Effects of autoantibody against M2-muscarinic acetylcholine receptor in peripartum cardiomyopathy patients on digoxin additional to standard treatment

doi:10.21203/rs.3.rs-199129/v1

Objectives

To evaluate the effects of autoantibodies against the M2-muscarinic receptor (anti-M2-R) on digoxin additional to standard treatment in peripartum cardiomyopathy (PPCM) patients.

Methods

107 PPCM patients, receiving digoxin and standard treatment regimen for HF, were enrolled between January 1998 and June 2020, who were separated into anti-M2-R negative (n = 59) or positive (n = 48) group according to the anti-M2-R reactivity. Echocardiography and serum digoxin concentration (SDC) were performed regularly. All-cause mortality, cardiovascular mortality and re-hospitalization for heart failure were compared.

Results

103 patients completed the final data analysis, including 46 in the anti-M2-R (+) group and 57 in the anti-M2-R (-) group. Heart rate of the positive group was lower than that of the negative group at baseline (102.7 ± 6.1 vs. 96.0 ± 6.4, p < 0.001). The initial SDC of patients in the positive group was higher than that of patients in the negative group with the same dosage of digoxin (1.25 ± 0.45 vs. 0.78 ± 0.24 ng/mL, p < 0.001). Anti-M2-R (-) patients had better tolerance to metoprolol (38.4 ± 4.6 mg b.i.d. vs. 27.4 ± 5.0 mg b.i.d., p < 0.0001) and digoxin (0.12 ± 0.02 mg/day vs. 0.08 ± 0.04 mg/day, p < 0.0001). The improvement of heart function was obvious in the first year, especially in the first half. Furthermore, anti-M2-R (-) patients showed better improvement than that in anti-M2-R (+) patients. Re-hospitalization for heart failure was decreased in the negative group, but not of all-cause or cardiovascular mortality.

Conclusions

Anti-M2-R maybe a predictor for vagus nerve overactivation and is associated with poor response to digoxin in PPCM patients.

Cardiac & Cardiovascular Systems

Anti-M2-muscarinic receptor

digoxin

peripartum cardiomyopathy

vagus nervous system

Peripartum cardiomyopathy (PPCM) is a rare idiopathic dilated cardiomyopathy defined by the signs and symptoms of heart failure (HF) in the last month of pregnancy through the fifth month postpartum [1]. The definition of PPCM states that there must be no previously known structural heart disease, and echocardiographic parameters must achieve one of the following: left ventricular ejection fraction (LVEF) < 45%, fractional shortening < 30%, or both, with a possible additive left ventricular end diastolic dimension > 2.7 cm/m² body surface area [2]. This disease is associated with a high morbidity and mortality but its aetiology remains unknown [1].

The excessive activation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system has been recognized as main pathogenesis of HF by most doctors. However, the importance of the vagus nervous system is relatively unfamiliar. The M2-muscarinic acetylcholine receptor is the most important receptor for the vagus nervous system to act on the heart. Autoantibodies against the M2-muscarinic receptor (anti-M2-R), have been found in HF patients of various etiologies including PPCM [3–5], which can interfere with radioligand binding on the target receptor, display agonist-like activities on the M2 receptors, and hence modulate cardiac function [6].

Digitalis is a positive inotropic agent, also has the function of vagus nervous system stimulation. Digoxin is the most commonly used oral digitalis in HF patients. So it could neutralize the over activation of the sympathetic system and RAAS, which is similar to the effect of anti-M2-R. While, what is the clinical significance of anti-M2-R? Are there any different responses to digoxin and β receptor blocker between patients negative and positive for anti-M2-R? What is the difference in clinical outcome between the two groups? In this study, we investigate the presence of anti-M2-R and cardiac function in response to digoxin additional to standard treatment regimen for HF (ACEI, β receptor blocker, furosemide and spironolactone) in PPCM patients.

Study population

This was an prospective observational study, which began in January 1998 and ended in June 2020. A total of 107 consecutive newly diagnosed PPCM patients, receiving digoxin and standard treatment regimen for HF, were enrolled at Beijing Chao-Yang Hospital and BENQ Medical Center. 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, and (4) no other identifiable causes for HF. 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 Chao-Yang Hospital and BENQ Medical Center. All the 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%.

Standard pharmacological regimen

All the patients received standard therapy regimens for HF (perindopril or losartan, metoprolol, furosemide, and spironolactone). Patients diagnosed prenatally were given furosemide and metoprolol firstly, and perindopril and spironolactone were added after fetus delivery. In patients with NYHA functional class III-IV, we prescribed digoxin at the beginning of treatment, and it was started when whose symptoms were still present after treatment for a month in patients with NYHA functional class II. The maximum tolerated heart rate and blood pressure were 60–75 bpm and 120/65 ± 10/5 mmHg, respectively. Perindopril was taken at an initial dose of 1-2 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 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 0.9-1.8 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 1.8 ng/mL. In addition, patients were advised to control their salt intake and body weight.

If the structural and functional of left ventricular returned to normal (LVEDD < 50 mm and LVEF > 50%) within one year, administration of digoxin will be stopped. If it occurred during the second year, we stopped metoprolol, spironolactone, furosemide and digoxin gradually, and keep perindopril for only.

Follow-up examination

All patients were assigned to a fixed investigator for two 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 the second year. Echocardiography and 6-minute walk tests were performed every 6 months. Clinical laboratory tests including SDC was tested in the first month and every 3 months thereafter. Serum anti-M2-R testing was detected once a year. 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 test for continuous variables, Wilcoxon rank‐sum test for nonparametric variables, and the chi-square test for categorical variables. 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. Chi-square statistics and log-rank test were used for all-cause mortality, cardiovascular mortality, and hospitalization for HF. 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).

Study characteristics

All the 107 patients were diagnosed as PPCM for the first time. 46 patients were primiparous, and 29 patients had multiple gestation. There were 41 patients who had pregnancy-induced hypertension, and 28 patients had gestational diabetes mellitus. There were 62 patients with symptoms in the postpartum period. At baseline, 35 patients were in NYHA functional class II, 50 patients were in class III, and 22 patients were in class IV.

According to the anti-M2-R reactivity, 59 patients were assigned to the negative group and the other 48 patients were assigned to the positive group. The baseline characteristics of the two groups were shown in Table 1. Four patients lost to follow-up during the first year (2 patients in the negative group and 2 patients in the positive group), and the other 103 patients completed the final data analysis, including 46 patients (46/48, 95.8%) in the positive group and 57 patients (57/59, 96.6%) in the negative group. Of all the parameters, only the mean resting heart rate of the negative group was higher than that of the positive group. We posit that anti-M2-R maybe influence heart rate via the activation of the vagus nervous system.

Drug dosages

All the patients received digoxin and standard pharmacological regimen for HF, which includes perindopril/losartan, metoprolol, spironolactone, and furosemide. In both groups, 4 patients were shifted to losartan respectively and all the dosages were 50mg/day. There were no differences between two groups regarding the dosages of perindopril, spironolactone and furosemide. The dosages of metoprolol and digoxin in the negative group was higher than that in the positive group, shown in Table 2.

Titration of metoprolol

Patients in the anti-M2-R (-) group demonstrated a better tolerance and a more rapid rate of up-titration of metoprolol than those in the anti-M2-R (+) group. The maximum tolerated dose of metoprolol for the anti-M2-R (-) group was 38.4 ± 4.6 mg b.i.d., which was higher than 27.4 ± 5.0 mg b.i.d. for the anti-M2-R (+) group (p < 0.001), as shown in Fig.1. The mean time to maximum tolerated dose of metoprolol was 67.5 ± 10.1 days in the anti-M2-R (-) group, which was shorter than 81.4 ± 11.1 days in the anti-M2-R (+) group (p < 0.001).

Serum digoxin concentration and the dose of digoxin

The target SDC was 0.5-0.9 ng/mL. We prescribed digoxin at an initial dose of 0.125 mg daily. The mean SDC was significantly higher in patients with anti-M2-R (+) than those negative for anti-M2-R for the first time detection (1.25 ± 0.45 vs. 0.78 ± 0.24 ng/mL, p < 0.001). The mean maintenance dose of digoxin was 0.12 ± 0.02 mg/day in the anti-M2-R (–) group, significantly higher than 0.08 ± 0.04 mg/day of the anti-M2-R (+) group ( p < 0.001).

Dynamic variation of the serum anti-M2-R

Sera positive for anti-M2-R was found in 44.9% (48/107) of the PPCM patients at enrollment. In positive cases, the mean titer of anti-M2-R was 1:120. In the first and second years after treatment, the positive rate of serum anti-M2-R dropped to 23.2% (23/99) and 10.1% (10/99), and the mean titer dropped to 1:56 and 1:53, respectively, which were significantly decreased compared to baseline (all p < 0.001) (Fig.2).

Adverse events

There were no obvious effects on blood glucose, serum lipid, or hepatic and renal function during the treatment and follow-up period. Only one patient in the positive group showed symptom of digoxin intoxication such as somewhat weakness and nausea. The SDC of this patient was 2.5 ng/mL, and the dosage of digoxin was reduced to 0.0625 mg every other day, and the above symptoms disappeared subsequently.

Cardiac function and 6-minute walk test

Clinical data, NYHA functional class, echocardiographic results, and 6-minute walk distance at baseline, 6-month, 1-year and 2-year were determined, shown in Fig 3. With low dose of digoxin additional to standard pharmacological treatment for HF, the LVEDD decreased from 60.43 ± 5.8 to 46.3 ± 2.0 mm in the anti-M2-R (-) group, and from 60.7 ± 5.6 to 47.6 ± 3.2 mm in the anti-M2-R (+) group. The LVEF increased from 38.2 ± 3.2 to 62.6 ± 5.9% in the anti-M2-R (-) group and from 38.0 ± 4.9 to 57.2 ± 4.1% in the anti-M2-R (+) group. Meanwhile, the 6-min walk distance increased from 199.8 ± 78.0 to 503.9 ± 58.1 m in the anti-M2-R (-) group and from 195.9 ± 83.2 to 463.1 ± 56.1 m in the anti-M2-R (+) group. Obviously, the structure and function of the left ventricular including a 6-minute walking distance improved greatly in the first year, especially in the first half. It is worth noting that anti-M2-R (-) patients showed better improvement than that in anti-M2-R (+) patients. The LVEF returned to normal in 89.3% (92/103) of patients at the first year and 94.2% (97/103) of patients at the second year. Laboratory data, including levels of hemoglobin, creatinine, glutamic pyruvic transaminase, and potassium were stable throughout the follow-up.

Primary endpoint events

During the follow-up, four patients died during hospitalization and the deaths were due to the progression of HF. One patient was in the anti-M2-R (-) group and the other three deaths were in the anti-M2-R (+) group. There were nineteen patients re-hospitalized for acute exacerbation of HF, including 5 patients in the anti-M2-R (-) group and 14 patients in the anti-M2-R (+) group (p = 0.006). Although it is not advised, three patients in the anti-M2-R (-) group with normalized LVEF had subsequent pregnancies safely without PPCM recurrence. There were no differences in all-cause mortality or cardiovascular mortality between the two groups (p > 0.05), as shown in Fig.4.

Major Findings

In this study, we observed 103 PPCM patients with low dose of digoxin additional to standard therapy for HF. We observed some characteristics. (Ⅰ) We found the heart rate of anti-M2-R (+) patients was lower than that in anti-M2-R (–) patients. (Ⅱ) Patients in the anti-M2-R (-) group had better tolerance to metoprolol and digoxin compared to patients positive for anti-M2-R. (Ⅲ) With the same dosage of digoxin, the mean SDC was higher in anti-M2-R (+) patients than that in the anti-M2-R (-) patients. (IV) LVEF ≥ 50% was observed in 89.3% (92/103) of patients at one year and 94.2% (97/103) of patients at two years. Anti-M2-R (-) patients showed greater reduction of left ventricular diameter and improvement in cardiac function compared to positive patients. (V) Four patients died due to the progression of HF. Risk of hospitalization for HF was decreased in the negative group. However, there were no differences in all-cause mortality or cardiovascular mortality between the two groups.

Digoxin and HF

Digoxin is a traditional drug for HF treatment for more than 200 years. It enhances LVEF by inhibiting the Na⁺-K⁺-ATPase activity on the cardiomyocytes and enhancing myocardial contractility. By inhibiting the activity of Na⁺-K⁺-ATPase in the afferent fibers of the vagus nerve, it can increase the activity of vagus nervous system and inhibiting the sympathetic activity. Besides, it can also increase the sensitivity of the carotid sinus baroreceptor to reduce the activity of the sympathetic and RAAS [9]. It is demonstrated that digoxin was able to improve maximum exercise endurance of patients with mild-moderate stable HF and reduce acute exacerbation of HF whether the basic treatment used diuretics alone or diuretics in combination with ACEIs [10]. It is important to note that the SDC was closely correlated with the prognosis of HF. Ahmed et al [11] reported that 0.5-0.9 ng/mL of SDC could reduce the mortality, all-cause hospitalization and HF-associated hospitalization, while SDC ≥ 1.0 ng/mL could only reduce the HF-associated hospitalization without impact on the mortality. Low-dose of digoxin (≤ 0.125 mg/day) was the strongest predictive factor of the low SDC. Digoxin should be added in the early stage of treatment in patients with severe HF, or those whose symptoms are still present after ACEIs and β receptor blockers have begun working [8]. In this study, 72 patients with NYHA functional class III-IV started digoxin administration at the beginning of treatment, and other 35 patients with NYHA functional class II started digoxin after treatment for a month. All the patients reacted well to low dose of digoxin. Symptoms of digoxin intoxication occurred when the SDC was significantly elevated, almost about 2.5 ng/mL. Regularly testing of the SDC was essential when using digoxin.

Anti-M2-R and PPCM

The M2 receptor is the main muscarinic acetylcholine receptor expression on cardiomyocytes [12]. Anti-M2-R was detected in the idiopathic dilated cardiomyopathy (IDCM) patients firstly [7]. It could induce ventricular enlargement and thinning of the walls, the typical changes of IDCM and PPCM in humans, by monthly immunization peptides in accordance to the sequence of the second extracellular loop of the M2 receptor in rabbits [13,14]. Gimenez et al [15] have immunized mouse by using plasmid DNA encoding entire M2 receptor proteins, which leads to cardiac remodeling and contractile dysfunction. Ribeiro et al [16] found immunization with plasmids encoding M2 receptor epitopes impairs cardiac function in mice and induces autophagy in the myocardium, indicating novel roles for the anti-M2-R. Our previous study suggested anti-M2-R not only existed in IDCM patients, but also in HF caused by different causes, including PPCM. We posit that serum anti-M2-R may be related to cardiac structural and functional changes, which need further studies to clarify [3].

Regulation of the autonomic system

Autonomic system has an important influence on the progression of PPCM. Elevated activities of sympathetic system is associated with an adverse prognosis. Activation of the vagus nervous system seems to be a double-edged sword. With the extensive use of ACEIs and β rceptor blockers, activities of sympathetic system was attenuated mainly, but the effects was poorly to the standard pharmacological regimen in a number of patients. For these patients, the tension of vagus nervous system may be activated pathologically. In anti-M2-R (+) patients, the chronic interaction between anti-M2-R and the M2 receptor causes a pathological activation of cardiac vagus nervous system. Therefore, these patients showed a slower heart rate and lower maximum tolerated dose of metoprolol and digoxin compared to anti-M2-R (-) patients. In the anti-M2-R (-) group, the enlarged left ventricular chamber could return to normal more rapidly, which maybe partially referred to different dosages of metoprolol and digoxin between the two groups.

The prognosis of PPCM

The standard treatment of PPCM is based on anti-HF treatment, focusing on controlling symptoms, inhibiting excessive activation of the neuroendocrine system, preventing thromboembolism and cardiac arrhythmia. The prognosis of PPCM is better than that of other cardiomyopathies with reduced LVEF, which maybe linked to the removal of hormonal toxins by the delivery of placenta and the termination of lactation. Reports on the prognosis of PPCM are various due to different research eras and geographic locations. In Haiti, Turkey and South Africa, only 21-43% of PPCM patients can return to normal LVEF, and the mortality rate is 15-30%, 30% and 28-40%, respectively [17]. A study included 85 PPCM patients and followed up for up to seven years, showed that 16% (14/85) of the patients died of PPCM or related problems in the United States [18]. The IPAC study enrolled 100 PPCM patients from 30 centers in North America. After one year of treatment, LVEF of 72% of PPCM patients returned to normal, and 12 patients had major cardiovascular events, including 4 deaths[19]. A worldwide registry study retrospectively enrolled 411 PPCM patients, and there were 10 deaths in one month, including 6 deaths from HF, 3 sudden cardiac deaths, and 1 stroke [20]. In this study, we found LVEF of 89.3% and 94.2% of PPCM patients returned to normal at 1-year and 2-year respectively, and the 2-year mortality was 3.9%, which was similar or better than other reports.

Limitations

There are some limitations in this study. Firstly, a relatively small number of patients accumulated in two hospitals in the past 20 years, and these patients are almost from Northern China and Eastern China which lacks regional differences and ethnic diversity. Secondly, due to factors such as population movement, urban demolition, and changes in contact information, four patients lost to follow-up during the first year. Thirdly, with the continuous updating of HF management concept in the past 20 years, the treatment strategies for PPCM patients are also different. Fortunately, the vast majority of cases in this study were selected between 2000 and 2018 and given standard neuroendocrine blockade therapy.

PPCM patients, with low dose of digoxin additional to standard treatment regimen, have a better prognosis, especially in the anti-M2-R (-) patients. The improvement in cardiac function may partially be related to the regulation of autonomic nervous system.

ACEI

Angiotensin-converting enzyme inhibitors

Anti-M2-R

Autoantibodies against the M2-muscarinic receptor

HF

Heart failure

IDCM

Idiopathic dilated cardiomyopathy

LVEDD

Left ventricular end-diastolic diameter

LVEF

Left ventricular ejection fraction

LVESD

Left ventricular end-systolic diameter

NYHA

New York Heart Association

PPCM

Peripartum cardiomyopathy

RAAS

Renin-angiotensin-aldosterone system

SDC

Serum digoxin concentration

Availability of data and materials

All of the data are available with reasonable request from the corresponding author.

Ethics approval and consent to participate

I confirm that I have read the Editorial Policy pages. The study protocol complied with the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Chao-Yang Hospital and BENQ Medical Center. All the patients provided written informed consent before study entry.

Consent for publication

All patient guardians signed a document of informed consent.

Competing interests

The authors declare that they have no competing interests.

Contributions

LZ and XYL conceived and designed the study and were responsible for performing data collection, analysis and interpretation and giving final approval of the version to be published. GLM was responsible for drafting the manuscript and revising it critically for important intellectual content, and made substantial contributions to follow-up of the patients and analysis of data. YY and LC performed the echocardiographic examination and were responsible for follow-up of the patients. WS, YSL and YDW were responsible for collection of patient and laboratory data, data analysis and interpretation and revision of the manuscript regarding content. FS and CS performed the ELISA for anti-M2-R and were responsible for follow-up and data analysis. All authors read and approved the final version of the manuscript for publication.

Acknowledgments

We thank all the patients and their families for their participation and follow-up.

Funding

This project was supported by the Natural Science Foundation of China (81370340).

Author information

Affiliations

Department of Cardiology, Beijing Key Laboratory of Hypertension, Beijing Chao-Yang Hospital, Capital Medical University, 8# Gong-Ti South Road, Chaoyang District, Beijing, 100020, China

Guiling Ma, Yin Yue & Yidan Wang

HTRM Cardiologist Group, Heart Center, BENQ Medical Center, Nanjing Medical University, 181# Zhuyuan Road, SuZhou City, JiangSu Province, 215000, China.

Long Chen, Xiyan Liu, Cheng Shi, Fei Song, Wei Shi, Yingshih Lo & Lin Zhang

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Table 1 Clinical characteristics of PPCM patients

	Anti-M2-R (+) group n = 48	Anti-M2-R (-) group n = 59	P value
Age (years)	30.1± 3.6	29.5 ± 3.7	0.383
Multiple gestation	14	15	0.665
Multiparity	28	33	0.803
Pregnancy complication
Pregnancy induced hypertension	19	22	0.808
Preeclampsia	9	10	0.808
Gestational diabetes mellitus	12	16	0.804
Postpartum diagnosed PPCM	29	33	0.640
Blood pressure (mmHg)
Systolic	137.2± 13.3	136.5 ± 14.5	0.779
Diastolic	88.0 ± 10.2	87.5± 10.7	0.797
Heart rate (bpm)	96.0 ± 6.4	102.7 ± 6.1	< 0.001
NYHA functional class	2.85 ± 0.73	2.88 ± 0.71	0.834
Echocardiographic data
LVEDD (mm)	60.7 ± 5.6	60.4 ± 5.8	0.823
LVESD (mm)	47.3 ± 6.1	47.0 ± 6.7	0.809
LVEF (%)	38.0 ± 4.9	38.2 ± 3.2	0.763
6-min walk distance (m)	195.9 ± 83.2	199.8 ± 78.0	0.802

Abbreviations: PPCM, peripartum cardiomyopathy; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; LVEF, left ventricular ejection fraction.

Table 2. Dosages of drugs

	Anti-M2-R (+) group	Anti-M2-R (-) group	P-value
Perindopril, mg	3.3 ± 1.0	3.2 ± 1.0	0.793
Metoprolol, mg	27.4 ± 5.0	38.4 ± 4.6	< 0.001
Spironolactone, mg	18.5 ± 3.6	19.1 ± 2.9	0.328
Furosemide, mg	21.9 ± 6.4	20.7 ± 6.8	0.366
Digoxin, mg	0.08 ± 0.04	0.12 ± 0.02	< 0.001

No competing interests reported.

Effects of autoantibody against M2-muscarinic acetylcholine receptor in peripartum cardiomyopathy patients on digoxin additional to standard treatment

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