Abstract
Background. Understanding the basis of myometrial and uterine artery smooth muscle contractility is crucial to understanding complications associated with pregnancy and childbirth. Mutations in ryanodine receptor 1 (RYR1) are associated with various phenotypes including malignant hyperthermia susceptibility (MHS), central core disease (CCD) and other congenital myopathies; highlighting the role of RYR1 in skeletal muscle function. Some patients with autosomal dominant RYR1 mutations have reported abnormal bleeding characterised by severe menorrhagia, post-partum bleeding and gum and postoperative bleeding. Excessive bleeding suggests a role of RYR1 in smooth muscle function, an observation experimentally tested by Lopez et al. (2016) who showed that Ryr1Y522S/+ mice had prolonged tail artery bleeding times compared to wild-type controls, an effect reversed by pre-treatment with dantrolene, a pharmacological antagonist of the RYR1 channel. Together these observations imply a contribution of RYR1 to vascular smooth muscle function and, the female reproductive system.Hypothesis. The presence of a gain-of-function Ryr1 mutation in pregnancy will lead to enhanced vasorelaxation of vascular smooth muscle cells and altered contractility of myometrium. This will impact on fetal and placental development and influence the length of gestation and parturition. The Ryr1 Y522S mutation will modify the cellular needs of the reproductive tissues resulting in altered global mRNA expression. The RYR1-variant carrying human cohort will demonstrate increased bleeding events, longer bleeding times, increased rate of menorrhagia and a shorter length of pregnancy.
Methods. Utilizing the Ryr1Y522S/+ mouse model, the impact of the gain-of-function mutation was investigated in late-stage pregnant mice. The impact of Ryr1 Y522S on vascular smooth muscle function in pregnant uterine arteries was investigated using wire myography with a pharmacological approach (calcium modulators and dantrolene). In addition, histological analyses of the pregnant uterine artery tissue explored changes in smooth muscle content and vessel morphology. The impact of the Ryr1 Y522S mutation on myometrial function was studied using video recordings to determine gestation length, isometric tension recordings of spontaneous myometrial contractions and RNAseq to investigate gene expression changes in the pregnant myometrial tissue. Fetal and placental weight measurements were made on gestation day 18.5. Placental morphology was studied using haematoxylin and eosin stains, and immunofluorescence staining. The abnormal bleeding phenomenon in RYR1 variant-carrying women was further investigated using an online questionnaire regarding generalised bleeding events, menorrhagia, and pregnancy/obstetric history.
Results. Uterine artery vasodilatory response was reduced in phenylephrine pre-constricted vessels from heterozygous Ryr1Y522S/+ (mixed litter) dams (a larger IC50) compared to vessels from wildtype (wildtype litter) dams, a response which was reversed by dantrolene. The contractile response of uterine arteries from heterozygous pregnant animals to phenylephrine was not statistically different to that of the wild-type pregnant uterine artery. Myometrial tissue from pregnant Ryr1Y522S/+ animals had a total of six differentially expressed genes (padj<0.3) compared to wild-type pregnant myometrium. Gestation length of Ryr1Y522S/+ mouse pregnancies was not statistically different to that of the wild-type mouse. The pregnant Ryr1Y522S/+ mouse had fewer fetuses in a litter, lower weight fetuses and heavier placentas, compared to wild-type littermates. The Ryr1Y522S/+ mouse placenta had a larger labyrinth, and smaller decidua, compared to the wild-type placenta. The RYR1 protein localised to the junctional zone of the placenta and Ryr1Y522S/+ placentas had more intense spongiotrophoblast staining of the junctional zone compared to wildtype.
Assessing bleeding symptoms in women, via a customized online questionnaire, revealed that women with RYR1-related disorders had higher bleeding scores and more often had pathological bleeding scores compared to control participants. In addition, RYR1-mutated participants more often reported having complications during a pregnancy, shorter pregnancies, planned Caesarean sections and gave birth to lower weight babies, in addition to gastrointestinal symptoms.
Conclusions. Through these studies we have shown that the maternal Ryr1 Y522S influences uterine artery vasodilation and myometrial contraction, suggesting a physiological role of RYR1 in smooth muscle function, which ultimately results in abnormal bleeding, altered fetal-placental growth and fetal survival. Findings in the animal model were corroborated by the findings from our online questionnaire study exploring a range of symptoms in RYR1-mutated females. This work highlights potentially modifiable phenotypes associated with pathogenic RYR1 variants.
| Date of Award | 1 Mar 2024 |
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| Original language | English |
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| Supervisor | Rachel Tribe (Supervisor) & Heinz Jungbluth (Supervisor) |