Increased Ca signaling through Ca 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone and cartilage development and homeostasis, knowledge about Ca signaling and the source of Ca signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca signaling through Ca 1.2 voltage-gated Ca channel in tendon formation. Using a reporter mouse, we found that Ca 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated mice that express a gain-of-function mutant Ca 1.2 channel (Ca 1.2 ) in tendon. We found that tendons in the mutant mice were approximately 2/3 larger and had more tendon fibroblasts, but the cell density of the mutant mice decreased by around 22%. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendon. Biomechanical testing revealed that the hypertrophic Achilles tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomics analysis reveals no significant difference in the abundance of major extracellular matrix (ECM) type I and III collagens, but mutant mice had about 2-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor of TGF-β family myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2 and cathepsin K. Taken together, these data highlight roles for increased Ca signaling through Ca 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.