ATP-sensitive potassium channels in zebrafish cardiac and vascular smooth muscle.

ATP-sensitive potassium channels (KATP channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, KATP 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 KATP expression and functional properties has only been performed in pancreatic ß-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 KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular KATP 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 KATP 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 KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular KATP 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 KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome.

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 zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

Genome-edited zebrafish model of ABCC8 loss-of-function disease.

ATP-sensitive potassium channel (KATP)gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. While transgenic mice expressing incomplete KATP LOF do reiterate mild hyperinsulinism, KATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical KATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human HI. Patch-clamp analysis confirmed the complete absence of channel activity in ß-cells from K499X (SUR1-/-) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish nor in homozygous SUR1-/- fish, mimicking findings in SUR1 knockout mice. Mutant fish did, however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent LOF mutations, these gene-edited animals provide valid zebrafish models of KATP -dependent pancreatic diseases.

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

Islet ß-cell membrane excitability is a well-established regulator of mammalian insulin secretion, and defects in ß-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 ß-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 (KATP -GOF) in ß-cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. KATP -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 Ca2+ , adult zebrafish islets recapitulate similar excitability-driven ß-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.

ABCC9-related Intellectual disability Myopathy Syndrome is a KATP channelopathy with loss-of-function mutations in ABCC9.

Mutations in genes encoding KATP channel subunits have been reported for pancreatic disorders and Cantú syndrome. Here, we report a syndrome in six patients from two families with a consistent phenotype of mild intellectual disability, similar facies, myopathy, and cerebral white matter hyperintensities, with cardiac systolic dysfunction present in the two oldest patients. Patients are homozygous for a splice-site mutation in ABCC9 (c.1320 + 1 G > A), which encodes the sulfonylurea receptor 2 (SUR2) subunit of KATP channels. This mutation results in an in-frame deletion of exon 8, which results in non-functional KATP channels in recombinant assays. SUR2 loss-of-function causes fatigability and cardiac dysfunction in mice, and reduced activity, cardiac dysfunction and ventricular enlargement in zebrafish. We term this channelopathy resulting from loss-of-function of SUR2-containing KATP channels ABCC9-related Intellectual disability Myopathy Syndrome (AIMS). The phenotype differs from Cantú syndrome, which is caused by gain-of-function ABCC9 mutations, reflecting the opposing consequences of KATP loss- versus gain-of-function.