Evidence of primary cilia in the developing rat heart

Sue R. McGlashan,Sarbjot Kaur,Marie-Louise Ward

doi:10.1186/s13630-018-0058-z

Abstract

BackgroundA transient increase in cytosolic Ca2+ (the “Ca2+ transient”) determines the degree and duration of myocyte force development in the heart. However, we have previously observed that, under the same experimental conditions, the Ca2+ transients from isolated cardiac myocytes are reduced in amplitude in comparison to those from multicellular cardiac preparations. We therefore questioned whether the enzymatic cell isolation procedure might remove structures that modulate intracellular Ca2+ in some way. Primary cilia are found in a diverse range of cell types, and have an abundance of Ca2+-permeable membrane channels that result in Ca2+ influx when activated. Although primary cilia are reportedly ubiquitous, their presence and function in the heart remain controversial. If present, we hypothesized they might provide an additional Ca2+ entry pathway in multicellular cardiac tissue that was lost during cell isolation. The aim of our study was to look for evidence of primary cilia in isolated myocytes and ventricular tissue from rat hearts.MethodsImmunohistochemical techniques were used to identify primary cilia-specific proteins in isolated myocytes from adult rat hearts, and in tissue sections from embryonic, neonatal, young, and adult rat hearts. Either mouse anti-acetylated α-tubulin or rabbit polyclonal ARL13B antibodies were used, counterstained with Hoechst dye. Selected sections were also labelled with markers for other cell types found in the heart and for myocyte F-actin.ResultsNo evidence of primary cilia was found in either tissue sections or isolated myocytes from adult rat ventricles. However, primary cilia were present in tissue sections from embryonic, neonatal (P2) and young (P21 and P28) rat hearts.ConclusionThe lack of primary cilia in adult rat hearts rules out their contribution to myocyte Ca2+ homoeostasis by providing a Ca2+ entry pathway. However, evidence of primary cilia in tissue from embryonic and very young rat hearts suggests they have a role during development.

Highlights

IntroductionA transient increase in cytosolic Ca2+ (the “Ca2+ transient”) determines the degree and duration of myocyte force development in the heart
A transient increase in cytosolic Ca2+ determines the degree and duration of myocyte force development in the heart
Methods relating to the cardiac myocyte isolation procedure are provided in the Additional file 1, together with details of the solutions, reagents, and chemicals used

Summary

Introduction

A transient increase in cytosolic Ca2+ (the “Ca2+ transient”) determines the degree and duration of myocyte force development in the heart. We hypothesized that the primary cilia may be lost during the enzymatic myocyte isolation process, leading to a net loss of intracellular [Ca2+] and reduced Ca2+ transients. The primary cilium is a solitary, non-motile organelle that is an extension of the basal body and is ubiquitously expressed in mammalian cell types [4, 5]. As sensory organelles, they transduce external forces, perhaps via intracellular Ca2+ signals [6] (but see [7]), coordinating multiple signalling pathways [8,9,10,11,12,13]. Primary cilia in adult cardiac myocytes, and their possible contribution to intracellular [Ca2+]i modulation, have not been fully investigated

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