Background
Cardiac resynchronization therapy (CRT) has been shown to be efficacious to improve cardiac function and become a standard treatment for patients with severe heart failure (HF) [
1]. However, approximately one-third of the patients did not obtain any benefit from this therapy [
2], and the mechanism remains unclear. To date, a limited number of studies have focused on finding specific biomarkers to predict the response to CRT. Myocardial imaging techniques assessing myocardial mechanical activation time and dyssynchrony are somewhat limited by low sensitivity and large observer variability to predict response to CRT [
3]. The evaluation of electrical dyssynchrony using QRS duration (QRSd) was more reliable to predict CRT outcome [
4]. Nonetheless, in patients with QRSd < 150 ms, there are limited data on markers of electrical dyssynchrony [
5]. Hence, recognition of patients who are likely to benefit from CRT before the device implantation is still challenging.
As an alternative strategy for biomarker discovery, metabolomics based on nuclear magnetic resonance (NMR) and mass spectrometry (MS) enables to identify the endogenous small-molecule metabolites that are sensitively associated with pathological alterations [
6], which is believed to have the potential to provide individualized and predictive information for disease progression and personalization of specific medical treatment [
7,
8]. Up to now, two published reports investigated the metabolite changes, contributing toward predicting response to CRT. In a study conducted based on 1H-NMR technique, the accuracy of discrimination between responders and non-responders remained low [
9]. In another report based on a wide range of targeted metabolite profiling with the use of gas chromatography-mass spectrometry (GC-MS) and 1H-NMR techniques, it was revealed that CRT responders may have a favorable metabolomic profile as a potential biomarker for predicting CRT outcome [
10]. It is also reported that the sensitivity of 1H NMR is relatively low when compared with MS [
11]. Therefore, it is meaningful to apply complementary liquid chromatography-mass spectrometry (LC-MS) based metabolomics platforms to identify novel biomarkers to predict CRT outcome.
In this study, we conducted a two-stage study to examine the role of the serum metabolite profile based on ultrahigh performance LC coupled with quadrupole-time-of-flight-MS (UHPLC-Q-TOFMS) to predict the response to CRT.
Discussion
Using UHPLC-Q-TOFMS-based serum metabolomics approach, we identified a panel of serum metabolites associated with response to CRT in Chinese population. The main findings of this study were as follows: (i) A total of 17 potential metabolites were found to have significant different serum concentrations between CRT responders and non-responders. (ii) Compared with non-responders, responders exhibited higher concentrations in valine, citric acid, isoleucine, phenylalanine, indoleacetic acid, lysoPC, and linoleic acid, while lower concentrations in hypoxanthine, inosine, tryptophan, sphingosine 1-phosphate, and carnitines. (iii) A combination of three serum metabolite biomarkers (isoleucine, tryptophan, and linoleic acid) was established as an ideal metabolite panel to distinguish responders from non-responders.
To understand the underlying molecular functions of these serum metabolite biomarkers, we further conducted metabolic pathway analysis. These 17 metabolites were found to be primarily involved in six metabolism pathways, including linoleic acid metabolism, valine, leucine and isoleucine biosynthesis, phenylalanine metabolism, citrate cycle (TCA cycle), tryptophan metabolism, and sphingolipid metabolism. This metabolic profile covered different aspects of pathogenesis, especially anomalous lipid and energy metabolism. The disturbed metabolic pathways are discussed in detail below.
Linoleic acid is a kind of n-6 polyunsaturated fatty acids and one of the oxidative stress biomarkers [
18]. A higher level of linoleic acid could prompt the fatty acid metabolism of myocardial cell, which plays an important role in suppressing cardiomyocyte hypertrophy [
19], while advanced left ventricle dilation was reported to be associated with poor CRT outcome [
20,
21]. In our study, CRT responders had a lower level of the left ventricular end diastolic dimension and a higher level of linoleic acid, reflecting that linoleic acid might improve the response to CRT by influencing the fatty acid metabolism of myocardial cell.
The high concentrations of valine and isoleucine, correlated with valine, leucine and isoleucine biosynthesis, were observed in CRT responders, which was consistent with Nemutlu et al.’s findings [
10]. Valine and isoleucine, two branched-chain amino acids essential for protein synthesis and metabolic signaling, may be important alternative energy substrates. The inhibition of the citrate cycle induced by the HF hypoxia could lead to the utilization of branched-chain amino acids as energy compensation [
11,
22]. On the other hand, Colak et al. [
23] discovered that genes involved in energy metabolic processes, such as the citric acid cycle and adenosine triphosphate synthesis, were upregulated in DCM patients. The results above heightened that metabolic activity may be a compensatory mechanism in the process of HF. The higher concentration of citric acid in our responder subjects indicated that responders may have a better compensatory adaptation, which might improve the response to CRT.
Phenylalanine is a precursor for tyrosine, including adrenaline and noradrenaline, and higher concentration of phenylalanine is observed in the progress of HF due to stress response to reduced cardiac output [
24]. Increased phenylalanine concentration has been identified in cross-sectional studies among individuals with established HF compared with normal controls [
24‐
26], indicating that phenylalanine metabolism may be related to the decline of cardiac function and therefore influences CRT outcomes.
Beyond the metabolites essential for protein synthesis and metabolic signaling [
27], we also observed the significant associations between the response to CRT and the following metabolites related to oxidative stress and inflammation. In our study, responders were found to have a lower level of tryptophan, which was reported to be reversely associated with incident cardiovascular disease in a randomized controlled trial [
28]. Indoxyl sulfate, a gut bacteria-derived product of tryptophan, could stimulate oxidative stress and, further contributes to the progression of cardiovascular disease, cardiac hypertrophy and fibrosis [
29]. The prognostic value of indoxyl sulfate has been proposed for DCM patients with normal renal function or mild-to-moderate chronic kidney diseases [
30], and high serum indoxyl sulfate was a significant predictor of cardiac events [
31]. Associations between indoxyl sulfate and overall mortality and cardiovascular disease were also reported [
31].
Our results showed that CRT responders had a lower level of sphingolipid than non-responders. A recent study revealed that sphingosine and sphinganine levels were decreased in patients with systolic HF due to ischemic or non-ischemic heart disease compared with healthy individuals [
32]. Sphingosine 1-phosphate is a bioactive sphingolipid with important functions in immunity, inflammation, and cardiovascular biology [
33]. It is associated with the impairment of LVEF, dyspnea, and causally involved in the pathophysiology of HF [
33]. Animal models has demonstrated that the deletion of cardiac sphingosine 1-phosphate receptor 1 could lead to the incident of cardiomyopathy and HF [
34]. Together with our results, these findings indicated that metabolites might influence the response to CRT via different pathways including energetic metabolism and oxidant stress.
Considering the moderate and unstable predictive ability of individual metabolite, a metabolite panel was constructed using three serum metabolites (isoleucine, tryptophan, and linoleic acid) to distinguish the indication of CRT and showed an ideal capacity to predict the response to CRT. Compared with QRSd, the metabolite panel increased about 50% of the prediction ability, which have a great clinical potential for both doctors and patients to reduce nonessential treatment and to lower medical costs.
This study had several limitations. First, metabolite profiles are variable in vivo because of human activities and changes in the external environment. However, the patients provided blood sample typically a day ahead of or during CRT implantation. In such a short time, the change of metabolite levels could be limited. Second, primary DCM is a genetically heterogeneous disease, and the genetic heterogeneity of the cases is a potential confounder that could influence the serum metabolome. Third, only patients with DCM were investigated in our study, therefore, the generalizability of our conclusion was limited, but this could minimize the impact of the type of disease on metabolite analysis results. Finally, the sample size of the study was relatively small. Consequently, further investigations in larger populations are warranted to explore the corresponding mechanisms of these metabolites related to CRT. Specific factors such as genetic polymorphisms to predict the response of CRT is also worthy of being investigated in the future.
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