Role of the DHPR calcium Current in Mammalian Skeletal Muscle

Voltage-gated Ca2+ channels mediate membrane depolarization-induced influx of extracellular Ca2+ into electrically excitable cells, including neurons, endocrine cells, sensory cells, and muscle cells. This Ca2+ influx drives critical cellular processes like rapid synaptic transmission, endocrine secretion, gene transcription, and cardiac muscle contraction. While cardiac excitation-contraction (EC) coupling is strictly Ca2+ influx-dependent, the skeletal muscle EC coupling mechanism is based on a Ca2+ influx-independent interaction between the sarcolemmal voltage-sensing L-type Ca2+ channel or 1,4-dihydropyridine receptor (DHPR) and the sarcoplasmic Ca2+ release channel (RyR1). It is understood that voltage-induced conformational changes of the DHPR trigger opening of RyR1 via interchannel protein-protein interaction to release Ca2+ from the sarcoplasmic reticulum (SR) stores for myofibril contraction. However, during this EC coupling process a small and slowly activating Ca2+ influx through the DHPR is found in mammalian skeletal muscle. The role of this regulated Ca2+ influx, which is not (immediately) required for EC coupling, is still enigmatic; possibly it might be crucial in Ca2+ homeostasis by, e.g., SR store filling. However, experimental attempts to evaluate the significance of this Ca2+ influx for mammalian muscle function in vivo have not been yet realizable due to the lack of an appropriate animal model system. Surprisingly, we recently could discover in patch-clamp studies on freshly dissociated skeletal muscle myotubes from zebrafish larvae, that zebrafish (and all higher teleost fish) lack such DHPR Ca2+ influx. The fact that teleost fishes express two distinct pore forming DHPRa 1S isoforms as well in red (superficial slow) as in white (deep fast) musculature, and that this is implemented by different point mutations, indicates an evolutionary pressure on the avoidance of Ca2+ influx in teleost skeletal muscle. Here we want to test the hypothesis that principal differences in the physiology of Ca2+ handling in mammalian and teleost skeletal muscle exist. We aim to thoroughly investigate this fascinating skeletal muscle DHPR Ca2+-conductivity / non-conductivity phenomenon via a range of state of the art biophysical, biochemical and whole-animal muscle performance approaches by using animal models (k.i. mouse and transgenic zebrafish) where the DHPR Ca2+ conductivity properties will be interchanged. Besides the evolutionary biologically interesting prospect that Ca2+ influx in mammals it is just an evolutionary tolerated remnant (vestigial Ca2+ current) from phylogenetic stages of early chordates (where DHPR Ca2+ influx was the exclusive signal for RyR activation in skeletal muscle), it is very feasible that Ca2+ influx is similarly detrimental in higher teleost fish muscle as it is essential in mammalian muscle. However, in mammalian skeletal muscle its character would have completely changed - from a crucial EC coupling signal transmitter to a supporter of Ca2+ homeostasis (exaptational Ca2+ current). From these novel model organisms we want to gain deeper insight into the role of the DHPR Ca2+ influx for muscle cell Ca2+ homeostasis. We aim to find out if a lack of influx in mammals might lead to unbalanced intracellular Ca2+ levels which could yield reduced muscle performance and on the long term might induce gross physiological effects like myotonia or paralysis. If this hypothesis can be verified, we might be able to identify so far unexplained human myopathies.

Skeletal muscle contraction is initiated by a depolarizing signal from the nervous system which is sensed by the muscle cell. This process is called excitation-contraction (EC) coupling. The voltage sensor for this excitation signal is the dihydropyridine receptor (DHPR) of the surface membrane of the muscle cell. In contrast to Ca2+ influx-dependent cardiac EC coupling, the skeletal muscle EC coupling mechanism is based on a Ca2+ influx-independent, inter-channel protein-protein interaction between the DHPR and the ryanodine receptor (RyR1) of the sarcoplasmatic reticulum (SR) membrane. Upon this physical interaction the voltage-induced conformational change of the DHPR is transmitted to the RyR1 channel, which subsequently opens and thus releases huge amounts of Ca2+ from the SR stores which then induces muscle contraction.One exciting phenomenon of skeletal muscle EC coupling is the duality of the DHPR as voltage sensor and Ca2+ channel. While its unique role as voltage sensor for conformational EC coupling is firmly established, its more conventional function as a Ca2+ channel is under debate for nearly half a century. It was questioned if this small and slowly activating Ca2+ influx during EC coupling is able to play any role for the muscle cell like for example for refilling the SR Ca2+ stores, or if it is just an evolutionary remnant of phylogenetic stages where also skeletal muscle EC coupling followed the cardiac (Ca2+ influx-dependent) mechanism? Or in other words: Is this DHPR current vestigial or an exaptation? This question could so far never be answered because an accurate animal model was missing.In establishing zebrafish as model system for EC coupling we found to our surprise that zebrafish and all other teleost fishes express non-conducting Ca2+ channels in their skeletal muscles. Thinking about a fast moving school of herrings it is pretty obvious that this lack of Ca2+ influx is irrelevant for them. But is this also true for the mammalian muscle? To this aim we engineered a point mutation, responsible for DHPR non-conductivity (n.c.) in zebrafish muscle into the mouse genome. No difference in locomotor activity (home-cage activity, voluntary wheel running), motor coordination (rotarod, beam walk), muscle strength (rotarod endurance test, wire-hang test, grip-strength test), and muscle fatigability (exhaustive treadmill running) was observed when comparing young (3-7 months) and old (18-22 months) homozygous (n.c.)DHPR mice to the corresponding WT-siblings. Similarly, no difference was found in force-frequency and fatigue tests on isolated EDL (fast twitch) and soleus (slow twitch) muscle, performed on both age groups, hence excluding putative age-related accumulative effects of lacking DHPR Ca2+-conductivity on muscle performance. Surprisingly, qPCR analyses of multiple key triadic proteins of the EC coupling machinery and Ca2+ homeostasis did not indicate any compensatory transcriptional regulation. All these compelling findings support the hypothesis of the DHPR current being just vestigial in mammalian skeletal muscle.