Cellular methods for studying Ca2+ channel mutations

Previously we have developed a muscle cell culture system, which allowed us to study the structure and function of genetically altered ion channels in their native cellular environment. This system proved to be of exceptional value for investigations of the role of calcium (Ca 2+) channels in the activation of muscle contraction - a process termed excitation-contraction (EC-) coupling. Here we propose to develop several new cell culture systems, which will extend this successful research strategy to a broader range of Ca2+ channels in muscle and nerve cells, and to the study of human disease mutations. It can be expected that these "homologous expression systems" will greatly contribute to basic and applied research in the field of EC-coupling, while at the same time reducing the need of animal experimentation. The novel approach of generating the cell lines is by cross-breeding available spontaneous null-mutant or knock- out mice, which lack an important Ca2+ channel, with the so-called ImmortoMouse. Cells derived from these mouse hybrids can be expanded over many generations under permissive culture conditions but undergo normal differentiation under non-permissive conditions. These properties make such cell lines suitable for stable transfection with recombinant Ca2+ channel constructs. Transfection with cDNA coding for the lacking wild-type channel restores normal function in the mutant muscle cells. Moreover, reconstitution of the deficient muscle cells with mutant channels enables us to analyze the effects of the experimentally introduced mutations on muscle development and function using a broad range of methods. This will yield important information on the properties of the Ca2+ channels, on the mechanism of EC-coupling, and on the pathophysiology of muscle disease. In order to assess the full potential of these cultures as cellular disease models, two cell lines expressing Ca2+ channel mutants, which in humans cause malignant hyperthermia, will be generated and analyzed in our own and several collaborating laboratories. Furthermore, we plan to generate null-mutant cell lines from brain tissue of the same mouse hybrids. Thus, this valuable approach to study Ca2+ channel mutants that has hitherto been limited to muscle cells shall now also be made possible for neurons.

The overall goal of this project was to develop new cellular expression models of muscle cells to study the structure and function of normal and mutant calcium channels in their native environment. To this end we cross- bred mice lacking the skeletal muscle calcium channels with the Immortomouse and isolated myoblasts which combined the properties of both mouse mutants. To allow genotyping of the mutant mice the position of the transgene in the genome of the Immortomice was identified and PCR-based assays were developed (Kern & Flucher, 2005). Using this approach several CaV1.1 and RyR1 calcium channel null-mutant cell lines were generated, and the potential to restore normal function by heterologous expression of the healthy copies of the affected genes was examined. Thorough characterization of these stably transfected muscle cell lines demonstrated an unexpected clone-to-clone variability in the properties of the muscle cell lines that precluded meaningful analysis of calcium channel disease mutants. In contrast, transient transfection of immortalized null-mutant cell lines proved to be suitable for investigation of structure-function relationships of mutated calcium channels (Kugler et al., 2004a and b; Weiss et al., 2004; Takekura et al., 2004; Schuhmeier et al., 2005). As an alternative to studying altered calcium channel function in reconstituted null-mutant cell lines, we investigated the effectiveness of siRNA gene silencing in striated muscle cells. This novel approach proved particularly useful for studying essential proteins for which knock-out animals are not viable. Therefore we used siRNA for the first time in muscle cells in a study of the function of the calcium channel a 2 d-1 subunit in controlling skeletal and cardiac muscle contraction (Obermair et al., 2004, 2005; Tuluc et al., 2007). Reconstituting our null-mutant muscle cell lines with the cardiac calcium channel a 1C subunit restored cardiac excitation- contraction coupling properties in the skeletal muscle system. Simulating the experimentally obtained effects of a 2 d-1 depletion in a computer model of cardiac myocytes indicated a disease-relevant prolongation of the cardiac action potential (Tuluc et al., 2007). Combining our unique null-mutant muscle cell systems with recombinant expression and siRNA techniques and, where applicable, with state-of-the-art computer models of heart cells, provides a powerful novel approach to investigate the consequences of experimental and disease mutation in the calcium channel genes on normal skeletal and cardiac muscle function. Thus, the new cell models and experimental strategies developed in the course of this project aid in understanding the calcium channel functions in normal and diseased muscle cells, and provide a valuable alternative to generating and experimentation with genetically modified animal models.