Journal of

Cardiovascular
Development and Disease

Article
Sex Differences in Circadian Clock Genes and Myocardial
Infarction Susceptibility
Ivana Škrlec 1, * , Jasminka Talapko 1 , Martina Juzbašić 1 and Robert Steiner 2,3

1 Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, HR-31000 Osijek, Croatia;
[email protected] (J.T.); [email protected] (M.J.)
2 Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Josipa Huttlera 4, HR-31000 Osijek, Croatia;
[email protected]
3 Clinical Department of Cardiovascular Diseases and Intensive Care, Clinic for Internal Medicine, University
Hospital Osijek, Josipa Huttlera 4, HR-31000 Osijek, Croatia
* Correspondence: [email protected]

Abstract: The growing body of evidence shows a significant difference in the circadian rhythm
of cardiovascular disease based on biological sex. The incidence of cardiovascular disease varies
between women and men. Additionally, biological sex is vital for the timely application of therapy—
chronotherapy, which benefits both sexes. This study aimed to examine the potential difference
of single nucleotide polymorphisms (SNPs) of the circadian rhythm genes ARNTL, CLOCK, CRY2
and PER2 in women and men with myocardial infarction. A cross-sectional study was conducted,
including 200 patients with myocardial infarction. Altogether, ten single nucleotide polymorphisms
in the ARNTL, CLOCK, CRY2 and PER2 genes were analyzed. The Chi-square test yielded statistically
significant differences in CLOCK gene rs11932595 polymorphism in a recessive genotype model





 between women and men with a p-value of 0.03 and an odds ratio 2.66, and a corresponding 95%
confidence interval of 1.07 to 6.66. Other analyzed polymorphisms of the circadian rhythm genes
Citation: Škrlec, I.; Talapko, J.;
Juzbašić, M.; Steiner, R. Sex
ARNTL, CRY2, and PER2 did not significantly differ between the sexes. According to the study’s
Differences in Circadian Clock Genes current results, the CLOCK gene’s genetic variability might affect myocardial infarction concerning
and Myocardial Infarction biological sex.
Susceptibility. J. Cardiovasc. Dev. Dis.
2021, 8, 53. https://doi.org/10.3390/ Keywords: biological sex; circadian rhythm; clock genes; myocardial infarction
jcdd8050053

Academic Editor: Andy Wessels

1. Introduction
Received: 24 February 2021
Circadian rhythm affects daily the physiological functioning that is important for the
Accepted: 7 May 2021
proper functioning of peripheral organs, including the heart [1]. The central molecular
Published: 8 May 2021
clock is located in the suprachiasmatic nucleus (SCN) and controls peripheral molecular
clocks located in almost all organs [2]. Thus, circadian rhythms are essential for normal
Publisher’s Note: MDPI stays neutral
physiology, and if there is a desynchronization between the central and peripheral clocks,
with regard to jurisdictional claims in
published maps and institutional affil-
disease develops [3,4]. The molecular clock’s basis consists of a transcriptional and trans-
iations.
lational feedback loop of four key transcription factors. CLOCK and ARNTL (BMAL1)
heterodimers form a positive loop that stimulates PER and CRY transcription and other
clock-linked genes. A negative feedback loop is composed of PER and CRY heterodimers,
which inhibits the transcription of CLOCK and ARNTL genes [5,6].
Circadian rhythm influences daily body functions such as temperature, hormone re-
Copyright: © 2021 by the authors.
lease, digestion, sleep, and mood [7,8]. Many cardiovascular functions are also regulated by
Licensee MDPI, Basel, Switzerland.
circadian rhythms, such as blood pressure, heart rate, endothelial function, and thrombus
This article is an open access article
distributed under the terms and
formation [3,9–12]. Circadian rhythm disorders can have negative consequences for the
conditions of the Creative Commons
whole organism, including the cardiovascular system, which leads to an increased risk of
Attribution (CC BY) license (https://
heart disease [13,14].
creativecommons.org/licenses/by/ Numerous studies have shown a significant difference in circadian rhythm on cardio-
4.0/). vascular disease (CVD) based on sex [3]. It has been shown that women are somewhat

J. Cardiovasc. Dev. Dis. 2021, 8, 53. https://doi.org/10.3390/jcdd8050053 https://www.mdpi.com/journal/jcdd

J. Cardiovasc. Dev. Dis. 2021, 8, 53 2 of 12

more sensitive to circadian rhythm disorders and have an increased risk of chronic dis-
eases affecting the reproductive and immune systems, central nervous system, endocrine
functions, and cardiovascular health [7,8]. Many physiological processes that are regulated
by circadian rhythms differ between women and men. Therefore, women have higher
systolic and diastolic blood pressure at night than men [15]. Women have a higher heart
rate [16], are more sensitive to sleep disorders than men [8], and have been shown to have
a shorter circadian period than men [17]. Many CVDs are associated with obesity, the onset
of which is mainly sex-related [18]. Sex plays a vital role in the circadian disorder of energy
homeostasis [19]. Women are more sensitive to insulin and have higher adiponectin and
leptin levels, while men have higher triglycerides levels in the blood [18–20]. Circadian
disorder in women stimulates adipose tissue storage, while fat stores’ mobilization is more
effective in men [18]. Men are less susceptible to circadian rhythm disorders [19]. Different
triggers could affect the various incidence of CVD between men and women. These triggers
may be due to exposure to multiple factors, sex-specific cardiac mechanisms, or both [21].
The circadian rhythm influences heart disease in a sexually dimorphic manner [9].
Women and men differ in their resistance to cardiovascular disease, with sex hormones and
sex chromosomes playing an important role [3,9,18,22–24]. Studies in rodents have shown
that female mice have a higher survival rate after myocardial infarction (MI) than male
mice [25]. Additionally, despite disturbed circadian rhythm, female mice are protected
from the development of metabolic changes and cardiomyopathy [13]. Sex hormones,
estrogens, are thought to play a protective role against CVD development, including
MI. Estrogens influence circadian molecular clock gene expression, which is essential in
regulating circadian rhythm via SCN [22]. Premenopausal women have less CVD than
men of the same age, but that changes when women reach menopause. Then, estrogen’s
cardioprotective effect is lost, and elevated testosterone levels lead to hypertension and
coronary artery disease in postmenopausal women [24,26]. Cardiolipin composition was
observed to differ in female and male mice’s heart cells, which may affect MI resistance
in females [9]. Cardiolipins are an essential part of mitochondria’s inner membrane and
play a crucial role in mitochondria’s bioenergetics, and their composition is altered in heart
disease [13]. Another possible explanation for the lower incidence of CVD in women is that
women may have unrecognized CVD symptoms, including myocardial infarction, and are
less likely to report chest pain associated with acute coronary syndrome [27]. Additionally,
it is known that a large number of women with acute MI die before hospitalization [27].
Studies have shown a significant reduction in morbidity and mortality from atherosclerosis
in men, while the rate of recurrent atherothrombotic events, including cardiovascular death,
has increased in women [28]. The mechanisms of circadian rhythm act differently in the
hearts of women compared to men. Women and men show flexibility in cardiovascular
disease, and biological sex is crucial for circadian therapy to benefit both sexes [9].
All of the mentioned above led to this study’s aim to investigate the potential difference
in ARNTL, CLOCK, CRY2, and PER2 circadian rhythm gene polymorphisms in women and
men with myocardial infarction.

2. Participants and Methods

2.1. Participants
A cross-sectional study was conducted, including 200 patients with myocardial infarc-
tion. Of these, 86 were women, and 114 were men. Subjects were patients with a history
of myocardial infarction hospitalized at the University Hospital Osijek, Croatia. Patients
with type I and II myocardial infarction with elevated cardiac troponin T above the 99th
percentile were included in the study and with one of the subsequent factors: electrocar-
diogram (ECG) changes, pathological Q waves, myocardial ischemia symptoms, loss of
viable myocardium or regional wall motion abnormalities, or coronary thrombus identified
by angiography [29,30]. Patients were excluded from this study if they had undergone
percutaneous coronary intervention (n = 35) or a coronary artery bypass (n = 42) because
those are not clinical characteristics of acute MI. Additionally, patients were excluded if
 identified by angiography [29,30]. Patients were excluded from this study if they had un-
dergone percutaneous coronary intervention (n = 35) or a coronary artery bypass (n = 42)
because those are not clinical characteristics of acute MI. Additionally, patients were ex-
cluded if they had cancer (n = 48), cardiomyopathy (n = 21), or other circadian rhythm-
J. Cardiovasc. Dev. Dis. 2021, 8, 53 regulated diseases, such as sleep disorders (n = 94) and mood disorders (n = 68), including
3 of 12
depression (Figure 1).
Data on the patient’s medical history and sociodemographic data were collected. All
data were additionally checked in the patient’s medical records.
they The
hadEthics
cancerCommittee
(n = 48), cardiomyopathy
of the University(nHospital
= 21), orOsijek
other circadian rhythm-regulated
(Ethical Approval Code: 25-
diseases, such as sleep disorders (n = 94) and mood disorders (n = 68), including
1: 3160-3/2012) approved this research. According to the Declaration of Helsinki depression
and its
(Figure 1).
amendments, the study was carried out. All participants signed informed consent forms.

Figure 1. Flowchart
Figure 1. of patient
Flowchart of patientselection.
selection.PCI—percutaneous
PCI—percutaneouscoronary
coronaryintervention,
intervention, CABG—coronary
CABG—coronary artery
artery bypass
bypass graft-
grafting.
ing.
Data on the patient’s medical history and sociodemographic data were collected. All
2.2.
dataGenotyping of Singlechecked
were additionally Nucleotide in Polymorphisms
the patient’s medical records.
Thethis
In Ethics Committee
study, SNPs wereof the determined
University Hospital
in four Osijek (Ethical
circadian Approval
rhythm genes:Code:
CLOCK,25-1:
3160-3/2012)
ARNTL, CRY2,approved
and PER2. this research.
A total of tenAccording
SNPs weretoanalyzed.
the Declaration of Helsinki
Three SNPs and its
in the ARNTL
amendments,
gene (rs3789327,the study was carried
rs4757144 out. All participants
and rs12363415), and CLOCK signed informed consent
(rs11932595, rs6811520 forms.
and
rs13124436). Meanwhile, two SNPs were analyzed in the CRY2 (rs2292912 and rs10838524)
2.2. PER2
and Genotyping
genesof(rs35333999
Single Nucleotide Polymorphisms
and rs934945). These central clock gene polymorphisms have
been In this study,
associated SNPs
with were determined
cardiovascular risk in four circadian
factors in earlierrhythm
researchgenes: CLOCK,
[14,31–33]. AllARNTL,
tested
CRY2, and PER2.
polymorphisms are located in the intronic region of the genes, except for PER2 genegene
A total of ten SNPs were analyzed. Three SNPs in the ARNTL pol-
(rs3789327, rs4757144 and rs12363415), and CLOCK (rs11932595, rs6811520 and rs13124436).
ymorphisms.
Meanwhile,
According two to
SNPsthe were
manufacturer’s the CRY2 (rs2292912
analyzed in standard andextraction
protocol, the rs10838524)ofand PER2 genes
genomic DNA
(rs35333999 and rs934945). These central clock gene polymorphisms
was made from peripheral blood lymphocytes (QIAamp DNA Blood Mini Kit, have been associated
Qiagen,
with cardiovascular
Hilden, Germany). All riskanalyses
factors inwere
earlier research by
conducted [14,31–33].
real-timeAll tested
PCR polymorphisms
using TaqMan probes. are
located in the intronic region of the genes, except for PER2 gene polymorphisms.
Allele discrimination analyses were performed with SDS 7500 software version 2.3 (Ap-
pliedAccording
Biosystems, toFoster
the manufacturer’s
City, CA, USA). standard protocol, the extraction of genomic DNA
was made from peripheral blood lymphocytes (QIAamp DNA Blood Mini Kit, Qiagen,
Hilden, Germany). All analyses were conducted by real-time PCR using TaqMan probes.
Allele discrimination analyses were performed with SDS 7500 software version 2.3 (Applied
Biosystems, Foster City, CA, USA).

2.3. Statistical Analyses

Statistical analyses were carried out with SPSS software (version 22.0, SPSS Inc.,
Chicago, IL, USA) for Windows. Quantitative data are shown as the mean and standard
deviation, while categorical data are shown as frequency and percentages. For each SNP, a
significant difference was calculated to compare allele frequency and genotype distribution
in women and men using the Chi-Square test (χ2) on contingency tables. An additional
J. Cardiovasc. Dev. Dis. 2021, 8, 53 4 of 12

level of genotyping quality control was achieved by applying the Chi-square goodness-of-
fit test and analyzing genotypes’ distribution with those expected in the Hardy–Weinberg
equilibrium. Genotype models were determined using the web tool SNPStats [34]. Logistic
regression was used to estimate the effect of ARNTL (rs3789327, rs4757144, rs12363415),
CLOCK (rs11932595, rs6811520, rs13124436), CRY2 (rs2292912 and rs10838524) and PER2
(rs35333999 and rs934945) genotypes on the patients’ sex. Age, smoking, history of hyper-
tension, dyslipidemia, type 2 diabetes mellitus, positive family history of CVD, history
of former CVD, systolic and diastolic blood pressure, and BMI were used as a covariate.
Kruskal–Wallis and Chi-square tests were utilized to define the association of cardiovas-
cular risk factors with polymorphisms of the analyzed molecular clock genes. In the case
where p ≤ 0.05, the association was considered significant. Significant value corrections
were made by the Benjamini–Hochberg method (false detection rate—FDR value) due to
several polymorphisms tested. Only q-values less than 0.05 were considered significant.
The most common haplotypes and the association between haplotypes and CVD risk fac-
tors were identified by the SNPStats web tool [34]. Only an additive model was considered
due to the low frequency of some haplotypes.

3. Results
Demographic and clinical characteristics of women and men are presented in Table 1.
The mean age of all participants was 66 ± 12 years, and 57% were men.

Table 1. Demographic and clinical characteristics of patients.

Variable Women Men p-Value

Number (%) 86 (43%) 114 (57%) -
Age (years) 69 ± 12 64 ± 12 0.002 *
Smoking 16 (18.6%) 25 (21.9%) <0.001 †
Hypertension 50 (58.1%) 57 (50%) 0.25 †
Dyslipidemia 17 (19.8%) 9 (7.9) 0.013 †
Type 2 diabetes mellitus 18 (20.9%) 26 (22.8%) 0.75 †
Positive family history of CVD 29 (25.4%) 18 (20.9%) 0.18 †
History of former CVD 82 (71.9%) 61 (70.9%) 0.33 †
Systolic blood pressure (mm Hg) 127.55 ± 18.21 126.14 ± 13.89 0.97
Diastolic blood pressure (mm Hg) 75.77 ± 10.86 77.27 ± 9.25 0.46
BMI (kg/m2 ) 29.31 ± 4.19 28.30 ± 5.07 0.09
* Mann–Whitney U test, † Chi-squared test, BMI—body mass index. The bold are statistically significant values.

After Bonferroni correction, a significant difference was found in the recessive geno-
type model of CLOCK rs11932595 polymorphism (GG vs. AG + AA) in women compared to
men (p = 0.03, the odds ratio of 2.66, 95% confidence interval 1.07 to 6.66). Other genotype
models of ARNTL, CRY2 and PER2 genes were not significantly different between women
and men (Table 2).
Logistic regression was adjusted to evaluate the independent impact of the chosen
polymorphism after modifying for age and cardiovascular risk factors. All tested SNPs
showed a significant interaction between age (p = 0.017), smoking (p < 0.001), and diastolic
blood pressure (p = 0.045) (Table 3).
The chi-square test compared the allelic frequency and genotype distribution between
women and men (Table 4). Women and men were not significantly different in tested
polymorphisms or genotype distribution in ARNTL, CLOCK, CRY2, or PER2 genes.
The association was found among cardiovascular risk factors and examined circadian
rhythm gene polymorphisms in women and men (Table 5). The SNP rs12363415 in the
ARNTL gene was associated with type 2 diabetes mellitus in women (p = 0.003). In
contrast, rs2292912 in the CRY2 gene and rs934945 in the PER2 gene were associated with
dyslipidemia in the men (p = 0.02, and <0.001, respectively). The CLOCK polymorphism
rs11932595 was associated with systolic and diastolic blood pressure in women (p = 0.005
and p = 0.006, respectively) and BMI in men (p = 0.024).
J. Cardiovasc. Dev. Dis. 2021, 8, 53 5 of 12

The haplotypes were examined in the four circadian clock genes. The frequencies of
the predicted haplotypes of the tested circadian clock gene polymorphisms in women and
men are shown in the supplementary material (Supplemental Table S1). The haplotype
CGA distribution at the ARNTL gene locus was statistically significantly different when
comparing haplotype frequency between women and men (p = 0.030, OR = 1.86, 95% CI
= 1.05–3.27). The CGA haplotype of the ARNTL gene was significantly associated with
dyslipidemia and hypertension under the additive model (p = 0.03, OR = 0.45, 95% CI
= 0.22-0.95, and p = 0.03, OR = 0.29, 95% CI = 0.09–0.88, respectively). Furthermore, a
significant difference was observed at the CRY2 gene locus when comparing the haplotype
GG frequency between women and men (p < 0.001, OR = 9.73, 95% CI = 9.71–9.76). The
CA and GA haplotypes of the CRY2 gene were significantly associated with dyslipidemia
under the additive model (p = 0.02, OR = 8.20, 95% CI = 1.42–47.45, and p = 0.03, OR = 6.44,
95% CI = 1.09–37.89, respectively).

Table 2. Genotype models of the ARNTL, CLOCK, CRY2, and PER2 polymorphisms.

Gene Codominant Model Dominant Model Recessive Model

p OR 95% CI p OR 95% CI p OR 95% CI
ARNTL
rs3789327 0.18 0.56 0.29–1.07 0.06 0.56 0.30–1.04 0.56 0.80 0.37–1.70
rs4757144 0.48 1.28 0.69–2.39 0.67 1.13 0.63–2.02 0.36 0.69 0.31–1.52
rs12363415 0.77 1.05 0.56–1.99 0.98 1.01 0.54–1.88 0.48 0.43 0.04–4.89
CLOCK
rs11932595 0.08 0.93 0.50–1.72 0.67 1.14 0.63–2.05 0.03 2.66 1.07–6.66
rs6811520 0.45 0.78 0.42–1.46 0.28 0.72 0.40–1.31 0.33 0.68 0.31–1.47
rs13124436 0.95 0.97 0.53–1.77 0.99 1.00 0.57–1.77 0.76 1.15 0.46–2.87
CRY2
rs2292912 0.99 0.97 0.54–1.76 0.94 0.98 0.55–1.75 0.96 1.05 0.17–6.60
rs10838524 0.30 1.46 0.75–1.97 0.46 1.26 0.68–2.36 0.28 0.67 0.33–1.38
PER2
rs35333999 0.89 1.24 0.42–3.72 0.79 1.15 0.41–3.21 0.77 0.66 0.04–10.76
rs934945 0.43 1.23 0.64–2.34 0.79 1.09 0.59–2.01 0.25 0.39 0.07–2.11
p-Values shown in the table are corrected for the multiple comparisons. The bold is statistically significant value.

Table 3. Odds ratios for the difference between sex, adjusted for age, and cardiovascular risk factors
included in the logistic regression model.

Risk Factor OR (95% CI) p Value

Age 0.96 (0.94–0.99) 0.017
Smoking 2.24 (1.45–3.47) <0.001
Hypertension 1.06 (0.53–1.44) 0.86
Dyslipidemia 2.36 (0.85–6.56) 0.10
Type 2 diabetes mellitus 0.85 (0.37–1.95) 0.71
Positive family history of CVD 1.20 (0.69–2.09) 0.51
History of former CVD 0.92 (0.59–1.44) 0.72
Systolic blood pressure (mm Hg) 0.97 (0.94–1.00) 0.07
Diastolic blood pressure (mm Hg) 1.05 (1.01–1.11) 0.045
BMI (kg/m2 ) 0.96 (0.89–1.03) 0.27
BMI—body mass index. The bold are statistically significant values.
J. Cardiovasc. Dev. Dis. 2021, 8, 53 6 of 12

Table 4. Allele and genotype distribution and frequencies of the ARNTL, CLOCK, CRY2, and PER2 polymorphisms.
Genotype Frequency, N (%)
Gene SNP Minor Allele MAF * Women MAF * Men p-Value q Value Genotype
Women Men p Value X2 q Value

TT 23 (26.7%) 44 (38.5%)

rs3789327 C 0.46 0.38 0.118 0.125 TC 47 (54.6%) 53 (46.4%) 0.210 3.11 0.216

CC 16 (18.6%) 17 (14.9%)

AA 35 (40.6%) 44 (38.5%)

rs4757144 G 0.38 0.37 0.823 0.835 AG 36 (41.8%) 55 (48.2%) 0.578 1.09 0.577
ARNTL
GG 15 (17.4%) 15 (13.1%)

AA 61 (70.9%) 81 (71%)

rs12363415 G 0.16 0.15 0.828 0.888 AG 23 (26.7%) 32 (28%) 0.698 0.72 0.757

GG 2 (2.3%) 1 (0.8%)

AA 32 (37.2%) 41 (35.9%)

rs11932595 G 0.35 0.41 0.214 0.255 AG 47 (54.6%) 52 (45.6%) 0.103 4.53 0.102

GG 7 (8.1%) 21 (18.4%)

CC 29 (33.7%) 46 (40.3%)

rs6811520 T 0.42 0.36 0.220 0.254 CT 41 (47.6%) 53 (46.4%) 0.465 1.53 0.486
CLOCK
TT 16 (18.6%) 15 (13.1%)

AA 9 (10.4%) 13 (11.4%)

rs13124436 A 0.32 0.33 0.846 0.914 AG 37 (43%) 49 (42.9%) 0.976 0.05 1

GG 40 (46.5%) 52 (45.6%)

CC 53 (61.6%) 71 (62.2%)

rs2292912 G 0.20 0.20 1 1 CG 31 (36%) 40 (35%) 0.982 0.03 0.999

GG 2 (2.3%) 3 (2.6%)
CRY2
GG 27 (31.3%) 30 (26.3%)

rs10838524 A 0.45 0.45 1 1 GA 40 (46.5%) 65 (57%) 0.327 2.23 0.317

AA 19 (22%) 19 (16.6%)

CC 79 (91.8%) 104 (91.2%)

rs35333999 T 0.05 0.05 1 1 CT 6 (6.9%) 9 (7.8%) 0.952 0.09 0.999

TT 1 (1.1%) 1 (0.8%)
PER2
CC 60 (69.7%) 78 (68.4%)

rs934945 T 0.18 0.16 0.722 0.789 CT 21 (24.4%) 34 (29.8%) 0.241 2.84 0.247

TT 5 (5.8%) 2 (1.7%)

* MAF—minor allele frequency, q value—corrected significant p-value by the Benjamini–Hochberg method.

J. Cardiovasc. Dev. Dis. 2021, 8, 53 7 of 12

Table 5. The association between cardiovascular risk factors and circadian clock gene SNPs.
Gene Age * Smoking † Hypertension † Dyslipidemia † Type 2 Diabetes Mellitus † Positive Family History of CVD † History of Former CVD † SBP * DBP * BMI *

Women Men Women Men Women Men Women Men Women Men Women Men Women Men Women Men Women Men Women Men

ARNTL

rs3789327 0.425 0.391 0.011 0.895 0.118 0.406 0.233 0.314 0.182 0.339 0.181 0.088 0.071 0.034 0.330 0.209 0.348 0.475 0.607 0.183
rs4757144 0.264 0.111 0.718 0.182 0.187 0.383 0.199 0.477 0.642 0.184 0.057 0.172 0.038 0.527 0.696 0.729 0.297 0.164 0.106 0.609
rs12363415 0.634 0.587 0.737 0.595 0.170 0.566 0.094 0.875 0.003 0.848 0.152 0.992 0.122 0.443 0.931 0.831 0.318 0.927 0.523 0.774

CLOCK

rs11932595 0.190 0.567 0.426 0.937 0.269 0.617 0.173 0.060 0.798 0.224 0.276 0.702 0.693 0.724 0.005 0.157 0.006 0.275 0.996 0.024
rs6811520 0.439 0.169 0.304 0.691 0.687 0.560 0.399 0.431 0.378 0.693 0.389 0.463 0.313 0.501 0.499 0.327 0.592 0.161 0.334 0.219
rs13124436 0.891 0.561 0.651 0.924 0.215 0.527 0.772 0.514 0.980 0.168 0.355 0.849 0.443 0.241 0.895 0.856 0.869 0.983 0.191 0.546

CRY2

rs2292912 0.818 0.366 0.811 0.562 0.169 0.178 0.768 0.020 0.188 0.908 0.840 0.579 0.139 0.303 0.749 0.503 0.750 0.515 0.531 0.834
rs10838524 0.135 0.544 0.215 0.776 0.338 0.138 0.288 0.175 0.606 0.224 0.879 0.575 0.095 0.857 0.202 0.814 0.500 0.844 0.274 0.080

PER2

rs35333999 0.194 0.035 0.101 0.632 0.624 0.574 0.391 0.896 0.365 0.860 0.274 0.029 0.713 0.525 0.607 0.149 0.368 0.511 0.423 0.217
rs934945 0.457 0.096 0.529 0.107 0.146 0.034 0.499 <0.001 0.933 0.162 0.819 0.785 0.129 0.004 0.305 0.195 0.264 0.668 0.345 0.449

The table presents * Kruskal–Wallis test p-value for numerical data and † Chi–squared test p-value for categorical data. CVD—cardiovascular disease, SBP—systolic blood pressure, DBP—diastolic blood pressure,
BMI—body mass index. The bold are statistically significant values.
J. Cardiovasc. Dev. Dis. 2021, 8, 53 8 of 12

4. Discussion
The genetic variability in the ARNTL, CLOCK, CRY2 and PER2 genes in this study
showed that variations in the circadian genes differ between women and men with myocar-
dial infarction. The present study found evidence of the difference between women and
men in the recessive genotype model of the rs11932595 in the CLOCK gene in the sample
of 200 patients with myocardial infarction. Circadian rhythm plays a vital role in cardiac
aging, which differs between men and women [9]. That is why biological sex is essential in
the right approach to studying circadian rhythm in cardiovascular diseases [9].
In this study, women had a higher prevalence of dyslipidemia. Moreover, the dyslipi-
demia prevalence was significantly higher in women than in men. Additionally, women
were significantly older than men which is similar to in the previous study [35]. Women
with CVD are usually older and have more risk factors than men. Thus, both hyperlipi-
demia and hypertension are more common in women with MI than in men [35], which is
evident in this study’s results. Some of the tested circadian rhythm genes were associated
with some of the CVD risk factors in women and men with MI. Thus, ARNTL rs12363415
was connected with type 2 diabetes mellitus in women, while CRY2 rs2292912 and PER2
rs934945 were connected with dyslipidemia in men. The CLOCK rs11932595 was associated
with systolic and diastolic blood pressure in women and BMI in men.
Circadian rhythm disorders in humans are associated with cardiometabolic dis-
eases [23]. Circadian rhythm is influenced by sex, and this interaction is evident throughout
most of life [23]. Women are more resistant than men to circadian rhythm disorders due
to sleep disorders and shift work [23]. Circadian rhythm disorders due to shift work or
sleep disorders are associated with an increased risk of cardiovascular disease, especially
in men [13]. Studies in rodents have found significant sex differences in heart size and
function, and sex influences cardiovascular aging [36]. Additionally, mutations in several
central clock genes have been linked to many cardiovascular diseases [36].
Mutations in the CLOCK gene lead to age- and sex-dependent dilated cardiomyopathy.
Aging female mice do not develop cardiomyopathy, even when the circadian rhythm
is disrupted due to CLOCK gene mutation [9]. However, ARNTL (BMAL1) knock-out
male mice show premature aging [9]. In mice with a CLOCK gene mutation, a circadian
period is prolonged. Female mice remain healthy for a long time and do not develop
cardiac dysfunction, unlike male mice that develop cardiac hypertrophy and dysfunction
quickly [23]. This effect is lost after ovariectomy. In this case, the cardiometabolic function
is impaired in female mice [23]. Female biological sex and estrogens lessen heart disease
development and have a cardioprotective effect [13].
The lower prevalence of the rs11932595 polymorphism in women (8.1% versus 18.4%
in men) could mean two things. First, more women with the GG genotype die before
surviving myocardial infarction and enrolling in the present study. Second, fewer women
with the GG genotype suffered a myocardial infarction and were hospitalized. Thus, one
cannot infer whether this is beneficial or detrimental. Nevertheless, patients carrying the
CLOCK rs11932595 GG genotype were 2.66 times likely to have myocardial infarction.
Genetic variations in the CLOCK gene are connected with blood pressure and car-
diovascular disorders in humans [37–40]. Here, an association of rs11932595, located in
the CLOCK gene’s intronic region, with systolic and diastolic blood pressure was found
in women and with BMI in men. CLOCK knock-out and CLOCK mutant mice show an
essential role of CLOCK in regulating blood pressure and kidney function [38–40]. Mice
with a knock-out or mutated CLOCK gene have lower blood pressure, plasma aldosterone
levels, changed urinary sodium excretion, and a higher incidence of diabetes insipidus
than control mice [37–40]. A possible mechanism contributing to this is the reduced
level of 20-hydroxyeicosatetraenoic acid in the CLOCK knock-out mice’s urine [37,39,40].
The 20-hydroxyeicosatetraenoic acid is essential in regulating blood pressure and blood
flow [37,39,40]. The CLOCK protein has also been associated with diabetes and obesity
due to a mutation that prevents CLOCK from binding to DNA and activating target
genes. Overweight women with a specific polymorphism in the 30 untranslated region
J. Cardiovasc. Dev. Dis. 2021, 8, 53 9 of 12

of the CLOCK gene have decreased gene expression activity, altered sleep patterns, and
circadian rhythmicity [39]. Studies conducted on CLOCK mutant mice have shown the
importance of CLOCK protein in daily pulse rate control, myocardial contractility, and
basal metabolism [41]. Mutation of the CLOCK gene in cardiomyocytes affects only the
circadian rhythm in cardiomyocytes and leads to physiological changes in cardiomyocytes.
Some of these changes are cardiac metabolism, heart rate, contractility, response to external
signals, cardiac growth, and regeneration [42].
In addition to CLOCK gene mutations that have been shown to affect sex-related CVD
development, research has shown that mutations in the PER1 gene also affect blood pres-
sure differently in men and women [25]. There is no dipping in blood pressure overnight in
PER1 knock-out male mice [25]. Such males develop non-dipping hypertension and have
an increased risk of developing CVD. PER1 knock-out female mice have a steady drop
in blood pressure overnight and are protected from hypertension without dipping [25].
Sex hormones contribute to sex differences in blood pressure and cardiovascular parame-
ters [25].
Some studies have shown that haplotypes of the ARNTL gene are associated with
hypertension and type 2 diabetes [43,44]. In the present study, the frequency of the CGA
haplotype of the ARNTL gene was 19% in women versus 11% in men, and patients carrying
the mentioned haplotype were 0.45 and 0.29 times less likely to have dyslipidemia and
hypertension, respectively. Kovanen et al. found the association of the CRY2 haplotype
with elevated triglycerides and metabolic syndrome [45]. This study’s haplotype analysis
further supports this phenotype association of dyslipidemia with the CRY2 polymorphisms
and haplotypes. It is observed that dyslipidemia is associated with rs2292912 CRY2 poly-
morphism in men, and patients carrying the CA or GA haplotypes of the CRY2 gene were
8.2 and 6.44 times likely to have dyslipidemia, respectively.
Understanding biological sex differences in cardiovascular function regulation by
circadian rhythm plays an essential role in applying drug therapy or chronotherapy [9,46].
Biological sex is a crucial factor in the timely administration of drugs in clinical cardiology [9].
There are sex differences in the incidence, pathophysiology, and clinical presentation of
heart disease. Women with ischemic heart disease have worse outcomes and are less
likely to receive timely treatment than men [47,48]. More women, after menopause, have
dyslipidemia than men [47], which is seen from the results of this study. Additionally, that
contributes to the higher incidence of CVD in women after menopause. Moreover, the
same diagnostic thresholds for cardiac troponin in both sexes could not be used because
women with myocardial infarction have a minor cardiac troponin increase. The sex-specific
diagnostic limits could improve the diagnosis of myocardial infarction in women. In women
with suspected ischemic heart disease, fewer tests are performed, and often the condition
remains undiagnosed. That is why women have a worse prognosis in ischemic heart
disease and die more often than men [47]. All of the above might be the reason why fewer
postmenopausal women are included in the study.
This study had several limitations. Sex-related differences in cardiovascular disease
disappear when women reach menopause. Since the average women’s age in this study
was 69 years, we did not find otherwise present sex-related differences in the CVD. An
additional limitation is the study sample size. A higher number of participants could yield a
better association between circadian clock gene polymorphisms and myocardial infarction.
Furthermore, probably, analyzed polymorphisms in the ARNTL, CRY2, and PER2 genes
are not functionally associated with myocardial infarction. In this case, functional SNPs
or a set of functionally relevant polymorphisms that could be strongly associated with
myocardial infarction should be named. This approach to the problem of myocardial
infarction provides an opportunity to continue research in multicenter studies with many
participants. This research’s advantage is that the participants involved were homogeneous
in demographic background characteristics such as ethnicity.
J. Cardiovasc. Dev. Dis. 2021, 8, 53 10 of 12

5. Conclusions
This study showed that genetic variation of the CLOCK gene was a biological sex
difference in myocardial infarction patients. Premenopausal women have a lower preva-
lence of cardiovascular disease than men of the same age due to estrogen’s protective effect.
However, after menopause, the prevalence of diseases like coronary heart disease and heart
failure increases dramatically and exceeds the prevalence in men of the same age. In this
study, postmenopausal women were older than men. This age difference might contribute
to a higher incidence of CVD in women with worse outcomes, which is a possible reason
why no more significant differences were observed between men and women in the present
study. The application of circadian biology in clinical practice represents a significant pos-
sibility of reducing patients’ morbidity or mortality after myocardial infarction. For both
sexes to benefit from CVD treatment, biological sex should be considered for chronotherapy.
Additional research and analysis of the sex-related circadian clock are possible.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/jcdd8050053/s1, Table S1: Frequencies and distribution of probable haplotypes in the women
and the men.
Author Contributions: I.Š., J.T., and M.J. were responsible for formal analysis, investigation, and
methodology. I.Š. was responsible for the visualization. R.S. was responsible for validation and
supervision the study. I.Š. wrote the manuscript. I.Š., J.T., M.J., and R.S. reviewed and edited the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by a grant from the Croatian Ministry of Science and Education
and dedicated to multi-year institutional financing of scientific activity at the Josip Juraj Strossmayer
University of Osijek, Osijek, Croatia, grant number IP8-FDMZ-2020.
Institutional Review Board Statement: The study was conducted according to the Declaration of
Helsinki’s guidelines and approved by the Ethics Committee of the University Hospital Osijek
(Ethical Approval Code: 25-1:3160-3/2012).
Informed Consent Statement: Informed consent was obtained from all patients involved in the study.
Data Availability Statement: The dataset generated during the study is available from the corre-
sponding author on reasonable request.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or
in the decision to publish the results.

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