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Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle.

Durcan PJ, Conradie JD, Van deVyver M, Myburgh KH - BMC Physiol. (2014)

Bottom Line: Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance.These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, Stellenbosch University, Private Bag X1 Matieland, 7602, Stellenbosch, South Africa. pdurcan@sun.ac.za.

ABSTRACT

Background: Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.

Results: We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.

Conclusion: The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

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Detection of Kirrel3 splice variants with primer set 1. A – PCR amplicons obtained with Kirrel3 primer set 1 to detect Kirrel3 A. mRNA was isolated from adult human skeletal muscle at rest and 4 hr and 24 hr post performance of a plyometric jumping (PLYO) exercise (S = subject) and culture human astrocytes. *Note - the unlabelled lane came from subject 1 baseline, however, its GAPDH level was very low and hence, it has not been commented on in this manuscript. B – Results obtained using a nested PCR approach with Kirrel3 primer set 1 and 2 on biopsy samples from subject 2 from Figure A. C – Additional PCR amplicon (500 nucleotides) was detected using Kirrel3 Primer set 1 from a subset of adult human skeletal muscle biopsies that were obtained from participants one and two days post a downhill run (DHR) exercise bout. D – Schematic of the exon structure of amplicons detected in A and B after excision of the bands from the gels and nucleotide sequencing.
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Fig2: Detection of Kirrel3 splice variants with primer set 1. A – PCR amplicons obtained with Kirrel3 primer set 1 to detect Kirrel3 A. mRNA was isolated from adult human skeletal muscle at rest and 4 hr and 24 hr post performance of a plyometric jumping (PLYO) exercise (S = subject) and culture human astrocytes. *Note - the unlabelled lane came from subject 1 baseline, however, its GAPDH level was very low and hence, it has not been commented on in this manuscript. B – Results obtained using a nested PCR approach with Kirrel3 primer set 1 and 2 on biopsy samples from subject 2 from Figure A. C – Additional PCR amplicon (500 nucleotides) was detected using Kirrel3 Primer set 1 from a subset of adult human skeletal muscle biopsies that were obtained from participants one and two days post a downhill run (DHR) exercise bout. D – Schematic of the exon structure of amplicons detected in A and B after excision of the bands from the gels and nucleotide sequencing.

Mentions: Primer set 1 was specific to Kirrel3 A with a forward primer targeting exon 12 and a reverse primer targeting exon 16 of Kirrel3 A (see Figure 1A for schematic on primer position). The expected amplicon is 391 nucleotides. Two distinct amplicons migrating at approximately 370 and 400 nucleotides were detected in the biopsy taken from subject 1 at 24 hr post plyometric exercise (Figure 2A). In contrast, only the approximately 370 nucleotide amplicon was detectable in the biopsy sample that was taken at the 4 hr post plyometric exercise time point from the same subject (Figure 2A). No biopsy from Subject 2 presented with either the approximately 370 or 400 nucleotide amplicon. Subject 3 had the approximately 370 nucleotide amplicon present at the baseline time point, but not at 4 hr or 24 hr post plyometric exercise (Figure 2A). The quality of cDNA template in all human skeletal muscle samples (except for subject 1 baseline) was confirmed as satisfactory via assessing for GAPDH mRNA expression (Figure 2A). A semi nested PCR was performed with primer sets 1 and 2 to assess if this approach would yield more consistent Kirrel3 mRNA detection. Primer set 2 contained a forward primer that was targeted towards exon 12 of Kirrel3 A with the reverse primer being the same as in primer set 1 (see Figure 1A for schematic of primer location). Three samples from subject 2 were negative for all amplicons in the first analysis. However, in one of these an amplicon of approximately 370 nucleotides was detected at the 4 hr time point using the semi-nested PCR (Figure 2B). In human astrocytes two amplicons were observed with primer set 1 at approximately 370 and 400 nucleotides (Figure 2A) thus matching those observed in human skeletal muscle. A much fainter amplicon was also detected in astrocytes at approximately 500 nucleotides (Figure 2A). We also analysed mRNA extracted from biopsies obtained from a previous study by our research group that investigated the effects of downhill running on skeletal muscle [32]. In some of these samples, an amplicon was detected that matched of the approximate 500 nucleotide amplicon observed in astrocytes (Figure 2C).Figure 2


Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle.

Durcan PJ, Conradie JD, Van deVyver M, Myburgh KH - BMC Physiol. (2014)

Detection of Kirrel3 splice variants with primer set 1. A – PCR amplicons obtained with Kirrel3 primer set 1 to detect Kirrel3 A. mRNA was isolated from adult human skeletal muscle at rest and 4 hr and 24 hr post performance of a plyometric jumping (PLYO) exercise (S = subject) and culture human astrocytes. *Note - the unlabelled lane came from subject 1 baseline, however, its GAPDH level was very low and hence, it has not been commented on in this manuscript. B – Results obtained using a nested PCR approach with Kirrel3 primer set 1 and 2 on biopsy samples from subject 2 from Figure A. C – Additional PCR amplicon (500 nucleotides) was detected using Kirrel3 Primer set 1 from a subset of adult human skeletal muscle biopsies that were obtained from participants one and two days post a downhill run (DHR) exercise bout. D – Schematic of the exon structure of amplicons detected in A and B after excision of the bands from the gels and nucleotide sequencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269076&req=5

Fig2: Detection of Kirrel3 splice variants with primer set 1. A – PCR amplicons obtained with Kirrel3 primer set 1 to detect Kirrel3 A. mRNA was isolated from adult human skeletal muscle at rest and 4 hr and 24 hr post performance of a plyometric jumping (PLYO) exercise (S = subject) and culture human astrocytes. *Note - the unlabelled lane came from subject 1 baseline, however, its GAPDH level was very low and hence, it has not been commented on in this manuscript. B – Results obtained using a nested PCR approach with Kirrel3 primer set 1 and 2 on biopsy samples from subject 2 from Figure A. C – Additional PCR amplicon (500 nucleotides) was detected using Kirrel3 Primer set 1 from a subset of adult human skeletal muscle biopsies that were obtained from participants one and two days post a downhill run (DHR) exercise bout. D – Schematic of the exon structure of amplicons detected in A and B after excision of the bands from the gels and nucleotide sequencing.
Mentions: Primer set 1 was specific to Kirrel3 A with a forward primer targeting exon 12 and a reverse primer targeting exon 16 of Kirrel3 A (see Figure 1A for schematic on primer position). The expected amplicon is 391 nucleotides. Two distinct amplicons migrating at approximately 370 and 400 nucleotides were detected in the biopsy taken from subject 1 at 24 hr post plyometric exercise (Figure 2A). In contrast, only the approximately 370 nucleotide amplicon was detectable in the biopsy sample that was taken at the 4 hr post plyometric exercise time point from the same subject (Figure 2A). No biopsy from Subject 2 presented with either the approximately 370 or 400 nucleotide amplicon. Subject 3 had the approximately 370 nucleotide amplicon present at the baseline time point, but not at 4 hr or 24 hr post plyometric exercise (Figure 2A). The quality of cDNA template in all human skeletal muscle samples (except for subject 1 baseline) was confirmed as satisfactory via assessing for GAPDH mRNA expression (Figure 2A). A semi nested PCR was performed with primer sets 1 and 2 to assess if this approach would yield more consistent Kirrel3 mRNA detection. Primer set 2 contained a forward primer that was targeted towards exon 12 of Kirrel3 A with the reverse primer being the same as in primer set 1 (see Figure 1A for schematic of primer location). Three samples from subject 2 were negative for all amplicons in the first analysis. However, in one of these an amplicon of approximately 370 nucleotides was detected at the 4 hr time point using the semi-nested PCR (Figure 2B). In human astrocytes two amplicons were observed with primer set 1 at approximately 370 and 400 nucleotides (Figure 2A) thus matching those observed in human skeletal muscle. A much fainter amplicon was also detected in astrocytes at approximately 500 nucleotides (Figure 2A). We also analysed mRNA extracted from biopsies obtained from a previous study by our research group that investigated the effects of downhill running on skeletal muscle [32]. In some of these samples, an amplicon was detected that matched of the approximate 500 nucleotide amplicon observed in astrocytes (Figure 2C).Figure 2

Bottom Line: Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance.These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, Stellenbosch University, Private Bag X1 Matieland, 7602, Stellenbosch, South Africa. pdurcan@sun.ac.za.

ABSTRACT

Background: Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.

Results: We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.

Conclusion: The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

Show MeSH
Related in: MedlinePlus