Myocardial Infarction Susceptibility and the MTNR1B Polymorphisms

Melatonin is a circadian hormone with antioxidant properties that protects against myocardial ischemia-reperfusion injury. Genetic variations of the melatonin receptor 1B gene (MTNR1B) play an important role in the development of type 2 diabetes, a risk factor for cardiovascular diseases. Accordingly, MTNR1B polymorphisms are crucial in numerous disorders of the cardiovascular system. Therefore, the aim of the present study was to investigate a possible association of MTNR1B polymorphisms with chronotype and susceptibility to myocardial infarction. The present case-control study included 199 patients with myocardial infarction (MI) (57% men) and 198 control participants (52% men) without previous cardiovascular diseases who underwent genotyping for the MTNR1B polymorphisms rs10830963, rs1387153, and rs4753426 from peripheral blood samples. Chronotype was determined using the Morningness-Eveningness Questionnaire (MEQ). As estimated by the chi-square test, no significant association was found in the distribution of alleles and genotypes between myocardial infarction patients and controls. In addition, there was no association between MTNR1B polymorphisms and chronotype in MI patients. As some previous studies have shown, the present negative results do not exclude the role of the MTNR1B polymorphisms studied in the development of myocardial infarction. Rather, they may indicate that MTNR1B polymorphisms are a minor risk factor for myocardial infarction.


Introduction
Melatonin, a neurohormone produced by the pineal gland [1] and controlled by the hypothalamic suprachiasmatic nucleus (SCN) [2], regulates the circadian rhythm [3]. This pleiotropic hormone has several different functions [4], but regulating circadian rhythm, including sleep and wakefulness, is considered the most important one [5]. Previous studies confirmed its fundamental antioxidant effects since it scavenges free radicals and stimulates antioxidant enzymes and molecules to prevent radicals [6,7], besides having antiinflammatory and possibly epigenetic regulatory functions [8][9][10]. Melatonin's circadian rhythm of secretion [11], through increased secretion during the night and decreased secretion during the day, coordinates physiological processes and behavioral activities [2,3]. Melatonin's effects on human diseases are numerous [6], mostly manifested through a membrane receptor [5]. Melatonin receptors are G-coupled proteins [8], and we distinguish three melatonin receptor subtypes, of which MT1 (melatonin receptor 1A, MTNR1A) and MT2 (melatonin receptor 1B, MTNR1B) have been identified in mammals, whereas MT3 (melatonin receptor 1C, MTNR1C) has been identified in amphibians and birds [5].
above categories or neither type [25,26]. Chronotype is related to the circadian rhythm of melatonin [27] and some CVD risk factors, such as hypertension and diabetes [11]. In addition, the evening chronotype is associated with higher morbidity, including higher rates of metabolic disorders and CVD [28] and higher mortality from CVD [25]. Research has also confirmed the association between chronotype and MTNR1B gene polymorphisms. The G allele of the rs10830963 polymorphism is associated with altered melatonin secretion and circadian phenotypes [2], and the evening type is associated with a higher risk of diabetes in individuals with the GG genotype of the rs10830963 MITNR1B gene polymorphism [27]. MITNR1B gene polymorphisms are associated with CVD risk factors such as type 2 diabetes. Moreover, cardiovascular diseases follow a circadian pattern of occurrence, and myocardial infarction most commonly occurs early in the morning or late in the evening. It has been shown that a person's chronotype can influence the occurrence of myocardial infarction [29]. In addition, melatonin levels fluctuate daily, and there might be a link between chronotype and MTNR1B gene polymorphisms, as the G allele of MTNR1B rs10830963 is associated with increased MTNR1B mRNA expression [3]. Increased levels of MTNR1B mRNA result in higher melatonin signaling levels [3]. In addition, the G allele is associated with a longer duration of elevated melatonin levels, a delayed phase of the circadian melatonin shift, and a delayed light-dark cycle [30]. Therefore, the MTNR1B gene polymorphisms might affect the synchronization between the melatonin phase and the light-dark cycle, which is associated with chronotype.
In this case-control study, we studied the association between melatonin receptor 1B gene polymorphisms and susceptibility to myocardial infarction in surviving patients. For this purpose, differences between patients and control participants in the frequency of individual polymorphisms and genotypes were analyzed. In addition, genotype models of MTNR1B gene polymorphisms and possible haplotypes were analyzed. Furthermore, it was investigated whether CVD risk factors were associated with the analyzed MTNR1B gene polymorphisms. Specifically, the standardized and validated Horne-Östberg Morningness-Eveningness Questionnaire (MEQ) was used to test whether participants' chronotype was associated with the MTNR1B gene polymorphisms, as well as the association of chronotype with CVD risk factors in MI patients. Previous studies showed that type 2 diabetes significantly affects myocardial infarction risk, and that MTNR1B gene polymorphisms are associated with increased diabetes risk, that the same receptor has a stronger protective effect against myocardial ischemia, and that MTNR1B gene polymorphisms are associated with circadian phenotype-chronotype. We aimed to investigate the association of MTNR1B polymorphisms with chronotype and susceptibility to myocardial infarction.

Characteristics of the Study Participants
The baseline characteristics of the study participants are shown in Table 1. There were a total of 397 participants, 199 MI cases, and 198 controls. The distribution according to gender was similar between the groups. Cases were significantly older than controls. The mean diastolic blood pressure was significantly higher in the control group. However, most MI cases had hypertension and a significantly higher body mass index (BMI) than control participants. There were more smokers among the control participants. The prevalence of type 2 diabetes, previous CVD, and family history of CVD was significantly higher in MI patients than in control participants.

Genetic Analyses
The analyzed genotype frequencies of polymorphisms were consistent with Hardy-Weinberg equilibrium (p > 0.05). In addition, the minor allele frequencies of all analyzed SNPs were consistent with a HapMap phase 3 CEPH reference population. Table 2 shows the distribution of genotypes and alleles of MTNR1B polymorphisms. There were no significant associations between rs10830963, rs1387153, and rs4753426 in the MTNR1B gene and MI. Genotype model analyses (recessive, dominant, and codominant) were also not significantly different between cases and controls (Supplementary Table S1). Table 3 shows the results of the logistic regression model fitted to estimate the independent effect of the analyzed polymorphism after adjustment for cardiovascular risk factors. SNP rs1387153 was associated with an increased risk of MI in the present study. In the recessive model, CC versus CT + TT, the p-value was 0.043 (OR = 0.30; 95% CI 0.09-0.96). A logistic regression model showed a significant association with age (p = 0.028), diastolic blood pressure (p = 0.001), systolic blood pressure (p = 0.003), BMI (p = 0.003), smoking (p < 0.001), and previous CVD (p < 0.001). The frequency of predicted haplotypes in MI patients and control participants is shown in Table 4. A statistically significant difference in haplotype distribution was confirmed for the CTT haplotype (p = 0.015) between MI patients and control participants. However, after correction for multiple tests, this difference was no longer significant (q = 0.060). There is linkage disequilibrium (LD) for all analyzed polymorphisms in the MTNR1B gene. The linkage disequilibrium between rs10830963 and rs1387153 was D' = 0.842 and r 2 = 0.701; between rs10830963 and rs4753426 was D' = 0.945 and r 2 = 0.368; and LD between rs1387153 and rs4753426 was D' = 0.819 and r 2 = 0.273. An association was found between some cardiovascular risk factors and MTNR1B gene polymorphisms in patients with MI ( Table 5). The rs10830963 and rs1387153 polymorphisms were associated with type 2 diabetes mellitus and previous CVDs.

Analysis of Chronotype and Polymorphisms
Analysis between MI patients and control participants revealed no association between chronotype and any MTNR1B polymorphisms tested at the allele level (Supplemental Table S2). The association between MTNR1B genotype frequency and chronotype in MI patients is shown in Table 6. There was no significant difference between individual genotypes and specific chronotypes in MI patients.  Table 7 shows the association between cardiovascular risk factors and overall chronotype score and chronotype in MI patients. Only one patient belongs to the evening chronotype, so it was not included in the analysis.

Discussion
Our results of the analysis of MTNR1B gene polymorphisms (rs10830963, rs1387153, and rs4753426) did not confirm the association between the analyzed variations of the melatonin receptor 1B gene and MI susceptibility. In addition, an association study was performed to determine which MTNR1B gene variants might be associated with chronotype in MI patients, and no association between MTNR1B gene variants and chronotype was found.
To the best of our knowledge, there are no comparable studies, so it is difficult to reconcile the results of this study with studies in patients with diabetes. However, the study by Huber et al. in hypertensive patients showed that the effects of MTNR1B gene polymorphisms and haplotypes did not differ between patients with and without myocardial infarction [6]. Moreover, although the frequency of the CTT haplotype was significantly different between the MI patient group and the control group in the present study, this difference was no longer significant after correction for multiple testing. Similarly, a study by Xue et al. showed that MTNR1B gene variations in patients with diabetes did not increase their risk of CVD mortality [12]. In addition, some of the analyzed cardiovascular risk factors, such as diastolic and systolic blood pressure, BMI, and smoking, were significantly associated with the analyzed polymorphisms in MI patients in this study. However, no significant association was found for the rs4753426 polymorphism in the present study. In addition, the rs10830963 and rs1387153 polymorphisms were associated with type 2 diabetes in MI patients, which is consistent with previous studies [31][32][33][34]. Furthermore, no association was found between chronotype and MTNR1B gene variants in individuals with myocardial infarction in the present study, and other studies also showed that chronotype was not significantly associated with increased risk of cardiovascular diseases or myocardial infarction [28,30,35,36].
Our results indicate no association between MTNR1B gene variants and myocardial infarction, unlike other studies that confirmed an association with increased LDL and triglyceride levels and arterial stiffness in people without CVD [37]. In addition, variations in the MTNR1B gene have been associated with its expression and signaling changes [6]. A commonly studied polymorphism of the MTNR1B gene is rs108390963, which is associated with an increased risk of diabetes. Diabetic patients who are carriers of the minor G allele have a 19% higher risk of myocardial infarction [12,17]. However, the minor G allele has not been associated with increased mortality from myocardial infarction or other cardiovascular diseases in people with diabetes [12]. In addition, the minor G allele has been shown to be associated with increased melatonin levels and disrupted circadian rhythms, i.e., a delayed light-dark cycle [3]. Indeed, the minor G allele rs10830963 is associated with increased mRNA expression of the MTNR1B gene and, thus, with increased melatonin levels [3]. Since melatonin controls circadian rhythm, increased melatonin secretion can lead to circadian rhythm disturbances associated with cardiovascular and metabolic disorders [3,[38][39][40].
A decrease in melatonin levels is associated with aging and is especially pronounced in some age-related diseases [41]. It is important to emphasize that melatonin levels have been reduced in cardiovascular diseases such as coronary artery disease, arterial hypertension, heart failure, and after myocardial infarction [37]. Previous studies confirmed decreased melatonin production in patients with coronary artery disease [42]. In contrast, melatonin synthesis increases in response to myocardial infarction, supporting the role of melatonin in protecting the heart after infarction [42]. Activation of the MTNR1B receptor on myocytes by melatonin leads to the attenuation of ischemic injury to the heart [19]. Animal studies have shown that melatonin inhibits the formation of reactive oxygen species and promotes the intracellular accumulation of calcium ions, which protects myocytes from damage caused by ischemia [8]. When melatonin was administered to patients with myocardial infarction shortly after the onset of initial symptoms, there was a significant reduction in infarct size [5]. Moreover, melatonin is a ligand for nuclear retinoid orphan receptors, including the RORα receptor, one of the circadian clock proteins involved in the control of the circadian rhythm itself [4]. In addition to the already known cardioprotective functions of melatonin, its association with the RORα receptor may be another way to protect against ischemic damage to the myocardium [4,43].
The association between MTNR1B gene variations and chronotype has also been studied, mainly in diabetic patients. The aging process affects chronotype, and older people tend to be morning types [35], and we also obtained similar results. The only association found in this study for a specific chronotype category was that neither type had significantly higher systolic pressure. However, no association was found between chronotype and MTNR1B gene variants in individuals with myocardial infarction. Thus, individuals belonging to the morning type and carriers of the minor G allele rs10830963 have been shown to have a higher risk of developing diabetes [27,36]. However, it is thought that a shift in circadian rhythm due to short sleep duration and early morning food intake increases the risk of diabetes, not MTNR1B gene variations [36]. On the other hand, another study showed that the minor G allele was associated with delayed melatonin secretion and an evening chronotype [3]. Shift work significantly affects an individual's chronotype, and some studies have shown that the G allele of the rs10830963 polymorphism is associated with a reduced risk of stroke. In contrast, the CC genotype of the same polymorphism has been associated with a higher risk of stroke in night shift workers [3]. However, another study showed that carriers of risk allele G of the rs10830963 polymorphism who work shift work or have a morning or evening chronotype do not have an increased risk of type 2 diabetes. In the aforementioned study, the authors showed that night shift work and morning or evening preference were associated with the prevalence of type 2 diabetes, but the rs10830963 polymorphism was not [36].

Study Limitations
This study has several limitations, but sample size is one of the most important. In previous studies of MTNR1B gene polymorphisms, the measure of association as estimated by OR ranged from 0.97 to 1.25 [44] and can be considered a small effect. Although the magnitude of the effect size is very likely, a larger number of participants could lead to a significant association between genotype and phenotype. In addition, the analyzed polymorphisms of the MTNR1B gene, which were selected based on previous publications, can be considered a limitation of the study. The selected polymorphisms were mainly associated with type 2 diabetes and fasting blood glucose levels, and the lack of association can be partly explained by the low prevalence of MI patients with diabetes-22.1%. In addition, because of the low frequency of some genotypes, it was not possible to statistically estimate associations. Similarly, we cannot exclude the possibility that statistically significant differences in baseline characteristics, such as hypertension and dyslipidemia, may influence the outcome of the study. The reproducibility of association studies is low, mainly because of the small effect size of individual polymorphisms. This explains the difference between our results and those of most other studies on the MTNR1B gene polymorphism. Circadian phenotype assessment and chronotype classification were based on a self-reported questionnaire and could be prone to bias. Nevertheless, this could not significantly affect the outcome of the study. In addition, because of the limited data collected retrospectively, we could not assess the potential influence of shift work schedules on chronotype.
The way cases were defined could introduce bias in the research. Since ischemic ECG changes were on the list of criteria that needed to be fulfilled, such a definition of outcome could potentially exclude non-STEMI patients. Myocardial infarction, as well as some other cardiovascular diseases, represent a continuum rather than a bivariant category. Still, the implementation of guidelines into clinical practice requires a definition of categories and the loss of some information in the process. Although no cardiovascular diseases were detected in the control group at the time of the study, we cannot exclude the possibility of cardiovascular events occurring later in life. However, the advantage of this study is the homogeneity of the sample in terms of demographic variables such as gender, ethnicity, and social environment. University Hospital Osijek is a tertiary-level medical center with a large catchment area of 258,026 inhabitants [45], and all patients with MI are referred to this center. Since this is a publicly funded hospital and there are no private hospitals in this county, the possible influence of social stratification can be excluded.

Participants
This case-control study was based on data collected in previously published studies of circadian clock variations in myocardial infarction patients [46][47][48]. The sample size consisted of 199 cases, 114 men and 85 women, with nonfatal acute myocardial infarction hospitalized at the Clinical Department of Cardiovascular Diseases and Intensive Care, University Hospital Osijek, Croatia, from 1 August 2012 to 31 December 2018. We reviewed the medical records of all participants.
Cases were included if they fulfilled the criteria for type 1 or type 2 MI, according to Thygesen et al. [49,50]. At least two out of three criteria had to be met: cardiac troponin T above the 99th percentile, chest pain symptoms for more than 30 min indicating ischemia, and ischemic electrocardiographic (ECG) changes.
The control group consisted of 198 participants, 103 men and 95 women, with no cardiovascular diseases noted in medical records. They were selected after a check-up by their primary care physician in the outpatient clinic.
Participants were excluded if they had severe heart disease or type 2 diabetes mellitus or were related to other participants in the study since chronotype traits are heritable [51,52]. Blood pressure in mmHg, height, and weight were measured in all participants.
The Ethics Committee of the University Hospital Osijek (No. 25-1:3160-3/2012) approved this study. It was performed in accordance with the Declaration of Helsinki and its revisions.

Polymorphysm Selection and Genotyping
Genotyping was performed using pre-designed TaqMan ® assays on the Applied Biosystems QuantStudio 5 real-time PCR system. Three single nucleotide polymorphisms (SNPs: rs10830963, rs1387153, and rs4753426) in the MTNR1B gene were analyzed using QuantStudio Design & Analysis Software version 1.5.2 (Applied Biosystems, Foster City, CA, USA). The above polymorphisms were selected because they had previously been associated with myocardial infarction risk, chronotype, arterial stiffness, and cardiovascular risk factors, particularly type 2 diabetes mellitus [2,19,27,33,37]. The polymorphisms analyzed are all located in the intronic region of the MTNR1B gene. Participants with missing genotyping data were excluded.
The SNPStats web tool was used to analyze recessive, dominant, and codominant genotype models independently [56]. In addition, the web tool SHEsis was used to reconstruct haplotypes from genotype data [57,58].

Statistical Analyses
Descriptive statistics were used to present data as numbers and percentages or as arithmetic means ± standard deviations. The chi-square test was used to calculate a significant difference for each polymorphism to compare allele frequency and genotype distribution between MI patients and control participants. Genotyping quality control was also performed using the chi-square test for goodness-of-fit and an analysis of genotype distributions compared with those expected by Hardy-Weinberg equilibrium. Logistic regression was employed to estimate the effect of MTNR1B rs10830963, rs1387153, and rs4753426 genotypes on the probability of myocardial infarction in patients. In addition, age, diastolic and systolic blood pressure, BMI, smoking, hypertension, dyslipidemia, family history of CVD, and previous CVDs were used as covariates. The chi-square test was used to determine the relationship between cardiovascular risk variables and MTNR1B polymorphisms. In addition, the chi-square test and Kruskal-Wallis test were used to determine the significance of the relationships between allele and genotype frequencies and chronotype. The association was considered significant if the p-value was equal to or less than 0.05. However, the Benjamini-Hochberg method (false discovery rate-FDR value) was used due to the three polymorphisms studied. Therefore, only q-values less than 0.05 were considered statistically significant. Statistical analyses were performed using SPSS software (version 26.0, SPSS Inc., Chicago, IL, USA).

Conclusions
Physiological processes are coordinated with the diurnal rhythm of the circadian hormone melatonin, for whose function the high-affinity membrane receptor MTNR1B is important. Through the MTNR1B receptor, melatonin plays an important role not only in the maintenance of cardiovascular homeostasis but also in the pathogenesis of disease. In this study, no association was found between MTNR1B gene variations (rs10830963, rs1387153, and rs4753426) and myocardial infarction because the effect size of a single gene on complex clinical parameters such as myocardial infarction is relatively small. In addition, no significant association was found between MTNR1B gene polymorphism and chronotype in patients with MI. However, some cardiovascular risk factors were associated with the analyzed polymorphisms and chronotypes in MI patients. Nevertheless, as some previous studies have shown, these negative results do not exclude the role of previously mentioned polymorphisms in the development of myocardial infarction. Rather, they may indicate that the MTNR1B gene polymorphisms are minor risk factors for susceptibility to myocardial infarction.