Renate J. Scheibe

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture.

The addition of cyclosporin A (500 ng ml−1) ‐ an inhibitor of the Ca2+‐calmodulin‐regulated serine/threonine phosphatase calcineurin ‐ to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 × 10−7m) was added to the medium, a partial fast‐to‐slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast‐to‐slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin‐regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin‐dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore‐ and electrostimulation‐induced fast‐to‐slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.

Fast-to-slow transformation and nuclear import/export kinetics of the transcription factor NFATc1 during electrostimulation of rabbit muscle cells in culture

Contractile activity imposed by chronic electrical stimulation of a primary skeletal muscle cell culture grown on microcarriers over several days led to an increase of slow myosin heavy chain I (MHCI) and a decrease of fast MHCII expression at mRNA and protein levels, indicating an ongoing fast‐to‐slow transformation. Only patterns with periods of continuous stimulation of > 5 min in a 45 min cycle were capable of inducing a fibre type transformation, and this was independent of the applied stimulation frequency over the range 1‐10 Hz. We have shown before that the calcineurin‐NFATc1 signalling pathway is indispensable in mediating MHCI upregulation during fibre type transformation. Therefore, subcellular localization of NFATc1 was studied immunocytochemically. This revealed that only one stimulation train lasting for > 5 min was sufficient to induce nuclear import of this factor, which was about complete after 20 min of continuous stimulation. For both induction of NFATc1 import and MHCI mRNA upregulation, the minimum stimulation interval of > 5 min was sufficient and stimulation frequency was not crucial between 1 and 10 Hz. Repetition of stimulation cycles, with pauses (< 40 min) shorter than the time required for complete export of NFATc1, led to an accumulation of NFATc1 in the nuclei with each cycle and thus to an amplification of the transformation signal during extended periods of electrostimulation. The temporal behaviour of NFATc import/export appears to determine the effectiveness of various electrostimulation protocols in inducing fast‐to‐slow fibre transformation.

Activation of the β myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway

Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast‐to‐slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T‐cells (NFAT) isoform c1, whose dephosphorylation by the calcium/calmodulin‐dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast‐type MyHCII protein undergo fast‐to‐slow transformation following calcium‐ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) β promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCβ promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc1, myocyte enhancer factor‐2D (MEF‐2D) and the myogenic transcription factor MyoD transactivated the MyHCβ promoter in calcium‐ionophore‐treated myotubes in a calcineurin‐dependent manner. To elucidate the mechanism involved in regulating MyHCβ gene expression, we analyzed the −2.4‐kb MyHCβ promoter construct for cis‐regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcoIP) assays, we demonstrated calcium‐ionophore‐induced binding of NFATc1 to a NFAT consensus site adjacent to a MyoD‐binding E‐box. At their respective binding sites, both NFATc1 and MyoD recruited the transcriptional coactivator p300, and in turn, MEF‐2D bound to the MyoD complex. The calcium‐ionophore‐induced effects on the MyHCβ promoter were shown to be calcineurin‐dependent. Together, our findings demonstrate calcium‐ionophore‐induced activation of the β MyHC promoter by NFATc1, MyoD, MEF‐2D, and p300 in a calcineurin‐dependent manner. J. Cell. Physiol. 211: 138–148, 2007.

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level

The adult fast character and a Ca2+‐inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. No non‐adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. The data show that a fast‐to‐slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3‐phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow‐to‐fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression.

Expression of Membrane-bound Carbonic Anhydrases IV, IX, and XIV in the Mouse Heart

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-α-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/β gene expression via acetylation of nuclear factor of activated T cells c1

The nuclear factor of activated T-cells (NFAT) c1 has been shown to be essential for Ca2+-dependent upregulation of myosin heavy chain (MyHC) I/β expression during skeletal muscle fiber type transformation. Here, we report activation of extracellular signal-regulated kinase (ERK) 1/2 in Ca2+-ionophore-treated C2C12 myotubes and electrostimulated soleus muscle. Activated ERK1/2 enhanced NFATc1-dependent upregulation of a −2.4 kb MyHCI/β promoter construct without affecting subcellular localization of endogenous NFATc1. Instead, ERK1/2-augmented phosphorylation of transcriptional coactivator p300, promoted its recruitment to NFATc1 and increased NFATc1–DNA binding to a NFAT site of the MyHCI/β promoter. In line, inhibition of ERK1/2 signaling abolished the effects of p300. Comparison between wild-type p300 and an acetyltransferase-deficient mutant (p300DY) indicated increased NFATc1–DNA binding as a consequence of p300-mediated acetylation of NFATc1. Activation of the MyHCI/β promoter by p300 depends on two conserved acetylation sites in NFATc1, which affect DNA binding and transcriptional stimulation. NFATc1 acetylation occurred in Ca2+-ionophore treated C2C12 myotubes or electrostimulated soleus. Finally, endogenous MyHCI/β gene expression in C2C12 myotubes was strongly inhibited by p300DY and a mutant deficient in ERK phosphorylation sites. In conclusion, ERK1/2-mediated phosphorylation of p300 is crucial for enhancing NFATc1 transactivation function by acetylation, which is essential for Ca2+-induced MyHCI/β expression.

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

The activity of membrane‐bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL‐60 myeloblastic leukemia cells was studied at physiological pH using p‐nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4–8.5. The value of apparent Km for pNPP in P19 and HL‐60 cells was 120 μM. Hydrolytic activity of the ecto‐enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (Ki P19 = 57 μM; Ki HL‐60 = 50 μM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL‐60 cells, induced levamisole‐sensitive ecto‐phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto‐kinase activity, a 98‐kDa membrane protein in P19 cells was found to be sensitive to ecto‐ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole‐sensitive dephosphorylation of the membrane phosphoproteins, while (R)‐(−)‐epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole‐sensitive phosphohydrolase activity on the cell surface is consistent with ecto‐ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto‐ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL‐60 myeloblastic leukemia cells. J. Cell. Biochem. 76:420–436, 2000.

T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

Mitogen-Activated Protein Kinase-Activated Protein Kinases 2 and 3 Regulate SERCA2a Expression and Fiber Type Composition To Modulate Skeletal Muscle and Cardiomyocyte Function

ABSTRACT The mitogen-activated protein kinase (MAPK)-activated protein kinases 2 and 3 (MK2/3) represent protein kinases downstream of the p38 MAPK. Using MK2/3 double-knockout (MK2/3−/−) mice, we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α). Using reporter gene and electrophoretic gel mobility shift assays, we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy-chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) gene. Elevated SERCA2a gene expression caused by a decreased ratio of transcription factor Egr-1 to Sp1 was associated with accelerated relaxation and enhanced contractility in MK2/3−/− cardiomyocytes, concomitant with improved force parameters in MK2/3−/− soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both reduced fatigability and enhanced force in MK2/3−/− mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.

The p38α/β Mitogen-activated Protein Kinases Mediate Recruitment of CREB-binding Protein to Preserve Fast Myosin Heavy Chain IId/x Gene Activity in Myotubes

In skeletal muscle, the transformation of fast into slow fiber type is accompanied by shifts in fiber type-specific gene expression that includes down-regulation of the adult fast fiber myosin heavy chain IId/x (MyHCIId/x) gene. Here, we report that the mitogen-activated protein kinases (MAPKs) p38α/β regulate MyHCIId/x gene expression. Electrical stimulation of rabbit skeletal muscle cells with a slow fiber type activity pattern and treatment of C2C12 myotubes with Ca2+-ionophore inhibited p38α/β MAPKs and reduced fast fiber type MyHC protein expression and promoter activity. Pharmacological inhibition of p38α/β also down-regulated MyHCII gene expression. In controls, binding of the myocyte enhancer factor-2 (MEF-2) isoforms C and D as a heterodimer to a proximal consensus site within the MyHCIId/x promoter and recruitment of a transcriptional coactivator, the CREB-binding protein CBP, were observed. Overexpression of wild type MEF-2C but not of a MEF-2C mutant that cannot be phosphorylated by p38 induced promoter activity. Mutation of the MEF-2-binding site decreased the inducing effect of overexpressed CBP. Inhibition of p38α/β MAPKs abolished CBP binding, whereas enforced induction of p38 by activated MAPK kinase 6 (MKK6EE) enhanced binding of CBP and increased promoter activity. Furthermore, knockdown of endogenous CBP by RNA interference eliminated promoter activation by MEF-2C or MKK6EE. In electrical stimulated and Ca2+-ionophore-treated myotubes, CBP was absent in complex formation at that site. Taken together, the data indicate that p38α/β MAPKs-mediated coactivator recruitment at a proximal MEF-2 site is important for MyHCIId/x gene regulation in skeletal muscle.