From excitation-contraction coupling to gene expression: Roles of RYR1 and Cav1.1 in myogenesis

The main function of differentiated skeletal muscle is contraction, allowing for movement. However, contraction also has important developmental roles and thus is indispensable for proper muscle formation and organization. On a molecular level, the initiation of skeletal muscle contraction relies on the interplay of two mechanically coupled Ca2+ channels - the principal subunit of the 1,4 dihydropyridine receptor (Cav1.1) and the type 1 ryanodine receptor (RYR1), the key event in the process of excitation-contraction coupling (ECC). While multiple functional and structural studies over the last decades have led to a deeper understanding of the roles of Cav1.1 and RYR1 in ECC, their specific involvement in muscle development and in gene expression remains obscure. The present work analyzes the morphological and global transcriptomic changes occurring in limb skeletal muscle from RYR1- and Cav1.1-deficient (RYR1-/- and Cav1.1-/-, respectively) mice at the beginning (E14.5) and at the end (E18.5) of secondary myogenesis. In both models initial muscle structure alterations are already observable E14.5. At this stage, increased apoptosis is observed only in Cav1.1-/- limb skeletal muscle. Microarray analyses reveal discrete transcriptomic changes in both mutants at E14.5, with downregulation of genes primarily associated with innervation and neuron development in RYR1-/-, and with muscle contraction in Cav1.1-/- skeletal muscle. At E18.5, both RYR1-/- and Cav1.1-/- skeletal muscles are characterized by more severe structural malformation, fibrosis, and signs of developmental retardation. At this stage a high number of the detected differentially expressed genes (DEGs) overlap i RYR1 /- and Cav1.1-/-. Both mutants display a failure to upregulate the expression of many genes involved in the buildup of the contractile machinery and exhibit changes in the expression of global signaling pathways and multiple microRNAs. Mutant-specific transcriptomic changes point to changes in the composition of the extracellular matrix in RYR1-/- muscle and in the lipid metabolism in Cav1.1-/- muscle. Finally, the absence of RYR1 in RYR1-/- mice alters the ratio of Cav1.1 splice variants at E14.5, and the total Cav1.1 mRNA levels at E18.5. Taken together, the results of this work highlight the importance of Cav1.1 and RYR1 for the proper execution of the developmental gene expression program during secondary myogenesis in mouse limb skeletal muscle. Furthermore, it provides insights into mutual but also specific roles of each Ca2+ channel during skeletal muscle development

Breaking the Barriers: ontological aspects of relation and mutuality in contemporary theories of atomic structure and in philosophy of personalism

Also published in Symposium Melitensia Vol. 15 (2019) p. 17-28The paper focuses on the interdisciplinary field between the sciences and the humanities. It explores the subject matter of breaking barriers and ‘individuality’ in terms of the subatomic particles as building blocks of the matter and the human beings according to existentialism and personalism as philosophical traditions. The ‘overcoming the barrier’ theme is considered in a twofold perspective in this paper: on the one hand, as an ontological, structural and organisational principle, and, on the other hand as a condition for interaction, relation and mutuality. Theoretical and conceptual references to some of the fundamental ideas and concepts of the Quantum Mechanical Model of the atom and the Standard Model of particles are made, emphasising the primary importance of the entanglement as the variation of relationality and mutuality in the field of natural sciences. Moreover, references are made as well as to Martin Buber’s and Emmanuel Lévinas’s philosophy. This paper will serve as a basis for the development of an interdisciplinary series of seminars in the Secondary school curriculum aiming to deepen the relation between the natural sciences (chemistry and physics) and the humanities.peer-reviewe

A mutation in Ca(v)2.1 linked to a severe neurodevelopmental disorder impairs channel gating

Ca2+ flux into axon terminals via P-/Q-type Ca(v)2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMjs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the 54 voltage-sensing helix of the fourth membrane-bound repeat of Ca(v)2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in Ca(v)2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in Ca(v)2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat Ca(v)2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an similar to 25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of Ca(v)2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMjs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type Ca(v)2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in Ca(v)2.1

Functional assessment of three Rem residues identified as critical for interactions with Ca2+ channel beta subunits

Members of the Rem, Rem2, Rad, Gem/Kir (RGK) family of small GTP-binding proteins inhibit high-voltage-activated (HVA) Ca2+ channels through interactions with both the principal alpha(1) and the auxiliary beta subunits of the channel complex. Three highly conserved residues of Rem (R200, L227, and H229) have been shown in vitro to be critical for interactions with beta subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to beta subunit-mediated RGK protein-dependent inhibition of HVA channels, we introduced alanine substitutions into all three positions of Venus fluorescent protein-tagged Rem (V-Rem AAA) and made three other V-Rem constructs with an alanine introduced at only one position (V-Rem R200A, V-Rem L227A, and V-Rem H229A). Confocal imaging and immunoblotting demonstrated that each Venus-Rem mutant construct had comparable expression levels to Venus-wild-type Rem when heterologously expressed in tsA201 cells. In electrophysiological experiments, V-Rem AAA failed to inhibit N-type Ca2+ currents in tsA201 cells coexpressing Ca(V)2.2 alpha(1B), beta(3), and alpha(2)delta-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed Ca(V)2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-beta interactions. Rem-dependent inhibition of Ca(V)2.2 channels was impaired to a much lesser extent by the R200A substitution. In contrast to the earlier work demonstrating that Rem H229A was unable to interact with beta(3) subunits in vitro, V-Rem H229A produced nearly complete inhibition of Ca(V)2.2-mediated currents

Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca2+ channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca2+ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing alpha(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary beta(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for beta(1a) has been difficult to test because beta(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, beta(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established. subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltageinduced myoplasmic Ca2+ transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca2+ store content. In contrast, a beta(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca2+ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca2+ release via its ability to interact with beta(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the beta(1a) subunit in skeletal-type EC coupling

Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RyR1 domain

The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an orthograde signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a retrograde signal from RyR1 increases the amplitude of the Ca2+ current produced by Ca(v)1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with Ca(v)1.1 by producing an N-terminal construct (RyR1(1)(:)(4300)) that lacks the C-terminal membrane domain. In Ca(v)1.1-null (dysgenic) myotubes, RyR1(1)(:)(4300) is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR-PM junctions containing endogenous Ca(v)1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR1(1)(:)(4300) is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR1(1)(:)(4300) stably associates with PM-SR junctions. Strikingly, expression of RyR1(1)(:)(4300) in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through Ca(v)1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that similar to 25% of RyR1(1)(:)(4300) in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR1(1)(:)(4300) is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DH PR tetrads is substantially increased by transfecting RyR-null myotubes with RyR1(1)(:)(4300) . In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR-PM junctions in myotubes only if Ca(v)1.1 is also present and is fully functional in retrograde signaling to Ca(v)1.1

Novel CB1-ligands maintain homeostasis of the endocannabinoid system in omega 3-and omega 6-long-chain-PUFA deficiency[S]

Mammalian omega 3- and omega 6-PUFAs are synthesized from essential fatty acids (EFAs) or supplied by the diet. PUFAs are constitutive elements of membrane architecture and precursors of lipid signaling molecules. EFAs and long-chain (LC)-PUFAs are precursors in the synthesis of endocannabinoid ligands of G(i/o) protein-coupled cannabinoid receptor (CB)1 and CB2 in the endocannabinoid system, which critically regulate energy homeostasis as the metabolic signaling system in hypothalamic neuronal circuits and behavioral parameters. We utilized the auxotrophic fatty acid desaturase 2-deficient (fads2(-/-)) mouse, deficient in LC-PUFA synthesis, to follow the age-dependent dynamics of the PUFA pattern in the CNS-phospholipidome in unbiased dietary studies of three cohorts on sustained LC-PUFA-free omega 6-arachidonic acid- and DHA-supplemented diets and their impact on the precursor pool of CB1 ligands. We discovered the transformation of eicosa-all cis-5,11,14-trienoic acid, uncommon in mammalian lipidomes, into two novel endocannabinoids, 20:3(5,11,14)-ethanolamide and 2-20:3(5,11,14)-glycerol. Their function as ligands of CB1 has been characterized in HEK293 cells. Labeling experiments excluded Delta 8-desaturase activity and proved the position specificity of FADS2. The fads2(-/-) mutant might serve as an unbiased model in vivo in the development of novel CB1 agonists and antagonists

Distinct transcriptomic changes in E14.5 mouse skeletal muscle lacking RYR1 or Ca(v)1.1 converge at E18.5

In skeletal muscle the coordinated actions of two mechanically coupled Ca2+ channels-the 1,4-dihydropyridine receptor (Ca(v)1.1) and the type 1 ryanodine receptor (RYR1)-underlie the molecular mechanism of rapid cytosolic [Ca2+] increase leading to contraction. While both [Ca2+](i) and contractile activity have been implicated in the regulation of myogenesis, less is known about potential specific roles of Ca(v)1.1 and RYR1 in skeletal muscle development. In this study, we analyzed the histology and the transcriptomic changes occurring at E14.5 -the end of primary myogenesis and around the onset of intrauterine limb movement, and at E18.5 -the end of secondary myogenesis, in WT, RYR1(-/-), and Ca(v)1.1(-/-) murine limb skeletal muscle. At E14.5 the muscle histology of both mutants exhibited initial alterations, which became much more severe at E18.5. Immunohistological analysis also revealed higher levels of activated caspase-3 in the Cav1.1(-/-) muscles at E14.5, indicating an increase in apoptosis. With WT littermates as controls, microarray analyses identified 61 and 97 differentially regulated genes (DEGs) at E14.5, and 493 and 1047 DEGs at E18.5, in RYR1(-/-) and Cav1.1(-/-) samples, respectively. Gene enrichment analysis detected no overlap in the affected biological processes and pathways in the two mutants at E14.5, whereas at E18.5 there was a significant overlap of DEGs in both mutants, affecting predominantly processes linked to muscle contraction. Moreover, the E18.5 vs. E14.5 comparison revealed multiple genotype-specific DEGs involved in contraction, cell cycle and miRNA-mediated signaling in WT, neuronal and bone development in RYR1(-/-), and lipid metabolism in Ca(v)1.1(-/-) samples. Taken together, our study reveals discrete changes in the global transcriptome occurring in limb skeletal muscle from E14.5 to E18.5 in WT, RYR1(-/-) and Ca(v)1.1(-/-) mice. Our results suggest distinct functional roles for RYR1 and Cav1.1 in skeletal primary and secondary myogenesis

Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca2+ release channel

In mature skeletal muscle, the intracellular Ca2+ concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca2+ release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1's potential roles in muscle development remain obscure. We used an established RyR1-null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca2+ signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca2+ signaling during muscle organ development