Skeletal myotube integration with planar microelectrode arrays in vitro for spatially selective recording and stimulation: a comparison of neuronal and myotube extracellular action potentials

Microelectrode array (MEA) technology holds tremendous potential in the fields of biodetection, lab-on-a-chip applications, and tissue engineering by facilitating noninvasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.     

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

Cytoskeleton-bound mRNA for a 40-kDa polypeptide in rat L6 cells is not always translated

The relationship between attachment of mRNA to the cytoskeletal framework and its translation was examined using the mRNA for a polypeptide of 40 kDa (P-40) which is translated in rat L6 myoblasts but not in the myotubes. In both myoblasts and myotubes this mRNA was found to be associated with the cytoskeletal framework. Furthermore, the stability of the association between P-40 mRNA and the cytoskeletal framework in absence of RNA and protein synthesis was examined by using actinomycin D and NaF to block RNA and protein synthesis, respectively. In absence of RNA synthesis portions of both nontranslated P-40 mRNA and translated actin mRNA of myotubes were released into the soluble fraction. In myoblasts, however, both mRNAs remained associated with the cytoskeletal framework following inhibition of RNA synthesis. Inhibition of protein synthesis, on the other hand, had a more dramatic effect on the association between the cytoskeletal framework and P-40 mRNA in myoblasts but not in myotubes. In contrast, the association between actin mRNA and cytoskeletal framework was unaffected by inhibition of protein synthesis in both myoblasts and myotubes. The results of these studies show that the molecular nature of association between cytoskeletal framework and mRNA may differ among mRNAs and may also depend on whether the cells are dividing or are terminally differentiated. Furthermore, no direct relationship between the translation of mRNA and its attachment to the cytoskeletal framework was observed.     

Human transformer 2beta and SRp55 interact with a calcitonin-specific splice enhancer

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. Sequences in the human calcitonin-specific fourth exon function as an exonic splice enhancer (ESE) which is required for incorporation of exon 4 into calcitonin mRNA. Deletion of these sequences from the rat calcitonin/CGRP gene was reported to have no effect on calcitonin splicing. We demonstrate that sequences in the rat calcitonin/CGRP fourth exon act as an ESE. In addition, we observed that three proteins in HeLa nuclear extract, of apparent molecular weights of 40, 55 and 85 kDa, specifically interact with the exon 4 ESE. The 40-kDa protein is human transformer 2beta (hTra2beta), a homolog of the Drosophila splice regulator transformer 2. hTra2beta is required for calcitonin splicing in vitro, one of the first biological functions identified for hTra2beta. The 55-kDa protein is SRp55, a member of the SR family of phosphoproteins. Binding of SRp55 to an ESE required for calcitonin mRNA splicing suggests that the different levels of SRp55 present in different cell types may regulate calcitonin/CGRP alternative splicing.     

Cloning and characterization of rat neuronal apoptosis inhibitory protein cDNA

The human neuronal apoptosis inhibitory protein (NAIP) gene was originally discovered because of its deletion in infantile spinal muscular atrophy (SMA), a childhood genetic disorder characterized by motor neuron loss and progressive paralysis with muscular atrophy. Although SMA is now known to be caused by deletions of survival motor neuron (SMN), the fact that NAIP is an anti-apoptotic protein is consistent with the NAIP gene modifying SMA severity. Here we report the cloning of a 1.5 kb rat NAIP cDNA fragment which contains BIR-3 (third baculovirus inhibitory repeat) domain. This fragment shows 78% homology to the human NAIP and 86% homology to the murine counterpart. We have investigated the distribution of NAIP mRNA expressing neurons by in situ RT-PCR technique in the rat central nervous system (CNS). Although all of the neurons appeared to express NAIP mRNA ubiquitously, pronounced elevation of NAIP mRNA expression was observed in the areas innervated by glutamatergic neurons after kainic acid (KA) injection. We have raised an anti-rat NAIP antiserum in rabbits using NAIP cDNA and recombinant rat NAIP, and carried out an immunohistological investigation. We observed highly immunoreactive neuronal subpopulations in the retinal ganglion, cerebral cortex, hippocampus, basal forebrain, thalamus, areas of midbrain, Purkinje cells of the cerebellum, and motor neurons in the spinal cord. Increased immunoreactivity of glutamatergic neurons was also observed broadly in the CNS after KA treatment. This study provides additional evidence that expression of mRNA and gene products of NAIP seem to be regulated in response to excessive stimuli or injuries in rat CNS, and these results are compatible with an anti-apoptotic role of NAIP in acute SMA as well as in brain injuries.     

Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes

BACKGROUND: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. METHODS: Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. RESULTS: We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. CONCLUSIONS: Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.     

Coordination of skeletal muscle gene expression occurs late in mammalian development

The acquisition of specialized skeletal muscle fiber phenotypes during development is investigated by systematic measurement of the accumulation of 21 contractile protein mRNAs during hindlimb development in the rat and the human. During early myotube formation in both species there is no coordination of expression of either fast or slow contractile protein isoform genes, but rather some slow, some fast, and some cardiac isoforms are expressed. Some isoforms are not detected at all in early myotubes. From Embryonic Day 19 in the rat, and after 14 weeks in the human, a strong bias toward fast isoform expression is evident for all gene families examined. This results in the establishment of a coordinated fast isoform phenotype at birth in the rat, and by 24 weeks in the human fetus. Unexpectedly, during secondary myotube formation in the rat we observe sudden rises and falls in contractile protein gene output. We interpret these fluctuations in terms of periods of myoblast proliferation followed by synchronized fusion into myotubes. The data presented indicate that each contractile protein gene has its own determinants of mRNA accumulation and that the different myoblast populations which contribute to the developing limb are not intrinsically programmed to produce particular coordinated phenotypes with respect to the non-myosin heavy chain contractile proteins.     

Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle

Glycogen-targeting PP1 (protein phosphatase 1) subunit G(L) (coded for by the PPP1R3B gene) is expressed in human, but not rodent, skeletal muscle. Its effects on muscle glycogen metabolism are unknown. We show that G(L) mRNA levels in primary cultured human myotubes are similar to those in freshly excised muscle, unlike subunits G(M) (gene PPP1R3A) or PTG (protein targeting to glycogen; gene PPP1R3C), which decrease strikingly. In cultured myotubes, expression of the genes coding for G(L), G(M) and PTG is not regulated by glucose or insulin. Overexpression of G(L) activates myotube GS (glycogen synthase), glycogenesis in glucose-replete and -depleted cells and glycogen accumulation. Compared with overexpressed G(M), G(L) has a more potent activating effect on glycogenesis, while marked enhancement of their combined action is only observed in glucose-replete cells. G(L) does not affect GP (glycogen phosphorylase) activity, while co-overexpression with muscle GP impairs G(L) activation of GS in glucose-replete cells. G(L) enhances long-term glycogenesis additively to glucose depletion and insulin, although G(L) does not change the phosphorylation of GSK3 (GS kinase 3) on Ser9 or its upstream regulator kinase Akt/protein kinase B on Ser473, nor its response to insulin. In conclusion, in cultured human myotubes, the G(L) gene is expressed as in muscle tissue and is unresponsive to glucose or insulin, as are G(M) and PTG genes. G(L) activates GS regardless of glucose, does not regulate GP and stimulates glycogenesis in combination with insulin and glucose depletion.