isolation-cardiac-vascular-smooth-muscle-cells-from-adult-juvenile

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Washington University in St. Louis

In pulmonary arterial hypertension (PAH), pathological changes in pulmonary arterioles progressively raise pulmonary artery pressure and increase pulmonary vascular resistance, leading to right heart failure and high mortality rates. Recently, the first potassium channelopathy in PAH, because of mutations in KCNK3, was identified as a genetic cause and pharmacological target.

Loss-of-Function ABCC8 Mutations in Pulmonary Arterial Hypertension.

Islet β-cell membrane excitability is a well-established regulator of mammalian insulin secretion, and defects in β-cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration-response relationship to mammalian β-cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose-dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain-of-function mutations in ATP-sensitive K channels (K -GOF) in β-cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. K -GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose-induced calcium responses. These results indicate that, although lacking tight cell-cell coupling of intracellular Ca , adult zebrafish islets recapitulate similar excitability-driven β-cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes.

Beta-cell excitability and excitability-driven diabetes in adult Zebrafish islets.

ATP-sensitive potassium channels (K channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, K channels are activated during ischaemia and potentially during adrenergic stimulation. In the vasculature, they are normally active at a low level, reducing vascular tone, but the ubiquitous nature of these channels leads to complex and poorly understood channelopathies as a result of gain- or loss-of-function mutations. Zebrafish (ZF) models of these channelopathies may provide insights to the link between molecular dysfunction and complex pathophysiology, but this requires understanding the tissue dependence of channel activity and subunit specificity. Thus far, direct analysis of ZF K expression and functional properties has only been performed in pancreatic β-cells. Using a comprehensive combination of genetically modified fish, electrophysiology and gene expression analysis, we demonstrate that ZF cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional K channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular K channels, ZF channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. The results provide a first characterization of the molecular properties of fish K channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome. KEY POINTS: Zebrafish cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional K channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular K channels, zebrafish channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. We provide a first characterization of the molecular properties of fish K channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome.

Article	Year
Loss-of-Function ABCC8 Mutations in Pulmonary Arterial Hypertension. Circulation. Genomic and precision medicine\| PubMed ID: 30354297	2018
Beta-cell excitability and excitability-driven diabetes in adult Zebrafish islets. Physiological reports\| PubMed ID: 31161721	2019
ATP-sensitive potassium channels in zebrafish cardiac and vascular smooth muscle. The Journal of physiology\| PubMed ID: 34820842	2022

Conor McClenaghan

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