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. 2015 Oct 29;6(10):e1944.
doi: 10.1038/cddis.2015.306.

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells

W Mughal  1   2 , L Nguyen  2 , S Pustylnik  3 , S C da Silva Rosa  1   2 , S Piotrowski  1   2 , D Chapman  2   4 , M Du  3 , N S Alli  3 , J Grigull  3   5 , A J Halayko  6   7 , M Aliani  8 , M K Topham  9 , R M Epand  10 , G M Hatch  2   11 , T J Pereira  2   11 , S Kereliuk  2   11 , J C McDermott  3 , C Rampitsch  12 , V W Dolinsky  2   11 , J W Gordon  1   2   4
Affiliations

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells

W Mughal et al. Cell Death Dis. .

Abstract

Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PKCδ inhibits the cooperation between MEF2 and SRF by direct phosphorylation. (a) C2C12 myoblasts were transfected with MEF2A, MEF2C, or SRF, as indicated. Following recovery, cells were differentiated in low serum media for 5 days and harvested for RNA. Quantitative PCR assays were performed using the ΔΔCT method, where RNU6 was used as an internal control. (b) C2C12 cells were transfected with shRNAs targeting MEF2C (shMEF2C) or SRF (shSRF), as indicated. Following 5 days of differentiation, cells were harvested and assayed as described above. (c) Schematic demonstrating the conservation surrounding the MEF2 threonine-20 and SRF threonine-160 phosphorylation motif. (d–f) SIM scans of the wild-type peptide (d and e) spanning the MADS-box motif of MEF2A. The unphosphorylated peptide (left) has 442 m/z, while the putative phosphorylation (right in b) showing an increased m/z of 20 that corresponds to PO3 (M=80.00 Da). (f) A SIM scan of a mutated peptide where threonine-20 is replaced with alanine is shown. On the right, phosphorylation of this mutate peptide is negligible at the predicted m/z that corresponds to the addition of a PO3 (M=80.00 Da). Both peptides are quadruply charged (z=4+). Data are represented as mean±S.E.M. *P<0.05 compared with control
Figure 2
Figure 2
Identification of threonine-20 as a putative PKCδ phosphorylation residue. (a) Inset: SIM scan of native peptide (442.72, z=4+) and putative phospho-peptide (462.48, z=4+), as shown in Figure 1. The mass shift of +19.76 m/z is consistent with that of a quadruply charged peptide ion. Both threonine-20 and threonine-22 are possible sites of phosphorylation. Main figure: CID MS2 spectrum of the native peptide showing a prominent neutral loss ion at 438.42 m/z with z=4+. This represents a −24.56 Da shift consistent with a neutral loss of phosphate from threonine-20 or threonine-22 of the native peptide. (b) Fragmentation of the phospho-peptide (462.48, z=4+) by ETD produced a near-complete c and z ion series with some y ions also present. Analysis of this fragmentation spectra confirmed that threonine-20 is the preferred phosphorylation residue. Inset: Schematic illustrating the z and c ions detected by ETD. (c) SIM scan of a peptide spanning the MADS-box motif of SRF (amino acids 154–167). The unphosphorylated peptide (left) has 581.58 m/z (z=3+), while the putative phosphorylation (right in a, m/z of 608.22) showing an increased m/z of 26.64 that corresponds to PO3 (M=80.00 Da). (d) CID MS2 spectrum of the phospho-peptide in (c) with m/z of 608.22, showing a prominent neutral loss ion at 575.5 m/z with z=3+. (e) ETD MS2 spectrum of SRF confirming phosphorylation of threonine-160
Figure 3
Figure 3
Mutational analysis of threonine-20. (a) HEK293 cells were transfected with MEF2A (MEF2 wt), or a plasmid containing MEF2A where threonine-20 is mutated to a neutral alanine (T20A) or phospho-mimetic aspartic acid (T20D), as indicated, and subjected to western blot (above). 10T1/2 cells were transfected, as above, along with MEF2-driven luciferase reporter gene (MEF2-luc). Extracts were subjected to luciferase assay, where β-galactosidase assay was used to correct for transfection efficiency (below). All assays were done in triplicate. (b) C2C12 myoblasts were transfected with MEF2C, SRF, or PKCδ, as indicated. Following recovery, cells were differentiated in low serum media for 5 days, harvested for RNA, subjected to qPCR assay. (c and d) Following 5 days of differentiation, C2C12 myotubes were treated with 200 μM palmitate conjugated to 2% albumin in low glucose media overnight. Control cells were treated with 2% albumin alone. Myotubes were harvested for RNA or protein, and assayed by immunoblot (c) or qPCR (d). (e) C2C12 myoblasts were transfected with MEF2-VP16 fusion where threonine-20 is mutated to a neutral alanine [MEF2(T20A)-VP16], or control plasmid. Cell was differentiated for 5 days and treated with 200 μM palmitate, as indicated. Myotubes were harvested for RNA and assayed by qPCR. (f) Wild-type (WT) or diacylglycerol kinase-δ knock-out (DGKδ KO) embryonic fibroblasts treated with palmitate, as described above. Extracts were immunoprecipitated (IP) with SRF antibody and probed using an antibody that recognizes phospho-serines/threonines with arginines at the –3 position (RXXpS/T) or immunoblotted (IB), as indicated. PKCδ=catalytic fragment of PKCδ. Data are represented as mean±S.E.M. *P<0.05 compared with control. **P<0.05 compared with palmitate treatment
Figure 4
Figure 4
Palmitate-induced PKCδ activation regulates mitochondrial membrane potential through miR-133a. (a) C2C12 myotubes were differentiated for 5 days (above), or hASMCs were differentiated for 2 days (below), and treated with 200 μM palmitate conjugated to 2% albumin in low glucose media with or without rottlerin (5 μM) overnight. Control cells were treated with 2% albumin alone. Cells were stained with TMRM, Hoechst, and MitoView Green, as indicated, and imaged by standard fluorescence techniques (20 × for C2C12s; 40 × for hASMCs). (b–d) Fluorescent intensities from myotubes in (a) were quantified using ImageJ software (NIH). (e) hASMCs were treated as described in (a), harvested for RNA and subjected to qPCR analysis for miR-133a. (f) Differentiated C2C12 myotubes were treated as in (a). Protein extracts were immunoprecipitated (IP) with an MEF2C antibody, and probed using an antibody that recognizes phospho-serines/threonines with arginines at the –5 and –3 positions (RXRXXpS/T), and immunoblotted (IB), as indicated. (g) C2C12 cells were transfected with a miR-133a inhibitor oligonucleotide or a control oligonucleotide. Cells were differentiated for 5 days and imaged at 20 × with TMRM, Hoechst, or MitoTracker Red CMXRos, as indicated. (h) Quantification of myotube fluorescence in (g). (i) H9c2 myoblasts were transfected with MEF2-T20A-VP16 (T20A-VP16) or miR-133a. Following 2 days of differentiation, cells were treated with 200 μM palmitate conjugated to 2% albumin or 2% albumin alone as a control. CMV-GFP was included to visualize transfected cells. Data are represented as mean±S.E.M. *P<0.05 compared with control
Figure 5
Figure 5
miR-133a regulates mitochondrial membrane potential through Nix. (a) Sequence alignment of mouse miR-133a and the 3' UTR of Nix. (b) H9c2 myoblasts were transfected with miR-133a or a shRNA targeting Nix (sh-Nix). Following protein extraction, samples were immunoblotted as indicated. (c) H9c2 cells were transfected with a miR-133a inhibitor (50 μM) or a scrambled control oligonucleotide. Extracts were immunoblotted, as indicated. (d) C2C12 cells were transfected with the catalytic isoform of PKCδ. Extracts were immunoblotted, as indicated. (e) Five-day differentiated C2C12 cells were treated with 200 μM palmitate conjugated to 2% albumin, or 5 μM rottlerin overnight, as indicated. Protein extracts were immunoblotted, as indicated. (f) C2C12 myoblasts were transfected with Nix, or an empty vector control. CMV-GFP was included to identify transfected cells. Cells were stained with TMRM and Hoechst and imaged by standard fluorescence microscopy. Arrows indicate GFP-positive cells. (g) H9c2 cells were transfected with Nix and Bcl-2, as indicated. CMV-dsRed was used to identify transfected cells. Cells were stained with calcein-AM with cobalt chloride (5 μM) to assess PTP opening. (h) H9c2 cells were transfected with shNix or a scrambled control shRNA. Following recovery, cells were treated with 200 μM palmitate conjugated to 2% albumin overnight, and stained with TMRM and Hoechst to evaluate mitochondrial membrane potential (above) or with calcein-AM with cobalt chloride (5 μM) to assess PTP opening (below). (i) Quantification of myotube fluorescence in (h). Data are represented as mean±S.E.M. *P<0.05 compared with control
Figure 6
Figure 6
Evaluation of mitochondrial respiration and glucose uptake. (a) Differentiated H9c2 cells were treated overnight with 200 μM palmitate conjugated to 2% albumin in low glucose media. Control cells were treated with 2% albumin alone. Oxygen consumption rate (OCR) was evaluated on a Seahorse XF-24. To evaluate mitochondrial function, cells were injected with oligomycin (1 μM) (a), FCCP (1 μM) (b), and antimycin A (1 μM) and rotenone (1 μM) (c). (b) H9c2 cells were transfected with a miR-133a mimic (50 μM) or a scrambled control oligonucleotide. Following recovery, OCR evaluated as in (a). (c and d) Calculated respiration rates from (a) and (b), respectively. (e and f) H9c2 cells were transfected as in (b) and treated as in (a). Insulin stimulated uptake (10 nM) was determined by 2NBDG fluorescence and quantified in (f). Data analyzed by two-way ANOVA, and represented as mean±S.E.M. #P<0.05 between groups, *P<0.05 compared with control
Figure 7
Figure 7
miR-133a expression in vivo. (a) Metabolomics analysis of 44 species of diacylglycerols from soleus muscle excised from LF, or HFS, and a normal pregnancy (Lean Dam), or gestational diabetes (GDM Dam) during development, as indicated. Five highly abundant diacylglycerols are highlighted in the chart. Blue=low abundance, yellow=medium low abundance, orange=medium high abundance, red=high abundance. (b–e) Rat soleus muscle or heart tissue was excised and total RNA was extracted, from rodents treated as described in (a). qPCR analysis was performed using the ΔΔCT method, where RNU6 was used as an internal control for miR-133a, and β-actin was used as a control for PCG-1α and mitofusin-2. (f) Protein extracts from rat soleus muscle were subjected to immunoblot analysis, as indicated. PKCδ catalytic fragment (PKCδ c.f.). Data are represented as mean±S.E.M. *P<0.05 compared with control
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