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Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the scarcity of methods that allow monitoring in tight inter-cell spaces of living tissues. Here we present high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (Kds) ranging from 0.8 mM to 2.9 mM, making them tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms., (© 2023. Springer Nature Limited.)

The phospholamban mutation Arg 9 to Cys (R9C) has been found to cause a dilated cardiomyopathy in humans and in transgenic mice, with ventricular dilation and premature death. Emerging evidence suggests that phospholamban R9C is a loss-of-function mutation with dominant negative effect on SERCA2a activity. We imaged calcium and cardiac contraction simultaneously in 3 and 9 days-post-fertilization (dpf) zebrafish larvae expressing plnb R9C in the heart to unveil the early pathological pathway that triggers the disease. We generated transgenic zebrafish lines expressing phospholamban wild-type (Tg(myl7:plnb wt )) and phospholamban R9C (Tg(myl7:plnb R9C )) in the heart of zebrafish. To measure calcium and cardiac contraction in 3 and 9 dpf larvae, Tg(myl7:plnb wt ) and Tg(myl7:plnb R9C ) fish were outcrossed with a transgenic line expressing the ratiometric fluorescent calcium biosensor mCyRFP1-GCaMP6f. We found that Plnb R9C raised calcium transient amplitude, induced positive inotropy and lusitropy, and blunted the β-adrenergic response to isoproterenol in 3 dpf larvae. These effects can be attributed to enhanced SERCA2a activity induced by the Plnb R9C mutation. In contrast, Tg(myl7:plnb R9C ) larvae at 9 dpf exhibited ventricular dilation, systolic dysfunction and negative lusitropy, hallmarks of a dilated cardiomyopathy in humans. Importantly, N-acetyl-L-cysteine rescued this deleterious phenotype, suggesting that reactive oxygen species contribute to the pathological pathway. These results also imply that dysregulation of calcium homeostasis during embryo development contributes to the disease progression at later stages. Our in vivo model in zebrafish allows characterization of pathophysiological mechanisms leading to heart disease, and can be used for screening of potential therapeutical agents., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)