Inducible Silencing of Junctophilins in Skeletal Muscle Leads to Reversible Remodeling of the Triad Junction Structure and Compromised Store-Operated Calcium Entry

Abstract

Junctophilins (JPs) play an essential role in muscle excitation-contraction (E-C) coupling by contributing to the formation of junctional membrane complexes (JMCs). However, the lethality associated with germ-line ablation of either JP1 or JP2 prevents physiological evaluation of their function in the maintenance of calcium homeostasis in adult muscle fibers. To investigate the physiological role of JP genes, we developed a novel transgenic system for tissue-specific and inducible control of gene expression in mouse models. This system employs a Tet-response CMV promoter that controls expression of a small-hairpin (sh) RNA against JP1 and JP2, which is non-functional until an interrupting reporter gene cassette is excised by the Cre recombinase. Insertion of the natural Dicer and Drosha-RNAse processing sites within the shRNA sequence allows for generation of specific siRNA probe for efficient knockdown of JP1 and JP2. Under the tight control of doxycycline (Dox), tissue- or lineage-specific expression of siRNA is achieved by the use of inducer mice that expresss Cre in a given tissue. Transgenic mice with muscle-specific expression of shRNA against JP showed no apparent change of JP expression before treatment with Dox; inducible and reversible knockdown of JP in skeletal muscle was achieved through feeding of animals with diet containing Dox. Dox-induced reduction of JP expression led to abnormal membrane structure and compromised store-operated Ca2+ entry in adult muscle fibers, consistent with JP's essential role in muscle development and function. This transgenic system can be applicable for inducible and reversible knockdown of different genes and in different tissues, as well as for control of transgene overexpression in an inducible and tissue-specific manner, thus providing a versatile system for eluciding the physiological gene function using viable animal models.