Background
Cardiovascular diseases (CVDs), the leading cause of death in humans, have emerged as a high socio-economic burden around the world with rising incidence. It is predicted that CVDs may increase by approximately 21.3 million events and 7.7 million deaths over 2010–2030 in China. Isoproterenol (ISO), a synthetic on selective β-adrenergic agonist, is widely used for inducing experimental CVDs such as myocardial ischemia, hypertrophy and infarction, cardiac fibrosis, and heart failure. Stimulation with ISO leads to the development of oxidative stress, calcium overload, myocardial inflammation and renin–angiotensin release, which ultimately cause CVDs [
1]. ISO-induced myocardial injury animal models are reported to be used for the evaluation of cardioprotective agents due to their advantages in lower mortality and higher reproducibility compared with other animal models [
2].
Environmental risk factors such as climatic change, are becoming a major public health concern, with increasing studies shown that extreme temperatures and humidity are associated with higher risks of mortality throughout the world [
3‐
5]. Hyperlipidemia, characterized by high serum lipids, is one of the well established risk factors of cardiovascular diseases. Phlegm-damp syndrome, as known as “Tanshi” in Chinese, is one of Traditional Chinese Medicine (TCM) syndrome differentiation, which has the highest incidence in Southern China. Phlegm-damp syndrome is characterized by hyperlipidemia combined with high temperature and high humidity in TCM. Previous researches have revealed the important role of phlegm-damp syndrome in the development of CVDs. However, the underlying mechanism remains unknown.
Metabolomics is a powerful approach for identification and quantification of small molecule metabolites that reflect molecular processes more proximal to disease states. Nontargeted metabolomics is commonly employed to capture the complexity of metabolic networks and reveal novel molecular alterations for its comprehensive analysis of the metabolome. Metabolomics received increasing attention in CVDs research for its help to better explain the biological mechanisms and identify novel biomarkers of CVDs [
6]. In present study, nontargeted metabolomics with LC–MS/MS was adopted to reveal serum metabolism profile in ISO-induced myocardial ischemia mice with high temperature high humidity and high-fat diet.
Discussion
ISO-induced myocardial injury animal models are characterized by myocardial hypertrophy, ischemia and even heart failure, which contributes effectively to understanding of the cellular alterations as well as the pathological changes in the heart [
10]. Compare with those animal models induced by surgical procedures (e.g., coronary artery ligation, aorta banding), ISO administration provided a simple and non-invasive method for inducing myocardial injury with lower mortality and higher reproducibility. In our present study, a myocardial injury mouse model was introduced by ISO administration combined with high temperature and high humidity and high fat diet to simulate the TCM syndrome of “phlegm-damp”. The results showed the levels of TC, TG and LDL-C in serum of Tanshi mice were significantly higher, and the values of IVS and LVPW were worse in Tanshi mice, which were fed with western diet and housed in room temperature of 35 ± 0.5 °C and relative humidity of 90 ± 5%, compared with the ISO mice. Moreover, mice in Tanshi group developed a significantly increase in cardiac index compared with ISO mice. Significantly more server myocardial fibrosis was also found in Tanshi mice compared with the ISO mice. All these results suggested that high-fat diet with high temperature and high humidity promotes in ISO-induced myocardial injury in mice.
Disorders in cardiac metabolism involved in the pathogenesis of most cardiovascular diseases since diverse sources such as carbohydrates, lipids, lactate, amino acids are needed for supply of energy [
11]. Moreover, disturbances in cardiac metabolism may affect systemic metabolism initiating a vicious cycle that accelerates the development of cardiovascular diseases. Metabolomics is downstream of transcriptional, translational, and posttranslational processes, and considered to be the most sensitive to environmental and dietary influences, including such as dietary intake, gut microbiota variation, physical activity [
12]. Circulating metabolites reflect immediate information of physiologic condition, and findings from metabolomics studies potentially offer key insights into cardiovascular diseases pathogenesis [
13,
14]. Accumulating experimental and clinical studies of metabolomics in cardiovascular diseases have been reported and the applications of which have been well summarized [
15,
16]. For instance, in vivo experiments on cardiac hypertrophy and heart failure using transverse aortic constriction (TAC) and myocardial infarction (MI) in mice revealed increased acylcarnitines and decreased tricarboxylic acid (TCA) cycle intermediates in heart tissue [
17], accumulation of branched-chain amino acids (BCAA) and branched-chain α-ketoacids (BCKA) in heart tissue and plasma [
18,
19], decreased cardiac fatty acid oxidation and increased ketone oxidation in heart tissue [
20,
21]. Two early clinical studies of patients with coronary artery disease (CAD) by using Targeted LC–MS showed that higher levels of BCAAs and urea cycle metabolites were associated with CAD [
22,
23]. Other clinical studies with patients undergoing elective cardiac catheterization respectively revealed that higher levels of trimethylamine-
N-oxide (TMAO) were associated with CVD [
24,
25], and long-chain dicarboxylacylcarnitines, BCAAs, and fatty acids were associated with death or MI [
26].
Metabolomic profiling of ISO-induced myocardial infarction has also been revealed in animal studies. By using the ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF–MS), 13 lipid biomarkers (e.g. Lyso-PCs and fatty acids) had been identified in serum of ISO-induced MI rats [
27]. An integrated UPLC-Q/TOF–MS and
1H NMR multiplatform based on a tissue-targeted metabonomic approach revealed 22 metabolites (14 down-regulated and 8 up-regulated) as potential biomarkers and 5 pathways (e.g. taurine and hypotaurine metabolism) as the most relevant pathways in ISO-induced MI rats [
28].
In our present study, PCA, PLS-DA and heatmaps of differential metabolites indicated clear differentiations among control mice, ISO mice and Tanshi mice, which were stimulated by ISO injection with high temperature and high humidity and high-fat diet. Our study also confirmed the presence of specific metabolites and disturbed metabolic pathways by comparing ISO mice and Tanshi mice. Our data suggested that impaired lipid metabolism (mainly glycerophospholipid metabolism), amino acid metabolism (mainly arginine–proline metabolism), digestive system (mainly protein digestion and absorption) and signal transduction (mainly sphingolipid signaling pathway) are associated with the promotion of ISO-induced myocardial injury in mice with high temperature and high humidity and high-fat diet. The lysophosphatidylcholines (lysoPCs), phosphatidylcholines (PCs), sphingomyelins (SMs) were the main groups of metabolites that showed a difference between ISO and Tanshi mice, which play deleterious roles in pathogenesis of ISO-induced myocardial injury.
The major classes of glycerophospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid [
29]. Phosphatidylcholines (PC), which constitutes approximately 40% of human cardiomyocytes, have received special attention in cardiovascular research. The activation of PC generation may lead to the accumulation of SM and Ceramide (Cer) which promote cell apoptosis. Increased PC in patients with angina pectoris and myocardial infarction had been demonstrated in previous clinical studies [
29,
30]. According to our results, two PCs were found higher (PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(18:3(6Z,9Z,12Z)/16:0), while three PCs were lower (PC(14:0/20:3(8Z,11Z,14Z)), PC(16:1(9Z)/18:1(11Z)), PC(18:1(9Z)/P-18:1(11Z))) in all three comparison groups. In addition, ten PCs were found higher in Tanshi mice compared with ISO and control mice (as shown in Fig.
4b). On the other hand, we found five decreased LysoPC (LysoPC(16:1(9Z)/0:0), LysoPC(20:5(5Z,8Z,11Z,14Z,17Z)), LysoPC(17:0), LysoPC(16:0), LysoPC(15:0)) in all three comparison groups and seven decreased LysoPC (LysoPC(18:2(9Z,12Z)), LysoPC(20:4(8Z,11Z,14Z,17Z)), LysoPC(20:1(11Z)), LysoPC(20:3(5Z,8Z,11Z)), LysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)), LysoPC(18:3(6Z,9Z,12Z)), LysoPC(22:5(7Z,10Z,13Z,16Z,19Z))) in Tanshi mice compared with ISO and control mice. LysoPC has proatherogenic roles in myocardial infarction by inducing monocyte recruitment, macrophage and smooth muscle cell proliferation, endothelial adhesion molecules expression and endothelial dysfunction. LysoPC may also participate in the oxidative stress process through the oxidase pathway and the activation of protein kinase C in blood vessels. The reduction of Lyso-PCs in Tanshi mice may be due to the activated Phospholipase A2 (PLA2) during the breakdown of membrane, which leads to the decreased generation of lysoPCs. The increased PC and decreased LysoPC showed in our study indicated an underlying lipid disturbance in ISO-induced mice model with high temperature and high humidity and high-fat diet that may be associated with the promotion of myocardial injury.
Sphingolipids, components of all eukaryotic cell membranes, have been shown to be related to regulation of various biological processes such as cell proliferation, migration and differentiation, which contribute to the pathogenesis of diverse diseases including cardiovascular diseases, neurodegenerative diseases and cancer [
31]. Sphingosine, the intermediate lipid between Ceramide (Cer) and S1P, can be converted to S1P by the action of sphingosine kinases (SphK), while Cer can be converted to sphingosine by the action of ceramidases [
32]. The balance between Cer and S1P is a crucial determinant of cellular responses to cytokines, inflammation and oxidative stress Increases in SM and sphingosine promote cell death such as apoptosis, whereas increases in S1P is beneficial to cell survival and proliferation [
33]. Sphingosine is also a signal transduction factor of TNF-α during myocardial injury, which reduced myocardial contractility, inhibited the transport of calcium ions in cardiomyocytes, and induced calcium overload [
34]. It was found in our present study that the levels of SM(d18:0/16:0), SM(d18:0/24:0), SM(d18:1/26:1(17Z)) and sphingosine were higher, while the level of S1P was lower in Tanshi mice compared with ISO and Con mice. These results indicated that the increase oxidative stress, calcium overload and cell death caused by alterations of all these sphingolipids might be the explanation for the promotion of ISO-induced myocardial injury in mice with high temperature and high humidity and high-fat diet.
Additionally, our study also showed that arginine–proline metabolism is one of the most relevant pathways in Tanshi versus ISO group. L-Arginine, a semiessential amino acid, is the substrate for nitric oxide synthase (NOS), which play a crucial role in regulation of nitric oxide (NO) generation [
35]. The decline of L-Arginine and disorder of its metabolism may cause dysfunction of endothelial NOS (eNOS), leading to decreased NO generation, which results in a sequent injury to vessels and organs including the heart. The activation of inducible NOS (iNOS), which leads to decrease of arginine, has been reported in ISO-induced myocardial apoptosis and injury by increasing reactive oxygen species (ROS), and arginine pretreatment can attenuate ISO-induced cardiac hypertrophy via regulating the expression of iNOS and eNOS [
36,
37]. In our study, a significant decrease of L-Arginine was found in Tanshi mice compare with ISO and Con mice, which might be the result of the underlying mechanism (e.g., eNOS dysfunction, cell apoptosis and oxidative stress) in the promotion of ISO-induced myocardial injury in mice with high temperature and high humidity and high-fat diet.
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