Biology subjects

Zawieja, S. D.

Publications and source records attributed to Zawieja, S. D..

4 recordsLinked to original sources

bioRxiv2023

Lymphatic muscle cells are the innate pacemaker cells regulating mouse lymphatic collecting vessel contractions

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. Here we combined immunofluorescence and scRNAseq analyses with electrophysiological methods to examine the cellular constituents of the mouse cLV wall and assess whether any cell type exhibited morphological and functional processes characteristic of pacemaker cells. We employed inducible Cre (iCre) mouse models to target specific cell populations including c-kitCreERT2 to target interstitial cells of Cajal like cells; Pdgfr{beta}CreERT2to target pericyte-like cells; PdgfrCreERTMto target CD34+ adventitial cells; and Myh11CreERT2to target lymphatic muscle cells (LMCs) directly. These inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light- activated cation channel rhodopsin2 (ChR2). Only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Further, optogenetic depolarization with ChR2 only induced propagated contractions in LMCs. Membrane potential recordings in LMCs demonstrated that the rate of diastolic depolarization significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking. ImpactLymphatic muscle cells, but not CD34+ adventitial cells, exhibited pacemaker behaviors such as pressure-dependent depolarization, pressure-dependent calcium mobilization during diastole, and propagated contraction waves induced by focal, optogenetic depolarization via enforced channel- rhodopsin2.

physiology↗

bioRxiv2022

A dual-clock-driven model emulating the effects of either Ano1 or IP3R knock-out on lymphatic muscle cell pace-making

Lymphoedema, a common dysfunction of the lymphatic system, results in fluid accumulating between cells. Fluid return through the lymphatic vascular system is primarily provided by contractions of muscle cells in the walls of lymphatic vessels, driven by electrochemical oscillations causing rhythmic action potentials and associated surges in intracellular calcium ion concentration. There is incomplete understanding of the mechanisms involved in these repeated events, restricting the development of pharmacological treatments for dysfunction. Previously, we proposed a model where autonomous oscillations in the membrane potential (M-clock) drove passive oscillations in the calcium concentration (C-clock). In this paper, to model more accurately what is known about the underlying physiology, we extend this model to the case where the M-clock and the C-clock oscillators are both active but coupled together, and thus both driving the action potentials. This extension results from modifications to the model for the IP3 receptor, a key C-clock mechanism. The synchronized dual-driving clock behaviour enables the model to match IP3 receptor knock-out data, resolving an issue with previous models. We also use phase-plane analysis to explain the mechanisms for the dual-clock coupling. The model has the potential to help determine mechanisms and find targets for pharmacological treatment of lymphoedema.

biophysics↗

bioRxiv2022

An Inducible Cre Mouse with Preferential Activity in Vascular Smooth Muscle Evades a Previously Lethal Intestinal Phenotype

All smooth muscle cell (SMC) restricted Cre mice recombine floxed alleles in vascular and visceral SMCs. We generated a new tamoxifen-inducible CreERT2 mouse, Itga8-CreERT2, and compared its activity to the widely used Myh11-CreERT2 mouse. Both CreERT2 mice showed similar activity in vascular SMCs; however, Itga8-CreERT2 displayed limited activity in visceral SMC-containing tissues (e.g., intestine). Myh11-CreERT2 (but not Itga8-CreERT2) mice displayed high levels of CreERT2 protein, tamoxifen-independent activity, and an altered transcriptome. Whereas Myh11-CreERT2-mediated knockout of Srf resulted in a lethal intestinal phenotype, loss of Srf with Itga8-CreERT2 (SrfItga8) revealed viable mice with attenuated vascular SMC contractile gene expression, but no evidence of intestinal pathology. Male and female SrfItga8 mice presented with vascular contractile incompetence; however, only male SrfItga8 mice showed systemic changes in blood pressure. These results establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre strains, including Myh11-CreERT2, where SMC gene loss results in visceral myopathies that obfuscate accurate phenotyping in vascular SMCs.

genetics↗

bioRxiv2021

Modelling the coupling of the M-clock and C-clock in lymphatic muscle cells

Lymphoedema develops due to chronic dysfunction of the lymphatic vascular system which results in fluid accumulation between cells. The condition is commonly acquired secondary to diseases such as cancer or the therapies associated with it. The primary driving force for fluid return through the lymphatic vasculature is provided by contractions of the muscularized lymphatic collecting vessels, driven by electrical oscillations. However, there is an incomplete understanding of the molecular and bioelectric mechanisms involved in lymphatic muscle cell excitation, hampering the development and use of pharmacological therapies. Modelling in silico has contributed greatly to understanding the contributions of specific ion channels to the cardiac action potential, but modelling of these processes in lymphatic muscle remains limited. Here, we propose a model of oscillations in the membrane voltage (M-clock) and intracellular calcium concentrations (C-clock) of lymphatic muscle cells. We modify a model by Imtiaz and colleagues to enable the M-clock to drive the C-clock oscillations. This approach differs from typical models of calcium oscillators in lymphatic and related cell types, but is required to fit recent experimental data. We include an additional voltage dependence in the gating variable control for the L-type calcium channel, enabling the M-clock to oscillate independently of the C-clock. We use phase-plane analysis to show that these M-clock oscillations are qualitatively similar to those of a generalised FitzHugh-Nagumo model. We also provide phase plane analysis to understand the interaction of the M-clock and C-clock oscillations. The model and methods have the potential to help determine mechanisms and find targets for pharmacological treatment of lymphoedema.

biophysics↗