The authors have declared that no competing interests exist.
Conceived and designed the experiments: LZ XY. Performed the experiments: JL YW MC XW HW ZZ. Analyzed the data: YW JZ LX WZ. Contributed reagents/materials/analysis tools: JL YW MC JC. Wrote the paper: YW LZ.
Peripartum cardiomyopathy (PPCM) is characterized by left ventricular systolic dysfunction and heart failure. However, its pathogenesis is not clear. Our preliminary study revealed that autoantibodies against β1-adrenergic receptors (β1R-AABs) and M2-muscarinic receptors (M2R-AABs) participated in heart failure regardless of primary heart disease. Whether β1R-AABs and M2R-AABs participate in the pathogenesis of PPCM is still unknown.
Totally 37 diagnosed PPCM patients and 36 normal pregnant women were enrolled in this study. Clinical assessment and 2-dimensional echocardiographic studies as well as the measurement of β1R-AABs or M2R-AABs by enzyme linked immunosorbent assay (ELISA) were performed.
The positive rates for β1R-AABs and M2R-AABs were 59.5% (22/37) and 45.9% (17/37) in PPCM patients, and 19.4% (7/36) (
The β1R-AABs and M2R-AABs reveal a significant elevation and are correlated with the increased left ventricular dimension and worse cardiac contraction function. The autoantibodies of cardiovascular receptors are independent risk factors for the onset of PPCM.
Peripartum cardiomyopathy (PPCM) is a serious disease with unknown etiology, which occurs between the last month of pregnancy and 5-month puerperium with significant morbidity and mortality. The diagnostic criteria include: (1) The disease occurs between the last month of pregnancy and 5-month puerperium; (2) There has no definite reason for heart failure; (3) LVEF <45%, LVFS <30%. The reported incidence fluctuates among geographic regions, but it is higher in Africa. It has been estimated to be 1∶3000–4000 in USA, 1∶400 in Haiti, and 1∶1000 in South Africa
The role of autoimmunity in cardiovascular diseases has become one of the focal points in the field of cardiomyopathy and heart failure. The β1-adrenergic and M2-muscarinic receptors belong to the family of cardiac G-protein-coupled receptors. Circulating autoantibodies against the second extracellular loop of β1-adrenergic receptors and M2-muscarinic receptors have been detected in a number of cardiovascular diseases characterized by heart failure including idiopathic dilated cardiomyopathy (IDCM)
The characteristics of the study population at the first presence in cardiac clinic (baseline) are shown in
Parameters | Value |
29±6 | |
1 (1–3) | |
18 (48.6) | |
II | 1 (2.7) |
III | 16 (43.2) |
IV | 20 (54.1) |
Ante partum | 16 |
1–3 month post-partum | 21 |
Systolic | 131±13 |
Diastolic | 86±8 |
100±19 | |
Left-ventricular EDD (mm) | 59±6 |
Left-ventriculer ESD (mm) | 47±7 |
Ejection fraction (%) | 38±7 |
Fractional shortening (%) | 19±4 |
Data were expressed as numbers (%), mean ± SD, or medians (interquartile range, IQR),
EDD = end-diastolic diameter, ESD = end-systolic diameter, NYHA = New York Heart Association.
Parameters | PPCM (n = 37) | NP (n = 36) | |
29±6 | 28±3 | 0.867 | |
Systolic | 131±13 | 115±7 | |
Diastolic | 86±8 | 73±6 | |
100±19 | 89±12 | ||
2901.00(1116.00,7078.00) | 113.42(95.24,162.00) | ||
Left-ventricular EDD (mm) | 59±6 | 45±5 | |
Left-ventriculer ESD (mm) | 47±7 | 27±4 | |
Ejection fraction (%) | 38±7 | 69±5 | |
Fractional shortening (%) | 19±4 | 41±4 |
Data were expressed as numbers (%), mean ± SD, or medians (interquartile range, IQR),
PPCM = peripartum cardiomyopathy, NP = normal pregnant, NT-proBNP
The positive sera for β1R-AABs were observed in 59.5% (22/37) of PPCM patients, while 19.4% (7/36) (
One patient (3%) died during the study period. The death was due to the progression of heart failure. Since the death was due to the progression of heart failure, patients who died were classified in FC IV at the latest follow-up examination. All patients received standard therapy for heart failure, which included daily 0.125 mg of digitalis, furosemide [median daily dose 40 mg (20–40 mg)], perindopril [median daily dose 4 mg (2–4 mg)], and betaloc [median daily dose 25 mg (6.25–50 mg)].
Patients who completed 12 months of standard therapy showed a significant reduction in heart rate, LV dimension, and significant improvement in echocardiographic LVEF and LVFS (
Baseline | 12 months | ||
I | 0 | 18 | |
II | 1 | 17 | |
III | 16 | 1 | |
IV | 20 | 0 | |
Systolic | 131±13 | 115±11 | |
Diastolic | 86±8 | 75±6 | |
100±19 | 72±8 | ||
2901 (1116, 7078) | 432 (34,1268) | ||
Left-ventricular EDD (mm) | 59±6 | 49±5 | |
Left-ventricular ESD (mm) | 47±7 | 34±6 | |
Ejection fraction (%) | 38±7 | 62±8 | |
Fractional shortening (%) | 19±4 | 31±4 |
Data were expressed as numbers (%), mean ± SD, or medians (interquartile range, IQR),
NYHA = New York Heart Association, EDD = end-diastolic diameter, ESD = end-systolic diameter, NT-proBNP
After 12 months treatment, the positive rate for β1R-AABs declined from 59.5% to 22.2% (
After 12-months treatment, the frequency and geometric titers of both β1R-AABs and M2R-AABs were decreased significantly (
There were 17 (46%) patients with double positive (DP) autoantibodies, 5 (14%) patients with single positive (SP) autoantibodies and 15 (40%) patients with double negative (DN) autoantibodies. Echocardiographic data among three groups had significant difference: left ventricular end diastolic dimension (LVEDD): 62.5±7.1 vs. 56.9±1.0 vs. 54.8±4.3, (χ2 = 14.411,
Echocardiographic data among patients with double positive (DP) autoantibodies, single positive autoantibodies (SP) and double negative (DN) autoantibodies had significant difference: LVEDD and LVESD: DP>SP>DN (
The levels of serum β1R-AABs and M2R-AABs (both frequency and titer of autoantibody) were positively correlated with left ventricular dimension, NT-proBNP and NYHA FC (Spearman coefficient differs from 0.378 to 0.590, all
Frequency of autoantibody | Titer of autoantibody | |||
Spearman coefficient | Spearman coefficient | |||
0.892 | 0.702 | |||
0.567 | 0.581 | |||
Left-ventricular EDD (mm) | 0.578 | 0.525 | ||
Left-ventricular ESD (mm) | 0.601 | 0.496 | ||
Ejection fraction (%) | −0.561 | −0.568 | ||
Fractional shortening (%) | −0.488 | −0.499 |
NYHA FC = New York Heart Association functional class, NT-proBNP
Frequency of autoantibody | Titer of autoantibody | |||
Spearman coefficient | Spearman coefficient | |||
0.736 | 0.625 | |||
0.541 | 0.554 | |||
Left-ventricular EDD (mm) | 0.607 | 0.530 | ||
Left-ventricular ESD (mm) | 0.622 | 0.536 | ||
Ejection fraction (%) | −0.526 | −0.532 | ||
Fractional shortening (%) | −0.498 | −0.528 |
NYHA FC = New York Heart Association functional class, NT-proBNP
Univariate analysis showed that several factors increased the risk for the onset of PPCM, including autoantibodies against cardiovascular receptors, advanced maternal age, multiple pregnancies, pregnancy-induced hypertension, and poor socioeconomic status. Multiple logistic analysis showed that the level of autoantibodies against cardiovascular receptors and pregnancy-induced hypertension were an independent predictor of the PPCM attack (autoantibodies against cardiovascular receptors: OR = 18.786, 95% confidence interval 1.926–183.262,
Univariate analysis | Multivariate analysis | |||||
OR | 95% CI | OR | 95% CI | |||
Autoantibody | 28.6 | 5.979–138.186 | 18.786 | 1.926–183.262 | ||
Advanced maternal age | 2.635 | 1.007–6.898 | ||||
Multiple pregnancies | 5.01 | 1.460–17.189 | ||||
Multiparity | 3.259 | 0.351–35.517 | 0.284 | |||
16.625 | 4.339–63.705 | 17.305 | 1.061–282.299 | |||
Poor socioeconomic status | 6.821 | 2.190–21.244 |
In this study, synthetic peptides corresponding to the sequences of the second extracellular loop of human β1 receptor (amino acid sequence number: 197–222) and M2 receptor (amino acid sequence number: 169–193) were used. We detected the autoantibodies against β1 and M2 receptors in patients’ sera with ELISA and demonstrated that the frequency and titer of serum β1R-AABs and M2R-AABs in patients with PPCM were higher than those of normal pregnant women. The comparison between β1R-AABs and M2R-AABs double positive group, single positive group and double negative group revealed that the expansion of left ventricular and decrease of heart function: double positive group> single positive group> double negative group. After treatment for 1 year, recovery of left ventricular dimension and heart function: double positive group< single positive group< double negative group. The test of β1R-AABs and M2R-AABs may have predicted value for the prognosis of PPCM. The levels (both frequency and titer) of serum β1R-AABs and M2R-AABs were positively associated with New York Heart Association functional class, left ventricular dimension and plasma NT-proBNP levels, and were negatively associated with left ventricular ejection fraction, left ventricular fractional shortening. Autoantibodies against cardiovascular receptors were independent predictors of the onset of PPCM. This study suggested that β1R-AABs and M2R-AABs play an important role in the pathogenesis of PPCM.
During the past few years, autoimmune mechanisms in heart disease have gained extensive attentions. Autoimmunity has been observed in several cardiovascular diseases such as dilated cardiomyopathy (DCM) and heart failure, rheumatic fever, myocarditis, atherosclerosis, and others
Our preliminary study revealed that β1R-AABs and M2R-AABs not only existed in IDCM patients, but also in heart failure caused by different cardiac diseases. Moreover, the serum level of autoantibody has no significant difference among different heart diseases, which suggested that the generation of β1R-AABs and M2R-AABs had no relationship with primary heart disease, but was correlated with the damage of cardiac function.
PPCM is characterized as systolic dysfunction and heart failure as well as IDCM. Its pathogenesis was still unclear. The autoimmune mechanism was believed to participate in the pathogenesis of PPCM. High titers of auto-antibodies against selected cardiac tissue proteins have been found in the majority of women with PPCM
Previous studies revealed that β1R-AABs and M2R-AABs might lead to cardiac remodeling in cardiomyopathy. In the present study, significantly higher levels of β1R-AABs and M2R-AABs in the serum of PPCM than normal pregnant women were observed. For PPCM patients, the enlarged left ventricular chamber may return to normal level, which seems to improve better than IDCM. In our study, the titer of autoantibodies revealed a decline, which is coincided with the decrease of left ventricular and the improvement of heart function after treatments. Therefore, β1R-AABs and M2R-AABs may participate in the pathological change of cardiomyocytes. What’s more, these two autoantibodies were the independent risk factors for the attack of PPCM and may influence the prognosis of PPCM patients. All of these investigations suggested that β1R-AABs and M2R-AABs might participate in the pathogenesis of PPCM.
It’s interesting to notice that, a hypothesis currently being widely entertained is that PPCM may be a vascular disease
PPCM is a diagnosis by exclusion. As the objects of this study are pregnant women, the coronary angiography, and right and left catheterization are not allowed for application to exclude other kinds of cardiac diseases. Because the morbidity of PPCM is not very high in China, especially in Beijing, the case number is insufficient. As all these patients enrolled in our study were Chinese Han and body mass index (BMI) was not mentioned in this article, the assessment of autoantibodies as an independent risk factor for PPCM were incomplete, which could result in the continuous improvement in the future. What’s more, the predictive value of autoantibodies against cardiovascular receptors for the prognosis of PPCM needs further studies.
This study has demonstrated that the presence of β1R-AABs and M2R-AABs were prevalent in a cohort of PPCM patients. The positive rates and the serum titers of β1R-AABs and M2R-AABs were positively correlated with left ventricular dimension, and negatively correlated with the cardiac contraction function of PPCM patients. The autoantibodies of cardiovascular receptors were independent risk factors for the onset of PPCM.
We propose that autoantibodies against cardiovascular receptors may be involved in the pathogenesis of PPCM. Further studies are needed to confirm these findings and dissect the underlying mechanisms for this novel observation.
The protocol as well as the consent form was approved by the Ethics Committee of Capital Medical University Beijing Chao-Yang Hospital and the study protocol complied with the Declaration of Helsinki. All participants provided written informed consent prior to admission into the study.
Since 1998, 37 Chinese Hanpatients with newly diagnosed PPCM were enrolled in Beijing Chao-Yang Hospital. The enrolled patients should meet following inclusion criteria: (1) age ≥16 or ≤40 years old, (2) New York Heart Association functional classes (NYHA FCs) II–IV, (3) symptoms of congestive heart failure developed in the last month of pregnancy or during the first 5 months post-partum, (4) no other identifiable causes for heart failure, (5) left ventricular ejection fraction (LVEF) <45%, left ventricular fractional shortening <30% by transthoracic echocardiography, (6) sinus rhythm and (7) eligible patients with high quality echocardiographic images. Exclusion criteria: (1) significant organic valvular heart disease, (2) systolic blood pressure (SBP) >160 mmHg and/or diastolic blood pressure >100 mmHg, (3) clinical conditions other than cardiomyopathy with increased autoantibody by screening the serum for rheumatoid arthritis and HIV and the evidence of sepsis, (4) severe anaemia (haemoglobin concentration <9 g/dL), (5) metabolic disorders affecting lipoprotein metabolism such as thyroid disease and (6) Clinical data are not complete. Clinical assessment, echocardiography and blood analysis were conducted at baseline and after 12 months of standard therapy. All patients received standard therapy for PPCM (β-blocker, angiotensin-converting enzyme inhibitors, diuretics and if indicated digoxin). Patients with an ejection fraction ≤25% or LV thrombus additionally received anticoagulation therapy. The follow-up examination at Beijing Chao-Yang Hospital was provided for all patients.
The autoantibodies against β1-adrenergic and M2-muscarinic receptors were measured in patient sera through an enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide corresponding to the sequence of the second extracellular loop of human β1 and M2 receptor (amino acid sequence number, β1∶197–222: H-W-W-R-A-E-S-D-E-A-R-R-C-Y-N-D-P-K-C-C-D-F-V-T-N-R; M2∶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). The peptide was synthesized by Biological Institute of the Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC) using a solid-phase method of Merrifield, and the purity was evaluated by high-pressure liquid chromatography (HPLC) on an automatic amino acid analyzer (Beckman instrument, Inc, USA). The procedures of ELISA were as follows: 50 µL of peptide (5 mg/L) in 100 mmol/L Na2CO3 solution (pH = 11.0) was incubated overnight at 4°C on NUNC (Kastrup, Denmark) microtiter plates. The wells were saturated with phosphate-buffered saline (PBS: 10 mmol/L phosphate, 140 mmol/L NaCl, pH 7.3–7.4) supplemented with 0.01% (w/v) Thimerosal (Sigma, St. Louis MO, USA), 0.1% (v/v) Tween-20 (Sigma, St. Louis, MO, USA) (PBS-T) and 10% (v/v) fetal bovine serum (PMT). A total of 50 µL of patient serum with gradient dilutions from 1∶20 to 1∶160 was added to saturated microtiter plates and incubated for 1 h at 37°C. After the wells were washed with PBS-T for 3 times, an affinity-purified biotinylated rabbit anti-human IgG (H+L) antibody (Jackson Laboratories, PA, USA) (1∶500 dilution in PMT) was added and allowed to react for 1 h. After washing the wells for 3 times with PBS-T, the bound biotinylated antibody was detected by incubating the plates for 1 h with streptavidin-peroxidase (2 ml/L) (Jackson Laboratories, PA, USA) solution in PMT, which was followed by washing for 3 times with PBS and the addition of substrate for peroxidase 2.5 mmol/L H2O2-2 mmol/L ABTS (Sigma, St. Louis, MO, USA). Optical density (OD) was recorded after 30 min at 405 nm in a micro plate reader (Molecular Devices Corp, Menlo Park, CA, USA). Positive rate was defined as positive/negative [P/N = (sample OD-blank OD)/blank OD)] ≥2.1
All studies were performed and interpreted by the same operator who was blinded to the protocol. Two-dimensional targeted M-mode echocardiography with Doppler color flow mapping was performed for every patient. Left ventricular dimension was measured according to the American Society of Echocardiography guidelines
The measured data were expressed as mean ± SD or median (interquartile range, IQR) and categorical data were expressed as percentage. Antibody titer was reported as geometric mean. Positive rate of autoantibody was described as the frequency of autoantibody. For two-group comparison, we used the 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. For three-group comparison, Kruskal-Wallis test was used. Correlation between the levels of serum anti-β1-AABs or anti-M2R-AABs and the echocardiographic and clinical indices were assessed by Spearman’s correlation. Risk factor analysis was performed by univariate and multivariate logistic analysis. All tests were 2-tailed, and statistically significant difference was considered at