Regulation of tendon formation by Ca2+ signaling through CaV1.2 L-type voltage-gated calcium channel

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PROJECT SUMMARY Intracellular Ca2+ influences major tendon signaling pathways. Although Ca2+ signaling has been studied extensively for its roles in muscle contraction, immune cell activation, hormone secretion, cell proliferation, neuronal regulation, and gene activation, Ca2+ signaling details in tendon and the channels responsible for Ca2+ influx into tendon fibroblasts are largely unknown. Using novel mouse models with a gain-of-function CaV1.2 mutant channel (CaV1.2TS), we observed potent regulatory effects of Ca2+ influx through CaV1.2 on tendons. CaV1.2TS channels carry a gain-of-function G406R mutation in the pore-forming CaV1.2 α1C subunit that impairs voltage-dependent inactivation and allows more Ca2+ influx into the cell. We observed that driving expression of CaV1.2TS specifically in tendon with Scleraxis-Cre (ScxCre) leads to a marked increase in tendon mass. Using a reporter mouse, we found that CaV1.2 is extensively expressed in tendon fibroblasts during tendon development. We therefore postulate that Ca2+ influx through CaV1.2 in tendon fibroblasts regulates tendon formation, a hypothesis that fits with our previous demonstration that CaV1.2 also functions in osteoblast precursor cells to regulate bone formation and homeostasis. In this proposal we seek to determine the cellular and molecular mechanisms of Ca2+ signaling through CaV1.2 that regulate tendon formation. Our specific hypothesis is that Ca2+ signaling through CaV1.2 regulates expression of tendon transcription factors that control tendon cell proliferation and/or tendon extracellular matrix (ECM) synthesis during tendon development and mechanical overload-induced adult tendon growth. In Aim 1, we will upregulate Ca2+ signals in tendon fibroblasts by using our CaV1.2TS gain-of-function mouse model and determine cell proliferation, extracellular matrix collagen synthesis, and expression of tendon transcription factors. We will determine the signaling cascades that mediate increased Ca2+ signals to upregulated tendon fibroblast proliferation and ECM synthesis in vitro in a tail tendon fibroblast culture system. In Aim 2, we will use our newly-developed Cav1.2 inducible conditional knockout mouse model to determine the physiological role of Ca2+ signaling through CaV1.2 during tendon development, postnatal tendon formation, and during mechanical overload-induced adult tendon growth. Successful completion of this study will provide a fundamental understanding of the role of Ca2+ signaling on tendon formation and a platform to identify new targets for developing therapeutic strategies for tendon diseases.