Serum SERCA2a levels in heart failure patients are associated with adverse events after discharge

Abstract

Calcium homeostasis imbalance is one of the important pathological mechanisms in heart failure. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), a calcium ATPase on the sarcoplasmic reticulum in cardiac myocytes, is a myocardial systolic-diastolic Ca2 + homeostasis regulating enzyme that is not only involved in cardiac diastole but also indirectly affects cardiac myocyte contraction. SERCA2a expression was found to be decreased in myocardial tissue in heart failure, however, there are few reports on serum SERCA2a expression in patients with heart failure, and this study was designed to investigate whether serum SERCA2a levels are associated with the occurrence of adverse events after discharge in patients hospitalized with heart failure. Patients with heart failure hospitalized in the cardiovascular department of the Second Affiliated Hospital of Guangdong Medical University, China, from July 2018 to July 2019 were included in this study, and serum SERCA2a concentrations were measured; each enrolled patient was followed up by telephone after 6 months (6 ± 1 months) for general post-discharge patient status. The correlation between serum SERCA2a levels and the occurrence of adverse events (death or readmission due to heart failure) after hospital discharge was assessed using multiple analysis and trend analysis. Seventy-one patients with heart failure were finally included in this study, of whom 38 (53.5%) were men and 33 (46.5%) were women (All were postmenopausal women). Multiple analysis revealed no correlation between serum SERCA2a levels and the occurrence of adverse events in the total study population and in male patients, but serum SERCA2a levels were associated with the occurrence of adverse outcome events after hospital discharge in female patients (OR = 1.02, P = .047). Further analysis using a trend analysis yielded a 4.0% increase in the risk of adverse outcomes after hospital discharge for each unit increase in SERCA2a in female patients (OR = 1.04; P = .02), while no significant difference was seen in men. This study suggests that serum SERCA2a levels at admission are associated with the occurrence of post-discharge adverse events in postmenopausal female patients hospitalized with heart failure.

1. Introduction

Heart failure (HF) is a structural and/or functional abnormality of the heart caused by multiple factors, resulting in increased intracardiac pressure and/or inadequate cardiac output at rest and/or during exercise, including major symptoms (e.g., dyspnea, ankle swelling and fatigue), and may be accompanied by physical signs (e.g., increased jugular venous pressure, pulmonary fissure and peripheral edema).^[1]

< Summary of China Cardiovascular Health and Disease Report 2020 > figures out the number of current patients with heart failure in China is 8.9 million, with an inpatient fatality rate of 4.1%, which will further push up the morbidity and mortality of heart failure with the widespread prevalence of lifestyle risk factors such as unhealthy diet, inadequate exercise, and smoking.^[2] With an aging population and increased risk of cardiovascular disease, heart failure has become a global public health problem affecting nearly 30 million people worldwide.^[3] Therefore, an in-depth study of the developmental process of heart failure to provide new insights and directions for the diagnosis, treatment and prognosis of heart failure and to reduce the prevalence and mortality remains the direction of our research.

Currently, heart failure is most commonly caused by myocardial dysfunction: systolic, diastolic or both.^[1] Imbalance of Ca2 + homeostasis is now considered to be one of the important pathophysiological mechanisms of heart failure, especially reduced ejection fraction heart failure (HFrEF). Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a regulatory enzyme of myocardial systolic-diastolic Ca2 + homeostasis that not only participates in cardiac diastole but also indirectly affects cardiac myocyte contraction. Previous studies have shown that the decreased expression and activity of SERCA2a in myocardial tissue during heart failure lead to a decrease in the concentration of calcium ions returning to the sarcoplasmic reticulum during diastole, which in turn leads to a decrease in the release of calcium ions during systole, and ultimately reduces the systolic function of the heart.^[4] Approaches to restore abnormal Ca2 + homeostatic imbalance have been developed for decades, among which SERCA2a gene therapy is one of the hot spots of research, and cardiac function in heart failure patients can be improved by SERCA2a gene therapy.^[5] However, the main studies of SERCA2a are the expression in myocardial tissue and its activity on heart failure and the feasibility of gene therapy, and there are few studies on SERCA2a expression in serum heart failure patients and whether it responds to the severity of heart failure patients consistent with the expression level in myocardial tissue. Our search revealed reports that SERCA2a serum levels may be associated with the occurrence of primary graft dysfunction and rejection after heart transplantation,^[6,7] so, is the concentration of SERCA2a serum levels also associated with the outcome of distant events in heart failure patients? Therefore, this study intends to investigate the correlation between SERCA2a serum levels at admission and their post-discharge adverse events in patients with heart failure.

2. Materials and methods

2.1. Sample collection

This was a single-center retrospective cohort study, and the included heart failure patients were admitted to the cardiovascular department of the Second Affiliated Hospital of Guangdong Medical University, China, from July 2018 to July 2019. Patients with heart failure included those admitted for acute heart failure episodes or acute exacerbation of chronic heart failure. Inclusion criteria: diagnosis according to Framingham criteria at admission^[8] and reconfirmation of diagnosis at discharge; voluntary participation in this study and signed informed consent; successful follow-up for 6 months (6 months ± 1 month) or adverse events during the follow-up period (adverse events after discharge were defined as readmission due to heart failure attack or death due to heart failure exacerbation); blood specimens retained within 12 hours of admission.

Exclusion criteria: suffering from malignant tumor, severe psychiatric disease, severe liver and kidney insufficiency and other uncontrollable systemic diseases; no blood specimen was left within 12h of admission; failure to complete follow-up and regular re-visit and medication after discharge as prescribed by the doctor. After strict inclusion and exclusion criteria, a total of 71 patients with heart failure were eventually enrolled in the study. The project was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Second Hospital of Guangdong Medical University (Approval NO. PJKT2022-003-01). All the participants provided signed informed content and agreed to attend this survey.

2.2. Serum SERCA2a determination

Blood specimens were collected within 12 hours of admission to the hospital by venipuncture, and venous blood was collected from the subject in a seated position. After centrifugation at 1500 rpm and 4ºC for 15 minutes, serum samples were separated and stored in frozen tubes at −80ºC until analysis, and thawed only once. After thawing, serum SERCA2a concentrations were determined by ELISA following standard protocol (Kit information) (Shanghai Jining Industrial Co., Ltd., China) according to the kit instructions, and the detection limit of SERCA2a test was 0.114 ng/mL. intra- and inter-batch coefficients of variation were <10% and 12%, respectively. There was no significant cross-reactivity or interference between SERCA2a and analogues. To mitigate detection errors during the assessment of serum SERCA2a levels, triplicate tests were conducted for each sample, and the mean value was subsequently adopted as the definitive outcome. We provide additional information on this matter in the Methods section.

2.3. Statistical methods

Empower(R) (www.empowerstats.com, X&Y solutions, Inc. Boston, MA) and R (http://www.R-project.org) were applied for all statistical analyses in the study. Data were expressed as mean ± standard deviation, proportion, or median (range), and the association between serum SERCA2a levels and the occurrence of adverse events after hospital discharge was assessed using multiple analysis and trend analysis. For the trend analysis, firstly, the patients were divided into 3 groups by trisection of SERCA2a levels, and then substitute the median of each group as a continuous variable into the equation to draw conclusions.

3. Results

3.1. Study population

As shown in Figure 1, a total of 151 patients hospitalized with heart failure in the Cardiovascular Department of the Second Hospital of Guangdong Medical University from July 2018 to July 2019 were included in the study. Fifty-two patients who did not retain serum specimens within 12 hours of admission; 14 patients with systemic diseases such as combined tumor and severe hepatic and renal insufficiency; and 14 patients who did not complete follow-ups and did not regular re-visit and medication after discharge as prescribed by the doctor were excluded, and 71 patients were finally included.

Figure 1.

Flow chart of study subjects screening. According to the inclusion and exclusion criteria, 71 patients were finally enrolled (38 males; 33 females).

The baseline characteristics of the participants in this study are shown in Table 1. There were 38 male patients (53.5%) with a mean age of 77.6 ± 10.2 years and 33 female patients (46.5%) with a mean age of 75.3 ± 12.0 years (All were postmenopausal women). The enrolled patients were dichotomized according to SERCA2a serum levels, and the mean body mass index (kg/m²) was higher in the low SERCA2a level group (≤314.31 ng/mL) than in the high SERCA2a level group (>314.31 ng/mL) (P = .71); left ventricular ejection fraction was higher in the low SERCA2a level group than in the high SERCA2a level group (P = .67); mean E/A was higher in the low SERCA2a group than in the high SERCA2a group (P = .15); smoking status was significantly different in the different SERCA2a level groups (P = .02) and not in the New York Heart Association classification (P = .28). The follow-up results showed no significant difference in the incidence of adverse events among different SERCA2a level groups (P = .88).

Table 1.

Basic clinical characteristics of the study subjects.

SERCA2A (ng/L)	Low (≤314.31 ng/mL)	High (>314.31 ng/mL)	P value
N (number)	35	36
Age (yr)	77.09 ± 9.89	75.67 ± 12.43	.85
Sex (%)			.12
Female	13 (37.14)	20 (55.56)
Male	22 (62.86)	16 (44.44)
BMI (kg/m2)	21.35 ± 4.44	20.87 ± 3.57	.71
Heart rate (beats/min)	89.33 ± 24.29	85.15 ± 24.30	.41
SBP (mm Hg)	146.3 ± 32.3	136.0 ± 24.7	.337
NT-ProBNP (Ng/L)*	3734.50 (1843.92–8048.25)	4664.00 (1817.75–6614.25)	.96
Creatinine (μmol/L)	144.08 ± 134.91	102.00 ± 69.43	.22
AST (U/L)	45.22 ± 42.89	30.13 ± 21.79	.40
ALT (U/L)	29.55 ± 32.10	26.64 ± 27.37	.30
CRP (mg/L)	13.12 ± 11.73	12.53 ± 12.36	.50
FPG (mmol/L)	8.9 ± 4.5	8.5 ± 3.5	.920
SUA (μmol/L)	381.2 ± 146.8	388.0 ± 214.4	.936
LDL-C (mmol/L)	2.6 ± 0.7	3.2 ± 1.2	.540
HDL-C (mmol/L)	1.3 ± 0.4	1.3 ± 0.2	.838
LVEF (%)	54.47 ± 10.25	51.00 ± 16.32	.68
E/A	0.88 ± 0.39	0.65 ± 0.17	.15
Alcohol consumption (%)			.002
No	27 (77.14)	36 (100.0)
Yes	8 (22.86)	0 (0.00)
Smoking (%)			.019
No	26 (74.29)	34 (94.44)
Yes	9 (25.71)	2 (5.56)
Hypertension (%)			.51
No	11 (31.43)	14 (38.89)
Yes	24 (68.57)	22 (61.11)
NYHA class (%)			.28
I/II	14 (40.00)	19 (52.78)
III/IV	21 (60.00)	17 (47.22)
Pulmonary disease (%)			.89
No	20 (57.14)	20 (55.56)
Yes	15 (42.86)	16 (44.44)
Renal insufficiency (%)			.15
No	29 (82.86)	34 (94.44)
Yes	6 (17.14)	2 (5.56)
ACE-I/ARB (%)			.56
No	14 (40.0%)	12 (33.3%)
Yes	21 (60.0%)	24 (66.7%)
Beta-blocker (%)			.165
No	10 (28.6%)	16 (44.4%)
Yes	25 (71.4%)	20 (55.6%)
Aldactone (%)			.257
No	5 (14.3%)	9 (25.0%)
Yes	30 (85.7%)	27 (75.0%)
Follow-up status (%)			.88
Health	13 (37.14)	14 (38.89)
Readmission/death	22 (62.86)	22 (61.11)

Value are mean (SD)

^*expressed as median (maximum, minimum).

ACE-I = Angiotensin-converting enzyme inhibitor, ALT = Alanine aminotransferase, ARB = Angiotensin II receptor blocker, AST = Aspartate aminotransferase, BMI = body mass index, CRP = C-reactive protein, FPG = fasting plasma glucose, HDL-C = high-density lipoprotein cholesterol, LDL-C = low-density lipoprotein cholesterol, LVEF = left ventricular ejection fraction, NT-proBNP = N-terminal pro-brain natriuretic peptide, NYHA = New York Heart Association, pulmonary disease = emphysema, pneumonia (non-severe pneumonia), chronic bronchitis, Renal insufficiency = chronic kidney disease stages 1–3, SBP = systolic blood pressure, SERCA2a = the sarcoplasmic reticulum Ca²⁺-ATPase, SUA = Serum Uric Acid.

3.2. Analysis of serum SERCA2a levels and post-discharge adverse events in patients with heart failure

In the multiple analysis as shown in Table 2, there was no correlation between serum SERCA2a levels on the occurrence of adverse events from the total study population (OR = 1.01, P = .398); there was also no correlation in men (OR = 0.98, P = .133). In female patients, we could observe that SERCA2a levels were associated with the occurrence of adverse outcome events after discharge, with a 2% increase in the risk of adverse events after discharge for every 1 unit increase in SERCA2a (P = .047). As shown in Table 2, trisect the SERCA2a levels and analysis, in female patients, we could observe that the risk ratio of developing adverse outcomes increased with increasing SERCA2a levels. The median of each of the 3 groups, divided by trisection of SERCA2a levels, was substituted as a continuous variable into the equation, and further analysis with trend analysis led to the conclusion, as shown in Table 3, in female patients we can see that for each unit increase in SERCA2a the risk of adverse outcome after discharge increases by 3.7% (1.006, 1.070; P = .02), and the trend analysis further validates the idea that high SERCA2a levels at admission may increase the risk of adverse events after discharge in female patients, with no significant difference seen in men.

Table 2.

Multiple regression analysis (OR, P value) of SERCA2a serum concentration levels and occurrence of adverse events in different genders.

	Female (n = 33)	Male (n = 38)	Total (n = 71)
SERCA2A (ng/L)	1.02, 0.047	0.98, 0.133	1.01, 0.398
SERCA2A (ng/L)
226.25–298.97	1	1	1
300.15–328.19	1.47, 0.70	0.23, 0.13	0.56, 0.35
328.25–410.12	17.23, 0.02	0.50, 0.48	2.63, 0.16

Adjust: age, NYHA class.

SERCA2a = the sarcoplasmic reticulum Ca²⁺-ATPase.

Table 3.

Trend analysis of SERCA2a serum concentration levels with the occurrence of adverse events in different genders (OR, P value).

	Gender = female (n = 33)	Gender = male (n = 38)	Total (n = 71)
SERCA2A (ng/L) group
226.25–298.97	1	1	1
300.15–328.19	1.468, 0.70	0.257, 0.13	0.559, 0.35
328.25–410.12	17.278, 0.02	0.504, 0.48	2.634, 0.16
SERCA2A (ng/L) group trend	1.037, 0.02	0.990, 0.44	1.012, 0.18

Adjust: age, NYHA class.

SERCA2a = the sarcoplasmic reticulum Ca²⁺-ATPase.

4. Discussion

The purpose of this study was to investigate the correlation between serum SERCA2a levels at admission and the occurrence of adverse events after discharge in patients with heart failure. From the study, we can find that in female patients, the risk ratio of developing adverse events increases with the increase of SERCA2A levels. However, the results of previous animal or clinical studies suggest that upregulation of myocardial tissue SERCA2a expression by gene transduction is effective in improving cardiac function and reducing the occurrence of adverse cardiovascular events.^[9,10] This differs from our observed results, which may be due to the difference in SERCA2a expression in myocardial tissue versus serum in heart failure patients, and serum levels of SERCA2a may be associated with increased release of SERCA2a from myocardial tissue injury, reflecting increased myocardial tissue injury and thus contributing to the occurrence of prognostic adverse events. As shown in Table 1: the mean values of left ventricular ejection fraction and E/A were higher in the group of patients with low serum SERCA2a heart failure than in the group with high serum SERCA2a levels, but none of them were statistically different, which we speculate is related to the small sample size of this study. Patients requiring mechanical circulatory support before heart transplantation have been found to have lower serum SERCA2a levels than clinically stable patients,^[11] and low serum SERCA2a levels have also been associated with a higher incidence of rejection after heart transplantation.^[6,7] This differs from our study suggesting that it is the female population of heart failure patients with high serum levels of SERCA2a that is more likely to cause the occurrence of prognostic adverse events, probably because the previous study population was mainly patients with advanced heart failure, whereas the population included in our present study was admitted with acute heart failure attack or acute exacerbation of chronic heart failure, with New York Heart Association classification of I and II in 46.4% (33). Secondly, the results we observed were presented in the female population, and the results of previous studies were discussed in terms of the total study population, lacking gender subgroups; therefore, are there gender differences causing the observed results to differ? This needs to be confirmed by more clinical studies in the future.

Also, in this study, we found that higher serum SERCA2a levels at admission in the female group were associated with an increased risk ratio of adverse events after discharge, and the trend analysis further confirmed this correlation (P = .02); while no such correlation was observed in male patients. We speculate that there is a gender difference between serum SERCA2a levels and the occurrence of adverse events after hospital discharge. The current study suggests that there are some gender differences in heart failure in terms of epidemiology, pathophysiology, treatment response, prognosis and biomarkers.^[12,13] For example, among the traditional risk factors for heart failure, obesity, diabetes mellitus and mental/psychological stress are of greater risk for women than men. Diabetes and obesity can drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation^[14] and are associated with a predilection for HFpEF in women. Among the etiologies of heart failure, coronary macrovascular disease and myocardial infarction predominate in men,^[13,15–17] while coronary microvascular dysfunction, hypertension and immune-inflammatory mechanisms predominate in women.^[13,18,19] This is consistent with a predilection for HErEF in male patients and HFpEF in female patients.^[20] Similarly, in the general population, natriuretic peptide levels are higher in women than in men, which may be related to the fact that testosterone decreases cardiac natriuretic peptide levels^[21] and estrogen can increase cardiac natriuretic peptide levels by directly increasing cardiac natriuretic peptide gene expression and release.^[22,23] In contrast, in the population of heart failure patients, women have lower natriuretic peptide levels than men, and this difference may be related to the difference in the prevalence of HF in men and women with reduced ejection fraction (HFrEF) versus preserved ejection fraction (HFpEF).^[12] In addition, echocardiographic studies have shown that women with HFpEF are more likely to have concentric LV remodeling, more severe diastolic dysfunction, including more severe LV diastolic injury, and higher LV filling pressures compared with men.^[24]

The current gender disparity in heart failure clinics is due to the fact that too few women are recruited to heart failure clinical trials (20–25% of cohorts) and therefore treatment guidelines are primarily based on data from men.^[13] There is a large knowledge gap regarding gender-specific mechanisms, optimal drug doses for women, and gender-specific criteria for device therapy. Gender differences, and in particular the lack of awareness of these differences, may lead to a gender bias in heart failure salvage and may result in a poor prognosis for women. In this study, serum SERCA2a levels may have been associated with the occurrence of post-discharge adverse events in postmenopausal female heart failure patients, whereas no such correlation was observed in the male population. The indication of gender differences in the correlation of serum SERCA2a levels on the occurrence of adverse events after hospital discharge in heart failure patients provides an additional perspective for researchers to understand the gender differences in the development of heart failure related to prognosis, and the underlying pathophysiological mechanisms need to be elucidated by further studies. Meanwhile, it also implies that the serum level of SERCA2a could serve as a potential marker for risk stratification of heart failure in postmenopausal women. Further investigation is warranted to explore the correlation between serum SERCA2a levels and the long-term prognosis of individuals with heart failure. This research direction is conducive to providing more robust evidence to assess the potential of serum SERCA2a levels as a novel biomarker for risk stratification and clinical decision-making in the management of heart failure.

5. Limitations of the study

The results of our present study must be interpreted in the context of a number of limitations. First, our study is a single-center study conducted in China, with a relatively limited representation of race and population demographics. A broader array of multicenter prospective studies is required to substantiate and establish the validity and prevalence of this viewpoint. Second, In the subgroup analysis, no statistically significant difference was observed in the male population, which could be attributed to the limited sample size leading to sample size causing type II errors. Third, the potential variability of serum SERCA2a levels must also be considered based on other unanalyzed parameters, such as medication and the time of evolution of heart disease. To our knowledge, our present study is the first time to examine the potential use of serum SERCA2a levels for the emergence of adverse events after hospital discharge in patients with heart failure, but the results of this study must be validated in a broader prospective cohort.

6. Conclusions

Our results confirmed serum SERCA2a levels at admission may be associated with the occurrence of post-discharge adverse events in postmenopausal female patients hospitalized with heart failure.

Acknowledgments

This manuscript has been edited and proofread by a professional English translation service. We would like to thank all volunteers who participated in the study.

Authors contributions

Conceptualization: Bilian Xu, Jinrong Xu.

Data curation: Yuesheng Huang, Xu Chen, Zhiye Wang, Xiaoxin Chen.

Formal analysis: Zhihui Hu, Biao Hao, Jiahao Li.

Funding acquisition: Jinrong Xu.

Investigation: Biao Hao, Yuesheng Huang, Xu Chen, Yingjie Guo, Zhiye Wang, Xiaoxin Chen, Miaoling Su, Weiwen Chen, Yinan Zhuo.

Project administration: Weiwen Chen, Yinan Zhuo, Jiahao Li, Shaofeng Wei.

Resources: Jinrong Xu.

Visualization: Panghe Chen.

Writing – original draft: Panghe Chen, Shudie Wu, Zhihui Hu.

Writing – review & editing: Panghe Chen, Shudie Wu, Zhihui Hu, Bilian Xu, Jinrong Xu.