Brief hypoxic cycles improve uterine contractile function after prolonged hypoxia in term-pregnant but not in nonpregnant rats in vitro

Accepted Manuscript Brief hypoxic cycles improve uterine contractile function after prolonged hypoxia in term-pregnant but not in nonpregnant rats in vitro Mohammed Alotaibi PII:

S0093-691X(18)30065-7

DOI:

10.1016/j.theriogenology.2018.02.011

Reference:

THE 14452

To appear in:

Theriogenology

Received Date: 17 November 2017 Revised Date:

29 January 2018

Accepted Date: 10 February 2018

Please cite this article as: Alotaibi M, Brief hypoxic cycles improve uterine contractile function after prolonged hypoxia in term-pregnant but not in nonpregnant rats in vitro, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.02.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Brief hypoxic cycles improve uterine contractile function after prolonged

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hypoxia in term-pregnant but not in nonpregnant rats in vitro

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During labour, the uterus itself is vulnerable to hypoxia/ischemia that can occur with

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each strong contraction and this may ultimately cause dysfunctional labour in some

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women. Periods of Intermittent re-oxygenations are beneficial to tissues subjected to hypoxia to wash out metabolic by-products that have been accumulated during

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hypoxic stresses which may affect the tissue viability. We proposed that short

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intermittent hypoxic episodes may protect the uterus from subsequent sustained long

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hypoxia. To investigate this, two sets of experiments were performed on term-

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pregnant and nonpregnant rat uterine tissues. In one set of experiment the uterus

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was subjected to sustained long hypoxia for 40 minutes and then allowed to recover

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in 100% O2. In the other set of experiment the uterus was subjected to 3 cycles of 2

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minutes hypoxia each separated by 20 minutes reoxygenation and followed by a

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sustained long hypoxia for 40 minutes and then allowed to recover. We found that

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challenging the uterine tissues with intermittent short hypoxic episodes improved the

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uterine contractility significantly after the sustained long hypoxia in term-pregnant but

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not in non-pregnant tissues. These results suggest that a mechanism of uterine

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tolerance (preconditioning) is confined to uterine tissues very close to labour and it is

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a protective phenomenon to improve the uterine activity despite the long-lasting

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paradoxical metabolic challenges that occur during the repeated strong labour

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contractions.

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Keywords: hypoxia, contraction, pregnant, uterus

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1. Introduction Strong uterine contractions that occur during labour are able to occlude the small

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uterine arteries traversing the myometria resulting in intermittent brief periods of

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hypoxia/ischemia to the contracting myometria [1, 2]. However, the decrease in

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uterine blood flow; and hence the local hypoxia/ischemia is proportional to the

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magnitude and the duration of uterine contraction [3] and are associated with

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oxytocin (OT) induction [4]. In addition, it has been showed previously in vivo that

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uterine contraction is associated with a decrease in uterine blood flow and pH, and

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the degree of hypoxia increases as the intensity of contraction becomes stronger [5].

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It was reported in rats and humans that pregnant uterus is more resistant to the

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effect of hypoxia compared to the nonpregnant uterus in vitro [6, 7]. It has been

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demonstrated in many tissues that sustained long hypoxia/ischemia could result in

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cellular dysfunction or irreversible cell damage [8-13]. Interestingly, preconditioning

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the tissues with repeated brief cycles of hypoxia/ischemia could decrease the cellular

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damage or dysfunction following the subsequent global hypoxia/ischemia in different

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tissues due to different cellular mechanisms [13-19]. This phenomenon of

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hypoxic/ischemic preconditioning was first described in the myocardium by Murry et

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al. in 1986 where brief cycles of coronary arteries occlusion separated by periods of

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reperfusion decreased the myocardial infarct size after sustained long ischemia [11].

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Studies have also shown that repeated episodes of brief hypoxia/ischemia improve

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the contraction and function of cardiac [10, 20, 21] and skeletal [22] muscles

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following global hypoxia/ischemia. Recently, we have demonstrated that repeated

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episodes of brief hypoxia increased the force of uterine contraction significantly and

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progressively in term-pregnant but not in non-pregnant uterine tissues and we

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termed this hypoxia-induced force increase (HIFI) [23]. Although we showed that

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HIFI could be part of labour to maintain the strong uterine contraction in the face of

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deleterious intermittent decrease in tissue oxygenation and pH, there is no empirical

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evidence if HIFI could also improve the uterine activity after long hypoxia that might

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occur in some women. Therefore, given that repeated uterine contractions and

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hypoxia occur as normal process of labour, we designed experiments to answer two

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basic questions. (1) What is the effect of sustained long hypoxia on term-pregnant

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and non-pregnant uterine contractility? (2) Does challenging the myometrium with

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repeated brief episodes of hypoxia improve the uterine contractility after subsequent

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long hypoxia and is this gestationally different?

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2. Methods 2.1 Animals and uterine tissues

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Experiments were performed on intact uterine tissues from adult female virgin non-

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pregnant (175-200g) and term-pregnant (22 day gestation) Wistar rats.The rats were

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housed in an environment with a temperature between 20–25˚C and had free access

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to food pellets and water ad libitum. At the day of experiments, animals were

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humanely killed by cervical dislocation under CO2 anaesthesia in accordance with

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the UK guidelines and legislations. The uteri were removed and immediately placed

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into physiological saline solution (Krebs) containing [in mM]: (154 NaCl, 5.6 KCl, 1.2

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MgSO4, 7.8 glucose, 10.9 HEPES and 2.0 CaCl2, pH7.4). Small longitudinal uterine

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strips (2mm × 10mm, width × length) without endometrium were dissected and

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mounted vertically in a 5-mL organ bath chamber containing HEPES-buffered Krebs

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solution pre-bubbled with 100% O2 at 37⁰C [24]. All animal care, treatments,

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sampling, and killing procedures were in accordance with the guidelines of the

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Institutional Animal Care Committee (IACC) of King Saud University.

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2.2 Tension measurement and experimental protocol

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All uterine strips were stretched to 1g standard resting tension and allowed to

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equilibrate for at least 30 minutes to obtain steady uterine contractions before

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performing any manoeuvre. As most spontaneous contractile activity became

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irregular after long time, all uterine strips were stimulated with 5nM OT [25] to obtain

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regular contractions for long period. Once steady contractions were observed under

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OT stimulation, the effect of sustained long hypoxia was investigated by exchanging

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the 100% oxygen in the bathing solution with 100% N2 for 40 minutes. In other

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experiments, uterine strips were challenged with brief and repeated 3 cycles of

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hypoxic episodes for 2 minutes separated by 20 minutes periods of re-oxygenation

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(to allow full recovery of uterine activity and to observe any changes in contractile

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parameters) [23]. After the 3rd recovery period, the uterine strips were subjected to

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prolonged hypoxia for 40 minutes follwed by a recovery period in 100% O2.

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2.3 Solutions

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The measurement of tension was made while the tissue was continually superfused

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with Krebs solution with OT. All chemicals and drugs were obtained from Sigma

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Aldrich, UK and were of analytical grades. OT was added directly to the Krebs

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solution and used at a final concentration of 5 nM.

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2.4 Data analysis and statistics

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Data were analyzed using Pro Origin Software (OriginLab, USA, V.9.1). The main

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parameters calculated were the force amplitude and the area under the curve (AUC).

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Data were analysed as follows: Contractile activity during the last 10 minutes in

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100% O2 preceding the application of long hypoxia was calculated and taken as

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100% control. The last 10 minutes of recovery period following the sustained long

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hypoxia was then analyzed and expressed as a percentage of this control. In other

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experiments where repeated brief hypoxic episodes were applied, data were

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analysed by calculating the last 10 minutes of recovery period after the 3rd hypoxic

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episode (100% control) and compared with the last 10 minutes during the recovery

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period following the sustained long hypoxia. Data are presented as Mean± standard

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error of the mean (SEM) using appropriate t-test or one way ANOVA. P values <

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0.05 were considered as statistically significant and “n” represents the number of

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uterine samples, one from each animal.

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3. Results

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3.1 The effect of sustained long hypoxia on term-pregnant uterine contractility

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Isolated uterine strips are able to produce regular contractions in vitro when

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superfused with buffered physiological saline solution and bubbled with 100% O2 at

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37 ⁰C. However, these contractions could last for long period when stimulated with

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oxytocin (Fig.1). Because the study protocol was lengthy, uterine strips were

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stimulated with oxytocin to keep the tissues contracting regularly. Recently, we

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demonstrated that application of single short hypoxia could significantly decrease or

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abolish the uterine contractility [23]. However, to investigate the effect of long

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hypoxia, term-pregnant uterine strips were challenged with 40mins long hypoxia by

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exchanging O2 for N2. A total number of 13 uterine strips were tested against the

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ACCEPTED MANUSCRIPT effect of long hypoxia. In 6 strips, long hypoxia resulted in a significant decrease in

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force amplitude (33± 3 %, p< 0.001) and AUC (38± 3 %, p< 0.001) during recovery

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period compared to100% control (Fig.2A). However, in 7 strips (Fig.2B), long

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hypoxia decreased the force amplitude slightly (96± 2 %, p> 0.05) and AUC

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significantly (92± 3 %, p< 0.05) during recovery period compared to100% control.

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3.2 The effect of repeated short hypoxic episodes on sustained long hypoxia

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in term-pregnant uterus

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In a paired experiment, a second uterine strip had 3 cycles of 2 mins hypoxia

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separated by 20mins re-oxygenation periods followed by sustained 40mins hypoxia

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(Fig.3A). Repeated brief hypoxic episodes significantly increased the force amplitude

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of uterine contractions during recovery periods. Surprisingly, after sustained long

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hypoxia (40mins), tissues were able to recover with significant increase in force

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amplitude (134± 7 %, p< 0.01, n=7, Fig.3Ai) and slight increase in AUC (108± 7 %,

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p< 0.05, n=7) during recovery period compared to100% control (Fig.3Aii).

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3.3 The effect of sustained long hypoxia on non-pregnant uterine contractility

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Challenging the uterine strips with long or short hypoxia always decreased the

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contractile activity. After sustained long hypoxia, uterine activity could recover in all

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non-pregnant uterine strips tested (n=4). However, these uterine activities were

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irregular and there was a significant decrease in force amplitude (91± 2 %, p< 0.05)

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and AUC (78± 3 %, p< 0.01) during recovery period compared to100% control

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(Fig.4A).

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3.4 The effect of repeated short hypoxic episodes on sustained long hypoxia

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in non-pregnant uterus

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To investigate if preconditioning the non-pregnant uterus with repeated brief hypoxic

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episodes could increase the force during recovery periods as seen in pregnant

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tissues, we challenged the non-pregnant uterine strips with 3 cycles of 2mins

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hypoxia separated by 20mins re-oxygenation periods and subsequently subjected

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the tissue to sustained long hypoxia (40mins). The force amplitude was

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progressively decreased during the 20mins re-oxygenation periods compared to

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control (prior initial hypoxia). However, after long hypoxia, tissues were able to

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recover with significant decrease in force amplitude (86± 2 %, p< 0.01, n=4) and

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AUC (65± 6 %, p< 0.01, n=4) during recovery period compared to100% control

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(Fig.4B).

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4. Discussion

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The presented data show that repeated short hypoxic episodes each separated by

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periods of recovery have a protective effect on uterine tissue subjected to

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subsequent long hypoxia. Consistent with our recent findings [23], this mechanism

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was only present in term-pregnant uterus very close to labour. After long hypoxia,

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some uterine strips did not recover well and the activity was significantly decreased

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compared to prior hypoxia. In addition, although some other strips were able to

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recover, the contractile activity did not significantly increase or improve. These data

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suggest that sustained long hypoxia may cause deleterious effects on contractile

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proteins. Andres et al. (1991) have found a significant decrease in myocardial

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contractile function after 20 minutes sustained ischemia suggesting that a

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modification of contractile apparatus may take place [26]. Moreover, Caron et al.

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(2009) found a significant increase in actomyosin breakdown and reduction in total

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protein content in L6 myotubes after severe hypoxia [27] and these proteins are

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known to be responsible for muscle contractility.

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During hypoxia the mitochondrial oxidative phosphorylation mechanism shuts rapidly

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resulting in stimulation of anaerobic metabolism and generation of lactic acids. It has

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been shown that acidosis can decrease the uterine contractility in women [28] and

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rats [29]. Surprisingly, previous studies have found that ischemic preconditioning of

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rat hearts with brief ischemic cycles can significantly decrease tissue acidosis and

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anaerobic glycolysis during the subsequent prolonged ischemic period [30].

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Sustained hypoxia can overload the concentration of intracellular calcium [Ca2+]i and

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subsequently may affect the calcium extrusion mechanisms. It has been

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demonstrated that hypoxia-increased [Ca2+]i can lead to irreversible damage to

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contractile proteins any may result in hypercontracture [8, 9]. We also found

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previously in rat myometrium that during hypoxia, the basal Ca2+ level was markedly

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elevated and returns to normal level during normoxia [23]. Therefore, the

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ACCEPTED MANUSCRIPT physiological level of [Ca2+]i must be maintained and the prolonged elevation of

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[Ca2+]i that usually occurs during long hypoxia must be avoided.

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Another mechanism by which hypoxia can cause cellular damage is the opening of

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mitochondrial permeability transition pore (mPTP). Previous works have found in rat

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myocardium that long ischemia/hypoxia can cause lethal reperfusion injury via

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opening of mPTP at the onset of reperfusion [31]. Blocking the mPTP with its specific

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inhibitors before ischemia could significantly reduce the cardiac damage associated

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with prolonged ischemia [32] .We cannot exclude the occurrence of any of these

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cellular changes in uterine tissues during long hypoxia as some uterine activity could

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not recover completely after the effect of long hypoxia.

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Interestingly, when the term-pregnant uterine strips were subjected to brief repeated

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cycles of hypoxia, the strips were able to recover after the subsequent long hypoxia

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with improved and increased contractions. This is consistent with the previous

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findings of improved cellular functions during preconditioning by brief cycles of

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hypoxia/ischemia in other tissues [20, 33-35].

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Hypoxic preconditiong describes a phenomenon in which brief preceding episodes of

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hypoxia improve the tolerance of the organ/tissue against the deleterious effect of

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subsequent prolong lethal hypoxia [11, 14-19]. Subsequent studies have confirmed

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the efficacy of this phenomenon in reducing the infarct size and improving the

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function of some smooth muscle cells [36-38]. Preconditioning can trigger a number

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of gene transcriptions and endogenous adaptive responses to hypoxic insults. The

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improvement of muscle contractility in our experiments may be related to the

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adaptation of uterine smooth muscles to the long hypoxia triggered by the preceding

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brief hypoxic insults. However, the exact mechanisms by which contractility is

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improved after the prolonged hypoxia are not fully understood. Studies have

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proposed that preconditioning with brief hypoxic/ischemic episodes protects the

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skeletal [17] and cardiac [12] muscles during subsequent long ischemia by

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promoting ATP-sparing. They speculated that ATP-sparing might have occurred

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during preconditioning manoeuvres which results in a reduction in ion pumping,

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lowering reliance on anaerobic metabolism (and hence reducing lactate production),

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and a tightening of muscle excitation-contraction coupling, all of which reduce the

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energy demand during sustained hypoxia/ischemia. If ATP-sparing has occurred in

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our experiments, then one might expect to see an improvement in uterine

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ACCEPTED MANUSCRIPT contractility after long hypoxia. It has also been shown in rat heart that preconditiong

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with short ischemic periods followed by recovery periods inhibits the opening of

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mPTP and confer cardiac protection against lethal long ischemia [39]. However, the

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mechanisms by which short repetitive cycles of ischemia can limit mitochondrial

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injury caused by long ischemia are being under extensive research.

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4.1 Conclusions

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In this study, we showed that repeated episodes of brief hypoxia can protect and

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improve the uterine contractility after sustained long hypoxia in term-pregnant uterus.

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This can be clinically relevant to labour to protect the uterus from the deleterious

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effect of the repeated hypoxic episodes that usually occur with repeated uterine

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contractions. However, some uteri are very sensitive to the uterotonins which could

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abnormally increase the uterine activity and hence the duration of local hypoxia. If

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this happens in vivo then the uterus would be able to withstand and maintain the

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strong uterine contractility despite the intermittent sustained hypoxic episodes until

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birth process is completed. Further studies are required to investigate the molecular

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mechanisms of improved contractions by repeated short hypoxic insults in pregnant

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uterus.

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Declaration of Conflicting Interests

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The author(s) declared no potential conflicts of interest with respect to the research,

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authorship, and/or publication of this article.

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References

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Fig.1. The effect of oxytocin on rat uterine contractions. A representative trace showing the response of uterine strip to 5nM oxytocin. Note that oxytocin increases the baseline, force amplitude, and frequency of contraction.

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Fig.3. The effect of repeated brief hypoxic episodes on uterine contractions after sustained long hypoxia in term-pregnant rats. A representative trace showing the effect of sustained 40mins hypoxia preceded by 3 cycles of 2mins hypoxia. Note the significant rebound increase in force after the effect of 40mins hypoxia. Means and standard error of the means (SEM) were blotted on the bar charts. Purple bars are amplitude and green bars are AUC. Control (uterine contractions after 3rd hypoxic episode), Recovery (uterine contractions after long hypoxia). ** p < 0.01

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Fig.2. The effect of sustained long hypoxia on uterine contraction in term-pregnant rats. A representative trace showing the effect of sustained 40mins hypoxia on recovery of uterine contraction with related bars chart means and standard error of means (SEM). Purple bars are amplitude and green bars are AUC. Control (uterine contractions before long hypoxia), Recovery (uterine contractions after long hypoxia). *** p < 0.001, * p < 0.05

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Fig.4. The effect of sustained long hypoxia and repeated brief hypoxic episodes on uterine contractions in non-pregnant rats. (A) A representative trace showing the effect of sustained 40mins hypoxia on the recovery of uterine contraction. (B) A representative trace showing the effect of 40mins sustained hypoxia preceded by 3 cycles of 2mins hypoxia. Note the significant decrease of force after the effect of 11

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40mins hypoxia. Purple bars are amplitude and green bars are AUC with means and standard error of the means (SEM). * p < 0.05, ** p < 0.01

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Long sustained hypoxia decreases or abolishes the uterine contractions.

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46% of uterine strips are severely affected by long hypoxia and are unable to recover when O2 is returned. Brief hypoxic episodes improve the uterine contractility after long hypoxia in term-pregnant

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but not in non-pregnant rats.

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