Single-cell mechanics and calcium signalling in organotypic slices of human myometrium.

Elucidation of cellular mechanisms regulating myometrial contractility is crucial for improvement in management of many obstetric abnormalities, such as premature delivery, uterine dystocia and post-partum haemorrhage. Myometrial contractions are triggered by periodic synchronous rises in intracellular calcium concentration ([Ca(2+)]i) elicited by spontaneously generated action potentials propagating throughout the entire myometrium. During labour, hormones like oxytocin and prostaglandins potentiate uterine contractions by increasing their duration, strength and frequency. The most informative approach to studying the mechanisms underlying hormonal modulation of uterine contractility is to record [Ca(2+)]i responses to hormones in intact myometrial samples that have not been subjected to enzymatic treatment for cell isolation or cell culture conditions. However, the spatio-temporal resolution of such recording is limited due to the motion artifacts occurring in contracting tissue. Here we describe the application of our newly developed motion correction algorithm to investigate the [Ca(2+)]i dynamics in control and oxytocin stimulated slices of human myometrium on a cellular level. We present evidence that oxytocin induces asynchronous [Ca(2+)]i oscillations in individual myocytes within intact myometrium which are similar to those observed in cultured cells. The oscillations occur between synchronous action potential-driven [Ca(2+)]i transients but appear to be unrelated to contractions. Furthermore, the oxytocin-triggered [Ca(2+)]i oscillations wane within 30-50min of hormone application, while the action potential induced [Ca(2+)]i transients remain augmented. We conclude that oxytocin-induced [Ca(2+)]i oscillations are not relevant to the acute regulation of myometrial contractility but may play a role in longer-term regulatory processes, for example, by triggering gene expression.

Fine spatiotemporal activity in contracting myometrium revealed by motion-corrected calcium imaging.

Successful childbirth depends on the occurrence of precisely coordinated uterine contractions during labour. Calcium indicator fluorescence imaging is one of the main techniques for investigating the mechanisms governing this physiological process and its pathologies. The effective spatiotemporal resolution of calcium signals is, however, limited by the motion of contracting tissue: structures of interest in the order of microns can move over a hundred times their width during a contraction. The simultaneous changes in local intensity and tissue configuration make motion tracking a non-trivial problem in image analysis and confound many of the standard techniques. This paper presents a method that tracks local motion throughout the tissue and allows for the almost complete removal of motion artefacts. This provides a stabilized calcium signal down to a pixel resolution, which, for the data examined, is in the order of a few microns. As a byproduct of image stabilization, a complete kinematic description of the contraction-relaxation cycle is also obtained. This contains novel information about the mechanical response of the tissue, such as the identification of a characteristic length scale, in the order of 40-50 µm, below which tissue motion is homogeneous. Applied to our data, we illustrate that the method allows for analyses of calcium dynamics in contracting myometrium in unprecedented spatiotemporal detail. Additionally, we use the kinematics of tissue motion to compare calcium signals at the subcellular level and local contractile motion. The computer code used is provided in a freely modifiable form and has potential applicability to in vivo calcium imaging of neural tissue, as well as other smooth muscle tissue.