Zebrafish as a model system of excitation-contraction coupling

To strengthen studies on the structure - function relationship of excitation-contraction (EC) coupling (i.e., the transformation of the electrical membrane depolarization signal to muscle contraction) we want to establish the zebrafish (Danio rerio) as a model system for the EC coupling field. // Zebrafish has several extraordinary features to make it a perfect model organism. It is easy to hatch, has a high reproduction rate together with a short generation time and it is relatively straightforward to generate transgenic fish. Another great advantage is the existence of a plethora of zebrafish mutants. Also mutants affecting the muscle motility exist. There is high evidence that one of those mutants is lacking a key element of ECC, namely the voltage gated L-type Ca2+ channel or dihydropyridine receptor (DHPR) of the muscle surface membrane. This DHPR is the "voltage sensor" which senses membrane depolarization. Subsequently this signal is transmitted to the intracellular ryanodine receptor (RyR) which in turn triggers Ca2+ release from intracellular Ca2+ stores - the initial step of muscle contraction. The aim of our study is to introduce transmuted DHPRs into this DHPR-null zebrafish to subsequently test this transgenic fish (in comparison to wildtype) as well in exactly monitored swimming experiments as with biophysical and immunohistochemical methods. One of those DHPR mutants studied in this way cause malignant hyperthermia (MH) in human - which can produce severe incidents during anesthesia. This fact explains sufficiently that the production of a MH model organism will be of highest interest. The second DHPR we want to investigate is lacking a molecular region previously postulated as an essential element for EC coupling. After experiments in a DHPR-null mouse cell line showed no effect of this mutant, we are eager to recheck this mutant on the intact (transgenic) organism. Our third projected zebrafish will express a DHPR carrying a tag to be recognized by a crosslinker. For future biochemical experiments we will use the transgenic approach for mass production of tagged DHPRs. In addition, an important part of our project will be to search for putatively new DHPR mutations on already existing zebrafish motility mutants. This is a potentially successful approach to open new insights into the EC coupling mechanism.

Zebrafish (ZF) emerged as a highly advantageous model organism for biomedical research during recent years. Our results present ZF now as a powerful system to also study skeletal-type excitation-contraction coupling (ECC). ECC is understood as the transformation of membrane depolarization to muscle contraction. Thereby the L-type Ca2+ channel or dihydropyridine receptor (DHPR) of the plasma membrane operates as a voltage sensor which transduces its conformational changes directly to the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR). Consequently, Ca2+ is released from SR stores, leading to muscle contraction by activation of the proteins of the sarcomere. For this allosteric DHPR-RyR1 interaction a physical touch of the RyR1 with a DHPR tetrad is essential. // By studying the paralyzed ZF relaxed we could show, that i) the DHPR subunit (SU) ß1a is responsible for this tetrad formation. Additionally, ii) only ~50% of the pore forming DHPR a 1S SU were expressed in the muscle membranes of this ß1 -null mutant, and those expressed were iii) not active as voltage sensors. By transfection of relaxed myotubes with ß1a cDNA we were able to fully restore DHPR voltage sensor function as well as tetrad re-establishment and thus ECC. In addition, we succeeded by ß1a cRNA zygote microinjection, to temporarily reconstitute the motile phenotype in relaxed larvae. With these two expression systems in our hands all opportunities are open now to study chimeric and point-mutated ß-constructs in skeletal muscle in-vitro and in- vivo thus analyzing structure-function relationships to explain ß actions as pointed out under i)-iii) (see above). // Surprisingly, the ZF DHPR did not show any Ca2+ conductance and thus functions as a pure voltage sensor and allosteric signal transmitter to the RyR. Thus, this represents the first in-vivo evidence that skeletal muscle ECC is independent from influx of extracellular Ca2+! Biophysical studies on chimeras and point mutants could identify a single charged amino acid (D636) in the a 1S SU pore, sufficient to impede Ca2+ conductance also in a mammalian DHPR. These results we consider not only important for better understanding Ca2+ channel gating, but also as a significant tool to analyze the so far enigmatic influx of extracellular Ca2+ during ECC. A corresponding knock-in mouse, as well as a transgenic ZF carrying a conducting Ca2+ channel, is underway. From both animal models we expect deeper insights into the role of the influx of extracellular Ca2+ during ECC and thus a better understanding of a suitable control of Ca2+ homeostasis for muscle performance. // The anticipated revelation of molecular regions of the ß1a SU responsible for the integration of the pore forming DHPR a 1S into membrane and tetrads, as well of ß-regions responsible for the central DHPR function of voltage sensing, make us hopeful for the development of ß SU-selective drugs in the future. If those functional molecular regions are even specific for ß isoforms of skeletal muscle, heart and brain, the development of a new generation of Ca2+ antagonists, operating on the expression level, would be feasible.