SDC). The target SDC was 0.5-0.9 ng/mL as suggested by the ACCF/AHA guideline for the management of HF [8]. If the SDC was 0.9-1.5 ng/mL, the dosage of digoxin was reduced to 0.0625mg/day. The dosage of digoxin was reduced to 0.0625mg every other day when the SDC was higher than 1.6 ng/mL. In addition, patients were advised to control their salt intake and body weight.
Follow-up examination
All patients were assigned to a fixed investigator for up to 5 years of follow-up after they were included in the study. The primary endpoint events were all-cause mortality, cardiovascular mortality, and re-hospitalization for HF. Patients received follow-up once a month for the first year and every 3–6 months for up to 5 years or until the primary endpoint. Echocardiography, 6-minute walk tests, and clinical laboratory tests including SDC were performed regularly. We collected heart rate, blood pressure, body weight, cardiac function, the presence of peripheral edema, drug dosages during the examinations. Subjects were also questioned and examined for the presence of any adverse drug reaction.
Statistical methods
Quantitative data are presented as mean ± SD, and categorical data are presented as percentage. Titers of serum anti-M2-R was reported as the geometric mean. For two groups comparison, we used Student’s t test or Mann-Whitney U test for continuous variables, and the chi-square test or Fisher’s exact test for categorical variables. Chi-square statistics and log-rank test were used for all-cause mortality, cardiovascular mortality, and hospitalization for HF. Data on the titration of metoprolol were fit to a variable slope sigmoidal equation (Y = Initial Dose + (Maximum Dose − Initial Dose)/(1 + 10(LogEC50 − X)* Slope)), in which the independent variable (X) is the log of the time of the dosage value (Y). The LogEC50 denotes the time that corresponds to halfway between the minimum and maximum dosages. All the tests were 2-tailed. P < 0.05 was considered to be statistically significant. Data were analyzed using SPSS 20.0 (SPSS, Chicago, Illinois, USA).