Stress
The International Journal on the Biology of Stress
ISSN: 1025-3890 (Print) 1607-8888 (Online) Journal homepage: http://www.tandfonline.com/loi/ists20
Sex-dependent effects of sleep deprivation on
myocardial sensitivity to ischemic injury
Phillip R. Zoladz, Anna Krivenko, Eric D. Eisenmann, Albert D. Bui, Sarah L.
Seeley, Megan E. Fry, Brandon L. Johnson & Boyd R. Rorabaugh
To cite this article: Phillip R. Zoladz, Anna Krivenko, Eric D. Eisenmann, Albert D. Bui, Sarah L.
Seeley, Megan E. Fry, Brandon L. Johnson & Boyd R. Rorabaugh (2016) Sex-dependent effects
of sleep deprivation on myocardial sensitivity to ischemic injury, Stress, 19:2, 264-268, DOI:
10.3109/10253890.2016.1152469
To link to this article: http://dx.doi.org/10.3109/10253890.2016.1152469
Published online: 08 Mar 2016.
Submit your article to this journal
Article views: 65
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ists20
Download by: [Ohio Northern University]
Date: 19 April 2016, At: 05:32
http://informahealthcare.com/sts
ISSN: 1025-3890 (print), 1607-8888 (electronic)
Stress, 2016; 19(2): 264–268
! 2016 Taylor & Francis. DOI: 10.3109/10253890.2016.1152469
SHORT COMMUNICATION
Sex-dependent effects of sleep deprivation on myocardial sensitivity to
ischemic injury
Phillip R. Zoladz1, Anna Krivenko1, Eric D. Eisenmann1, Albert D. Bui2, Sarah L. Seeley2, Megan E. Fry1,
Brandon L. Johnson1, and Boyd R. Rorabaugh2
Department of Psychology, Sociology & Criminal Justice and 2Department of Pharmaceutical & Biomedical Sciences, Ohio Northern University,
Ada, OH, USA
Downloaded by [Ohio Northern University] at 05:32 19 April 2016
1
Abstract
Keywords
Sleep deprivation is associated with increased risk of myocardial infarction. However, it is
unknown whether the effects of sleep deprivation are limited to increasing the likelihood of
experiencing a myocardial infarction or if sleep deprivation also increases the extent of
myocardial injury. In this study, rats were deprived of paradoxical sleep for 96 h using the
platform-over-water method. Control rats were subjected to the same condition except the
control platform was large enough for the rats to sleep. Hearts from sleep deprived and control
rats were subjected to 20 min ischemia on a Langendorff isolated heart system. Infarct size and
post ischemic recovery of contractile function were unaffected by sleep deprivation in male
hearts. In contrast, hearts from sleep-deprived females exhibited significantly larger infarcts
than hearts from control females. Post ischemic recovery of rate pressure product and + dP/dT
were significantly attenuated by sleep deprivation in female hearts, and post ischemic recovery
of end diastolic pressure was significantly elevated in hearts from sleep deprived females
compared to control females, indicating that post ischemic recovery of both systolic and
diastolic function were worsened by sleep deprivation. These data provide evidence that sleep
deprivation increases the extent of ischemia-induced injury in a sex-dependent manner.
Sleep deprivation, heart, infarct, cardiac
ischemia, ischemic injury, heart attack
Introduction
Methods
Average sleep duration in the United States has significantly
decreased over the course of the past several decades
(Adenekan et al., 2013). Sleep deprivation has a significant
effect on cardiovascular function. Inadequate sleep is
associated with impairment of the baroreceptor reflex
(Almeida et al., 2014), increased risk of hypertension
(Palagini et al., 2013), endothelial dysfunction (Calvin
et al., 2014), and increased formation of pro-atherosclerotic
foam cells in the vasculature (Carreras et al., 2014).
Inadequate sleep also increases the risk of having a myocardial infarction (Hsu et al., 2015; Laugsand et al., 2011).
However, it is unknown whether sleep deprivation alters the
extent of myocardial injury that occurs during an ischemic
insult. The goal of this study was to determine whether sleep
deprivation alters myocardial sensitivity to ischemic injury
and whether sleep deprivation produces similar effects in
male and female ischemic hearts.
Subjects
Correspondence: Boyd R. Rorabaugh, Ohio Northern University, 525 S.
Main St, Ada, Ohio, 45810, USA. Tel: +1 4197721695. Fax: +1
4197721695. E-mail: b-rorabaugh@onu.edu
History
Received 11 November 2015
Revised 5 February 2016
Accepted 6 February 2016
Published online 7 March 2016
This study utilized 19 male and 18 female Sprague-Dawley
rats obtained from a breeding colony established at Ohio
Northern University. All rats were 10–12 weeks of age.
Starting body weights of males in the control (331 ± 18 g) and
sleep deprived (344 ± 16 g) groups were similar prior to
initiation of sleep deprivation. Likewise, control (289 ± 9 g)
and sleep deprived (270 ± 9 g) females also had similar body
weights. All experiments were approved by the Institutional
Animal Care and Use Committee of Ohio Northern
University.
Sleep deprivation
Rats were subjected to paradoxical sleep deprivation using the
platform-over-water method (Mendelson et al., 1974). Rats in
the sleep deprived group were placed on a circular platform
(6 cm diameter) located 1 cm above the surface of the water
(21–22 C) in a polycarbonate cage (43 22 29 cm).
Muscle atonia caused the rats to fall off the platform and
awaken whenever paradoxical sleep occurred. This method is
a well-established model of paradoxical sleep deprivation
Sleep deprivation worsens cardiac injury
DOI: 10.3109/10253890.2016.1152469
(Mendelson et al., 1974; Maloney et al., 2000; Mathangi
et al., 2012; Sharma et al., 2015). Brain electrophysiology and
markers of stress were not monitored in this study. However,
prior electrophysiological studies demonstrate that this
method selectively suppresses paradoxical sleep without
significantly altering slow wave sleep (Mendelson et al.,
1974; Maloney et al., 2000). Control rats were placed in an
identical cage on a platform located 1 cm above the surface of
the water. However, the platform used for control animals was
large enough (13 cm diameter) for the rats to lie down and
sleep. All animals had free access to food and water
throughout the experiment.
Downloaded by [Ohio Northern University] at 05:32 19 April 2016
Langendorff isolated heart experiments
Following 96 h in the sleep deprivation or control chambers,
rats were anesthetized with pentobarbital (50 mg/kg, i.p.).
Hearts were rapidly removed and cannulated while bathed in
ice-cold Krebs-Henseleit solution (118 mM NaCl, 4.7 mM
KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4,
0.5 mM Na2EDTA, 11 mM glucose, 2.5 mM CaCl2 and pH
7.4) as previously described (Rorabaugh et al., 2016). Krebs
solution was perfused through the aortic cannula at a constant
pressure of 80 mmHg. Contractile function of the left
ventricle was measured using an intraventricular balloon
connected to a pressure transducer. Data were continually
recorded using a Powerlab 4SP data acquisition system (AD
Instruments, Colorado Springs, CO). Hearts were equilibrated
for 25 min prior to the onset of 20 min ischemia and 2 h of
reperfusion. Pre-ischemic contractile function was measured
immediately prior to ischemia. Post-ischemic recovery of
contractile function was measured following 1 h of reperfusion. Infarct size was measured as previously described
(Rorabaugh et al., 2016).
Western blots
Hearts were isolated and the atria were quickly removed.
Ventricular tissue was immediately flash frozen in liquid
nitrogen and stored at 80 C. ERK phosphorylation and
protein kinase C-e expression were measured in ventricular
homogenates as previously described (Rorabaugh et al.,
2016).
Statistical analysis
We and others have previously shown that a 20 min ischemic
insult differentially effects infarct size in male and female
hearts (Rorabaugh et al., 2016). Thus, data from male and
265
female rats were analyzed separately using independent
samples t tests. Alpha was set at 0.05 for all analyzes.
Results
Females
Sleep deprivation had no significant effect on pre-ischemic
contractile function in female hearts (Table 1). Exposure to
20 min ischemia produced significantly larger infarcts in
hearts from sleep deprived female rats compared to hearts
from control animals, t(15) ¼ 2.60, p ¼ 0.019 (Figure 1A).
Sleep deprivation also significantly attenuated post ischemic
recovery of rate pressure product, t(15) ¼ 2.20, p ¼ 0.044
(Figure 1B), and + dP/dT, t(15) ¼ 2.40, p ¼ 0.028 (Figure 1C).
End diastolic pressure (Figure 1D) was significantly elevated
in sleep deprived females compared to control animals,
t(15) ¼ 2.80, p ¼ 0.015, indicating that the heart was unable to
adequately relax during diastole. Recovery of dP/dT was
also decreased in hearts from sleep deprived females
(Figure 1E), but this did not reach statistical significance,
t(15) ¼ 2.00, p ¼ 0.061. These data indicate that sleep
deprivation increases infarct size and worsens recovery of
both systolic and diastolic parameters of contractile function
in female rats.
Males
In contrast to female hearts, sleep deprivation had no effect on
infarct size in male hearts subjected to an ischemic insult,
t(17) ¼ 1.10, p ¼ 0.28 (Figure 1A). Post ischemic recovery of
rate
pressure
product,
t(15) ¼ 0.80,
p ¼ 0.47
(Figure 1B), +dP/dT, t(15) ¼ 0.60, p ¼ 0.56 (Figure 1C),
dP/dT, t(15) ¼ 0.90, p ¼ 0.37 (Figure 1E), and end diastolic
pressure, t(15) ¼ 0.60, p ¼ 0.99 (Figure 1D), were also
unaffected by sleep deprivation. These data provide evidence
that sleep deprivation worsens myocardial sensitivity to
ischemic injury in a sex-dependent manner.
Effect of sleep deprivation on ERK phosphorylation
and PKC-e expression
The roles of extracellular signal-regulated kinase (ERK) and
PKC-e in protecting the heart from ischemic injury are well
established (Baines et al., 2002). In light of the observation
that sleep deprivation worsens ischemic injury in female
hearts (Figure 1), we assessed the impact of sleep deprivation
on ERK phosphorylation and the expression of PKC-e. ERK
phosphorylation was significantly decreased in ventricles
from sleep deprived female rats compared to control rats,
t(8) ¼ 3.70, p ¼ 0.006 (Figure 2A). In contrast, PKC-e
Table 1. Parameters of pre-ischemic contractile function in female and male hearts.
Female control
Female sleep deprived
Male control
Male sleep deprived
Rate pressure product
(mmHg bpm)
+dP/dT
(mmHg/sec)
dP/dT
(mmHg/sec)
EDP
(mmHg)
Coronary flow
rate (ml/min)
35.938 ± 1942
32.983 ± 1962
34.783 ± 1851
32.421 ± 1460
4.921 ± 279
5.149 ± 288
4.377 ± 179
4.184 ± 264
3.158 ± 203
3.302 ± 190
2968 ± 150
2.756 ± 166
6 ± 0.4
6 ± 0.4
6 ± 0.7
5 ± 0.5
12 ± 0.9
11 ± 0.8
15 ± 0.9
16 ± 0.9
Male and female data were analyzed separately by independent t test. Sleep deprivation had no significant effect (p50.05) on pre-ischemic parameters
of contractile function in either male or female hearts.
266
P. R. Zoladz et al.
Stress, 2016; 19(2): 264–268
Downloaded by [Ohio Northern University] at 05:32 19 April 2016
Figure 1. Effect of sleep deprivation on
infarct size and post ischemic recovery of
contractile function in male and female
hearts. Hearts from sleep-deprived female
rats exhibited increased infarct size (panel A),
and decreased contractile recovery (panels
B–E) compared to hearts from control female
rats. Sleep deprivation had no effect on
infarct size (panel A) or post ischemic
recovery of contractile function (panels B–E)
in male hearts. Data represent the
mean ± S.E.M. of 8–9 hearts for each group.
expression was unaffected by sleep deprivation, t(8) ¼ 0.70,
p ¼ 0.47 (Figure 2B).
Effect on weight gain
Control and sleep deprived males gained similar amounts of
body weight over the course of the 96 h period (5 ± 2% and
3 ± 1% gain in body weight for control and sleep deprived
male rats, respectively). Control and sleep deprived females
also gained similar amounts of body weight (9 ± 2% and
7 ± 1% gain for control and sleep deprived rats, respectively).
Discussion
The primary finding of this study is that sleep deprivation
increases myocardial sensitivity to ischemic injury in a sexdependent manner. Paradoxical sleep deprivation had no
significant effect on male hearts subjected to an ischemic
insult. However, sleep deprivation significantly increased
infarct size and worsened post ischemic recovery of contractile function in female hearts. These data extend previous work
by demonstrating that inadequate sleep not only increases the
likelihood of experiencing a myocardial infarction
Sleep deprivation worsens cardiac injury
Downloaded by [Ohio Northern University] at 05:32 19 April 2016
DOI: 10.3109/10253890.2016.1152469
267
Alternatively, the decreased sensitivity to ischemic injury in
female hearts might be due to the cardio protective effects of
estrogen or sex-dependent differences in other hormones
(Murphy & Steenbergen, 2007). Regardless of the underlying
cause, our data indicate that the mechanisms responsible for
decreased myocardial sensitivity to ischemic injury in female
hearts can be overcome by sleep deprivation.
Some studies indicate that sleep deprivation disproportionately increases the incidence of cardiovascular disease
related mortality in women. Meisinger et al. (2007) reported
that short sleep duration is associated with an increased
incidence of myocardial infarction in women but not in men.
Furthermore, data from the Whitehall II study indicates that
short sleep duration and disrupted sleep both independently
increase the risk of cardiovascular disease-related death in
women but not in men (Rod et al., 2014). Our data suggest
that this sex-dependent effect of sleep deprivation on
cardiovascular-related mortality might result from the ability
of sleep deprivation to increase the amount of infarcted tissue
in the female heart.
The cardio protective impact of ERK signaling in the
ischemic heart is well established (Baines et al., 2002). Our
finding that ERK phosphorylation is suppressed in hearts
from sleep deprived females is consistent with the observation
that these hearts exhibit worsened ischemic injury. However,
it is difficult to establish whether suppression of ERK
signaling is the cause of increased myocardial sensitivity to
ischemia. Further work is needed to understand the mechanism by which sleep deprivation worsens ischemic injury in the
female heart.
In conclusion, this is the first study to demonstrate that
sleep deprivation increases the extent of myocardial ischemic
injury in a sex-dependent manner. One limitation of this study
is that we measured contractile recovery in hearts that were
isolated from the autonomic nervous system. This is an
important limitation in light of previous work demonstrating
that sleep deprivation differentially influences autonomic
function in male and female rats (Matos et al., 2013).
Nevertheless, these data emphasize the importance of proper
sleep for minimizing myocardial ischemic injury.
Figure 2. Effect of sleep deprivation on ERK phosphorylation and PKCe expression in female hearts. ERK phosphorylation was significantly
decreased in sleep-deprived female rats compared to control rats (panel
A). Sleep deprivation had no effect on PKC-e expression (panel B). Data
represent the mean ± S.E.M. of 5 hearts for each group.
Acknowledgments
The authors thank Joseph Lawson and Lauren Stoner for their
assistance with sleep deprivation.
Declaration of interest
(Hsu et al., 2015; Laugsand et al., 2011), but also increases
the extent of myocardial injury.
Control female hearts exhibited significantly smaller
infarcts than hearts from control males. This is consistent
with recent work in our own laboratory (Rorabaugh et al.,
2016) as well as the work of others (Bae & Zhang, 2005). The
mechanism by which female hearts are more resistant than
male hearts to ischemic injury is unclear (Ostadal & Ostadal,
2014). The existence of endogenous signaling pathways that
protect the heart from ischemic injury is well established. It is
possible that there are sex-dependent differences in cardioprotective signaling pathways that render a greater degree of
protection to female hearts (Ledvenyiova et al., 2013).
The authors report no conflicts of interest.
References
Adenekan B, Pandey A, Mckenzie S, Zizi F, Casimir GJ, Jean-Louis G.
(2013). Sleep in America: role of racial/ethnic differences. Sleep Med
Rev 17:255–62.
Almeida FR, Perry JC, Futuro-Neto HA, Almeida VR, Sebastiao RM,
Andersen ML, Tuflik S, et al. (2014). Cardiovascular function
alterations induced by acute paradoxical sleep deprivation in rats. Clin
Exp Hypertens 36:567–71.
Bae S, Zhang L. (2005). Gender differences in cardioprotection against
ischemia/reperfusion injury in adult rat hearts: focus on Akt and
protein kinase C signaling. J Pharmacol Exp Ther 315:1125–35.
Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli
R, et al. (2002). Mitochondrial PKC-e and MAPK form signalng
Downloaded by [Ohio Northern University] at 05:32 19 April 2016
268
P. R. Zoladz et al.
modules in the murine heart: enhanced mitochondrial PKC-e-MAPK
interactions and differential MAPK activation in PKC-e-induced
cardioprotection. Circ Res 90:390–7.
Calvin AD, Covassin N, Kremers WK, Adachi T, Macedo P,
Albuquerque FN, Bukartyk J, et al. (2014). Experimental sleep
restriction causes endothelial dysfunction in healthy humans. J Am
Heart Assoc 3:e001143.
Carreras A, Zhang SX, Peris E, Qiao Z, Gileles-Hillel A, Li RC, Wang Y,
Gozal D. (2014). Chronic sleep fragmentation induces endothelial
dysfunction and structural vascular changes in mice. Sleep 37:1817–24.
Hsu CY, Chen YT, Chen MH, Huang CC, Chiang CH, Huang PH, Chen
JW, et al. (2015). The association between insomnia and increased
future cardiovascular events: a nationwide population-based study.
Psychosom Med 77:743–51.
Laugsand LE, Vatten LJ, Platou C, Janszky I. (2011). Insomnia and the
risk of acute myocardial infarction: a population study. Circulation
124:2073–81.
Ledvenyiova V, Pancza D, Matejikova J, Ferko M, Bernatova I,
Ravingerova T. (2013). Impact of age and sex on response to ischemic
preconditioning in the rat heart: differential role of the PI3K-AKT
pathway. Can J Physiol Pharmacol 91:640–7.
Matos G, Tenorio NM, Bergamaschi CT, Campos RR, Cintra F, Tufik S,
Andersen ML. (2013). More than hormones: sex differences in
cardiovascular parameters after sleep loss in rats. Prog
Neuropsychopharmacol Biol Psychiatry 44:34–8.
Maloney KJ, Mainville L, Jones BE. (2000). C-Fos expression in
GABAergic serotonergic, and other neurons of the pontomedullary
reticular formation and raphe after paradoxcial sleep deprivation and
recovery. J Neurosci 20:4669–79.
Mathangi DC, Shyamala R, Subhashini AS. (2012). Effect of REM sleep
deprivation on the antioxidant status in the brain of wistar rats. Ann
Neurosci 19:161–4.
Stress, 2016; 19(2): 264–268
Mendelson WB, Guthrie RD, Frederick G, Wyatt RJ. (1974). The flower
pot technique of rapid eye movement (REM) sleep deprivation.
Pharmacol Biochem Behav 2:553–6.
Meisinger C, Heier M, Lowel H, Schneider A, Doring A. (2007).
Sleep duration and sleep complaints and risk of myocardial
infarction in middle-aged men and women from the general
population: the MONICA/KORA Augsburg cohort study. Sleep
30:1121–7.
Murphy E, Steenbergen C. (2007). Gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury.
Cardiovasc Res 75:478–86.
Ostadal B, Ostadal P. (2014). Sex-based differences in cardiac ischaemic
injury and protection: therapeutic implications. Br J Pharmacol 171:
541–54.
Palagini L, Bruno RM, Gemignani A, Baglioni C, Ghiadoni L, Riemann
D. (2013). Sleep loss and hypertension: a systematic review. Curr
Pharm Des 19:2409–19.
Rod NH, Kumari M, Lange T, Kivimaki M, Shipley M, Ferrie J. (2014).
The joint effect of sleep duration and disturbed sleep on cause-specific
mortality: results from the Whitehall II cohort study. PloS One 9:
e91965.
Rorabaugh BR, Seeley SL, Bui A, Sprague L, D’Souza MS. (2016).
Prenatal methamphetamine differentially alters myocardial sensitivity
to ischemic injury in male and female adult rat hearts. Am J Physiol
Heart Circ Physiol 310:H516–23.
Sharma A, Muresanu DF, Lafuente JV, Patnaik R, Tian ZR, Buzoianu
AD, Sharma HS. (2015). Sleep deprivation-induced blood brain
barrier breakdown and brain dysfunction are exacerbated by sizerelated exposure to Ag and Cu nanoparticles. Neuroprotective effects
of a 5-HT3 receptor antagonist ondansetron. Mol Neurobiol 52:
867–81.