Effects of new polymorphisms in the bovine myocyte enhancer factor 2D (MEF2D) gene on the expression rates of the longissimus dorsi muscle

Myocyte enhancer factor 2D (MEF2D), a product of the MEF2D gene, belongs to the myocyte enhancer factor 2 (MEF2) protein family which is involved in vertebrate skeletal muscle development and differentiation during myogenesis. The aim of the present study was to search for polymorphisms in the bovine MEF2D gene and to analyze their effect on MEF2D mRNA and on protein expression levels in the longissimus dorsi muscle of Polish Holstein–Friesian cattle. Overall, three novel variations, namely, insertion/deletion g.−818_−814AGCCG and g.−211C<A transversion in the promoter region as well as g.7C<T transition in the 5′untranslated region (5′UTR), were identified by DNA sequencing. A total, 375 unrelated bulls belonging to six different cattle breeds were genotyped, and three combined genotypes (Ins-C-C/Ins-C-C, Del-A-T/Del-A-T and Ins-C-C/Del-A-T) were determined. The frequency of the combined genotype Ins-C-C/Ins-C-C and Del-A-T/Del-A-T was varied between the breeds and the average frequency was 0.521 and 0.037, respectively. Expression analysis showed that the MEF2D variants were highly correlated with MEF2D mRNA and protein levels in the longissimus dorsi muscle of Polish Holstein–Friesian bulls carrying the three different combined genotypes. The highest MEF2D mRNA and protein levels were estimated in the muscle of bulls with the Ins-C-C/Ins-C-C homozygous genotype as compared to the Del-A-T/Del-A-T homozygotes (P < 0.01) and Ins-C-C/Del-A-T heterozygotes (P < 0.05). A preliminary association study showed no significant differences in the carcass quality traits between bulls with various MEF2D combined genotypes in the investigated population of Polish Holstein–Friesian cattle

Body mass (mean+sem) for <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading followed by seven days of reloading (n = 6 per genotype and day).

<p>Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

Arbitrary concentrations (mean+sem) of MyoD, Myf5 and Myogenin mRNA in the <i>B. femoris</i> muscles of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading (n = 6 per genotype and day).

<p>Asterisks denote differences between genotypes on days shown (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 and ***<i>P</i>&lt;0.001). Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

The amount of 4E-BP1 bound to eIF4E.

<p>(A) Representative western blots of 4E-BP1 bound to eIF4E and (B) the ratio (mean+sem) of 4E-BP1 bound to isolated eIF4E purified by an m⁷GTP-sepharose pull-down assay in the <i>gastrocnemius</i> muscle of <i>Mstn</i>(−/−) and wild-type mice (n = 6 per genotype and day) before and after two days of unloading. There were main effects of day (<i>P</i>&lt;0.05) and genotype (<i>P</i>&lt;0.05), but no day × genotype interaction.</p

Changes in the composition of MyHC and the cross-sectional area of muscle fibres.

<p>(A) Representative gels stained with coomassie blue showing the myosin heavy chain (MyHC) protein isoforms in the <i>B. femoris</i> muscle of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading. A mixture of 1∶1 <i>soleus</i> and <i>Extensor digitorum longus</i> served as a ladder. (B) The change (mean+sem) in the relative abundance of type IIb MyHC protein in the <i>B. femoris</i> muscles is shown for <i>Mstn</i>(−/−) and wild-type mice (n = 6 per genotype and day) during seven days of unloading and seven days of reloading. There were main effects of day (<i>P</i>&lt;0.001) and genotype (<i>P</i>&lt;0.01), but no day×genotype interaction. Asterisks denote significant differences between genotypes (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01). (C) Cross-sectional area (mean+sem) of myofibres in the <i>gastrocnemius</i> muscle of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading. The cross-sectional area was significantly reduced in both genotypes at d7 (P&lt;0.05), before being restored to pre-unloading areas at d14. Asterisks denote significant differences between genotypes (***<i>P</i>&lt;0.001). Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

Arbitrary concentrations (mean+sem) of LC3b, Gabarapl1 and Atg4b mRNA in the <i>B. femoris</i> muscles of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading (n = 6 per genotype and day).

<p>The asterisks denote differences between genotypes on days shown (**<i>P</i>&lt;0.01, ***<i>P</i>&lt;0.01). Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

Muscle mass (mean+sem) expressed as a percent of the initial body mass at d0 for <i>Mstn</i>(−/−) and wild-type mice at days 0, 2 and 7 of unloading and days 8, 10 and 14 of reloading.

<p>The asterisks denote differences from d0 within genotype at the days noted (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 and ***<i>P</i>&lt;0.001). The asterisks (**<i>P</i>&lt;0.01) in the data for soleus indicates that muscle mass has been lost equally from both genotypes at d7. EDL = <i>Extensor digitorum longus</i>, Gast = <i>gastrocnemius</i>, Quad = <i>Quadriceps femoris</i>.</p