A shortcut to a skeletal muscle DHPR knock-in?

Excitation–contraction (EC) coupling in mammalian skeletal muscle relies on intermolecular communication between the L-type Ca2+ channel (or 1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine receptor (RyR1). Conformational rearrangements in the DHPR that occur in response to transverse tubular membrane depolarization are transduced to RyR1 via a physical coupling between the two channels that is largely independent of any Ca2+ entry via the DHPR (reviewed in Karunasekara et al. 2009). In addition to this ‘orthograde’ signal transmitted from the DHPR to RyR1, conformational coupling also supports a ‘retrograde’ signal from RyR1 to the DHPR which increases macroscopic L-type current amplitude (Nakai et al. 1996). Although there are many other components of the macromolecular signalling complex that support this mode of communication (e.g. triadin, JP-45, Homer, FKBP12, junctophilins, DHPR γ and α2δ-1 subunits, etc.), only RyR1 and the DHPR α1S and β1a subunits are to date known to be essential for EC coupling. Mice null for any one of these three proteins die perinatally from asphyxia resulting from diaphragm paralysis. Needless to say, the inability of these neonatal mice to contract their diaphragms has precluded investigation of the basic mechanism of EC coupling in adult skeletal muscle. Fortunately, myotubes can be easily cultured from fetal or neonatal pups, and for this reason, have proven to be an exceptionally useful model system for the study of the interaction between the DHPR and RyR1. This in vitro system has enabled the expression of modified RyR1, α1S and β1a subunits in true null backgrounds, thereby facilitating identification and functional characterization of regions of these essential proteins that support communication between the DHPR and RyR1. Despite the many advantages of using null myotubes to study EC coupling, the obvious shortcoming of this system is that myotubes growing in a plastic culture dish are not differentiated muscle fibres. In particular, the fully developed triad junctions formed by the membranes of the transverse tubules and the sarcoplasmic reticulum (SR) of adult muscle are absent in cultured myotubes, which have less organized plasma membrane–SR junctions. Since conformational coupling is critically dependent on junctional ultrastructure, mechanistic differences in the DHPR–RyR1 interaction between myotubes and adult fibres may exist. In light of these potential differences, there is a need for an experimental system in which modified α1S, β1a or RyR1 clones can be expressed and evaluated in adult fibres without interference from the respective endogenous proteins. Not too long ago, Julio Vergara and colleagues introduced a technique –in vivo electroporation – in which cDNA plasmids can be delivered to an isolated set of muscles in the distal hindlimb of mature mice (see DiFranco et al. 2007). In effect, they created a transgenic system that in some respects is superior to genetically engineered mice. Specifically, expression of the plasmid of interest was limited to one or two muscles in the paw of the mouse and optimal expression was achieved in less than 2 weeks. Moreover, the technique is extremely cost-effective compared to the generation of a transgenic mouse strain. Recently, Pietri-Rouxel and colleagues (2010) similarly employed local adenoviral delivery of siRNA with the aim of ablating expression of the endogenous DHPR α1S subunit in adult tibialis anterior muscle. The attempt to create effectively dysgenic adult fibres was not entirely successful because of the inability of the siRNA to quash α1S expression completely (an inherent weakness of siRNA-based strategies), but a key role for DHPR expression in maintaining healthy muscle mass was revealed. In the study discussed below, the Vergara laboratory (DiFranco et al. 2011) has now taken the in vivo electroporation strategy to the next level; they attempt to create pharmacologically inducible α1S‘knock-in’ fibres by silencing the endogenous protein with a dihydropyridine (DHP) antagonist and replacing it with a DHP-insensitive counterpart. Their success would provide EC coupling investigators with a suitable system in which to investigate DHPR–RyR1 conformational coupling in fully differentiated muscle fibres. The first obstacle DiFranco and colleagues encountered in their shortcut to a DHPR knock-in was whether an EGFP-α1S subunit fusion construct could actually be overexpressed at reasonable level using in vivo electroporation. The Vergara laboratory has had good success expressing non-fused EGFP and an EYFP-β1a fusion construct in adult fibres (239 and 789 residues, respectively) but expression of the considerably larger EGFP-α1S construct (∼2100 residues) was by no means a trivial matter. However, much of the uncertainty concerning EGFP-α1S expression was relieved when successfully transfected flexor digitorum brevis (FDB) or interosseus (IO) fibres were identified by the fluorescence in the transverse tubules generated by the EGFP tag (DiFranco et al. 2011, Fig. 2). As one might expect with overexpression of the α1S subunit, transfected fibres had enhanced maximal gating charge movement, which indicated the presence of more DHPRs within the transverse tubule system of transfected fibres (Fig. 8). Interestingly, there was not a corresponding increase in either L-type conductance or maximal depolarization-induced SR Ca2+ release (Figs 3 and 9, respectively). The authors posited that even though there were more DHPR channels present at triad junctions in transfected fibres, there was no increase in RyR1 expression. Therefore, the ‘orphan’ DHPRs newly present in the transverse tubules were not capable of engaging EC coupling and existed in a low open probability gating state because they lacked both orthograde and retrograde coupling interactions with an RyR1 (Nakai et al. 1996). Such an explanation is quite reasonable based on previous work which has demonstrated that the retrograde influence of RyR1 substantially upregulates DHPR current density in myotubes. However, this conclusion would be considerably more sound if RyR1 expression had been tested and found to be unaltered in muscle fibres overexpressing EGFP-α1S. Having demonstrated that EGFP-α1S can be efficiently overexpressed in FDB/IO fibres, the authors overexpressed a relatively DHP-insensitive mutant α1S subunit (EGFP-α1S T935Y). The rationale for this manoeuvre was that any DHPR function (i.e. L-type Ca2+ current, voltage-sensor for SR Ca2+ release) remaining following application of a DHP antagonist could be attributed entirely to the transfected DHP-insensitive point mutant. Somewhat surprisingly, L-type current amplitude was decreased in fibres transfected with EGFP-α1S T935Y even though charge movement was increased by 44% (Fig. 8). This observation is consistent with the idea that some of the endogenous wild-type channels were displaced from the influence of RyR1 by overexpressed EGFP-α1S T935Y DHPRs that do not conduct L-type current as well as the wild-type channel (Table 2). When applied at 1 μm, the neutral DHP antagonist isradipine blocked ∼60% of the peak control L-type current in EGFP-α1S T935Y transfected fibres and ∼95% of the peak current in control fibres (naive fibres and those overexpressing EGFP-α1S, pooled; Fig. 7). From this observation, the authors concluded that the DHP-insensitive mutant was integrated into triad junctions and constituted the predominant component of the total L-type current in isradipine-treated EGFP-α1S T935Y-transfected fibres in an 8:1 ratio relative to the endogenous channel. However, the flip side of this interpretation is that the endogenous channel supported a small, but significant, portion (∼10–15%) of the DHP-insensitive current in EGFP-α1S T935Y-transfected fibres. Thus, the persistence of a contribution from the endogenous α1S subunit to DHPR function in fibres expressing EGFP-α1S T935Y is problematic for an expression system which requires a true null background. In conclusion, the work of DiFranco et al. (2011) represents a step forward in the development of an adult model for the study of conformational coupling between the DHPR and RyR1, but the current approach requires refinement to be truly useful. Perhaps species-specific sequence differences between the transfected rabbit α1S clone and the endogenous mouse α1S subunit could be exploited. If there are such regions of low conservation, then siRNA directed against the latter might be employed to eliminate the pesky isradipine-insensitive component of the native L-type current. Even though the strategy to create a pharmacologically inducible α1S knock-in fibre has not been perfected here, the in vivo electroporation strategy represents a progressive approach to the study of intrinsic muscle excitability. The great potential of the technique is the ability to overexpress or knock-down proteins that play supporting roles in EC coupling in a short period of time and at low expense in fully differentiated muscle fibres. In addition, in vivo electroporation presents an opportunity to study recessive muscle diseases such as malignant hyperthermia and central core disease by mimicking heterozygous channel expression in the muscle of afflicted individuals. For those of us who study these α1S/RyR1-linked disorders and who chase the elusive molecular underpinnings of EC coupling, the collective work of DiFranco and colleagues provides a powerful tool for these pursuits.