Patrick K. Umeda

Strong expression of foreign genes following direct injection into fish muscle

We report here for the first time direct injection of genes into fish muscle in vivo. Plasmids used contain either SV40 early promoter, rabbit β‐cardiac myosin heavy chain promoter, human MxA promoter of an artificial promoter, fused to a chloramphenicol acetyltransferase (CAT) or β‐galactosidase reporter gene. CAT assays revealed that most gene constructs were highly expressed. Histochemical analysis showed that β‐galactosidase was strongly expressed at the site of injection within muscle fibres. This method provides an excellent system for testing expression of gene constructs, including those of mammalian origin, in fish muscle in vivo and has the potential for fish vaccination.

Conformation of the Regulatory Domain of Cardiac Muscle Troponin C in Its Complex with Cardiac Troponin I

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca2+-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca2+. However, in the presence of bound cardiac TnI (cTnI), activator Ca2+induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC·cTnI complex has properties favorable for the Ca2+-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca2+-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.

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.

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.

Indicators of Delayed Maturation of Rat Heart Treated Prenatally with Dexamethasone

We investigated the effects of prenatal dexamethasone treatment on indicators of cardiac maturation: heart weight/body weight ratios, myosin heavy chain (MHC) expression, cell proliferation, and extracellular matirix. We administered dexamethasone, a synthetic glucocorticoid (approximately 48μg/d, 3-wk slow release pellets), to pregnant rats (n = 8) beginning at 17 d postconception. Control dams were unmanipulated (n= 8). After approximately 4-5 d of dexamethasone exposure, hearts were collected from neonatal rats (12-24 h after birth). The prenatal dexamethasone treatment produced smaller pups with larger heart/body weight ratios, accompanied by a higher proliferative index and a reduction in extracellular matrix in the ventricles (with lowest values in the septal region) compared with control pups. We also report that, although there were no sex differences in body mass or heart and heart/body weight ratios, females had a greater proportion of cells synthesizing DNA in the heart. In addition, ventricles of male pups treated with dexamethasone contained lower levels of α-MHC mRNA, as reflected in a sex by treatment interaction. The changes in each parameter are consistent with delayed maturation. Our findings suggest that exposure to excess glucocorticoids in utero can affect cardiac development in potentially detrimental ways and that assessment of cardiac function should be closely monitored when such circumstances arise.

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.

Pharmacological evidence for Orai channel activation as a source of cardiac abnormal automaticity.

Calcium transport through plasma membrane voltage-independent calcium channels is vital for signaling events in non-excitable and excitable cells. Following up on our earlier work, we tested the hypothesis that this type of calcium transport can disrupt myocardial electromechanical stability. Our Western and immunofluorescence analyses show that left atrial and ventricular myocytes express the Orai1 and the Orai3 calcium channels. Adding the Orai activator 2-aminoethoxydiphenyl borate (2-APB) to the superfusate of rat left atria causes these non-automatic muscles to contract spontaneously and persistently at rates of up to 10 Hz, and to produce normal action potentials from normal resting potentials, all in the absence of external stimulation. 2-APB likewise induces such automatic activity in superfused rat left ventricular papillary muscles, and the EC(50)s at which 2-APB induces this activity in both muscles are similar to the concentrations which activate Orais. Importantly, the voltage-independent calcium channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) suppresses this automaticity with an IC(50) of 11 ± 0.6 μM in left atria and 6 ± 1.6 μM in papillary muscles. 1-(5-Iodonaphthalene-1-sulfonyl)-hexahydro-1,4-diazepine (ML-7), a second voltage-independent calcium channel inhibitor, and two calmodulin inhibitors also prevent 2-APB automaticity while two calmodulin-dependent protein kinase II inhibitors do not. Thus an activator of the Orai calcium channels provokes a novel type of high frequency automaticity in non-automatic heart muscle.

Evidence that 2-aminoethoxydiphenyl borate provokes fibrillation in perfused rat hearts via voltage-independent calcium channels.

We tested whether 2-aminoethoxydiphenyl borate (2-APB) induces arrhythmia in perfused rat hearts and whether this arrhythmia might result from the activation of voltage-independent calcium channels. Rat hearts were Langendorff perfused and beat under sinus rhythm. An isovolumic balloon inserted into the left ventricle was used to record mechanical function while bipolar electrograms were recorded from electrodes sutured to the base and the apex of hearts. Western and immunofluorescence analyses were performed on rat left ventricular protein extracts and left ventricular frozen sections, respectively. Rat ventricular myocytes express Orai 1 and Orai 3, and ventricle also contains the Orai regulator Stim1. Rat hearts (n=5) perfused with Krebs-Henseleit (KH) alone maintained sinus rhythm at 4.8 ± 0.1 Hz and stable mechanical function. By contrast, perfusing hearts (n=5) with (KH+22 μM 2-APB) provoked a period of tachycardic ectopy at rates of up to 10.8 ± 0.2 Hz. As perfusion with (KH+22 μM 2-APB) continued, the rate of spontaneous ventricular depolarization increased to 21.8 ± 1.2 Hz and became disorganized. Heart mechanical function collapsed as developed pressure decreased from 87 ± 8.8 to 3.5 ± 1.9 mm Hg. Flow rate did not change between normal (16.6 ± 0.9 ml/min) and fibrillating (17.4 ± 0.8 ml/min) hearts. The addition of 20 μM 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) to (KH+22 μM 2-APB) perfusates (n=4) restored sinus rhythm and heart mechanical output. These data indicate that activating myocardial voltage-independent calcium channels, possibly the Orais, may be a novel cause of ventricular arrhythmia.

Gene regulation mediating fiber-type transformation in skeletal muscle cells is partly glucose- and ChREBP-dependent.

Adaptations in the oxidative capacity of skeletal muscle cells can occur under several physiological or pathological conditions. We investigated the effect of increasing extracellular glucose concentration on the expression of markers of energy metabolism in primary skeletal muscle cells and the C2C12 muscle cell line. Growth of myotubes in 25mM glucose (high glucose, HG) compared with 5.55mM led to increases in the expression and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a marker of glycolytic energy metabolism, while oxidative markers peroxisome proliferator-activated receptor γ coactivator 1α and citrate synthase decreased. HG induced metabolic adaptations as are seen during a slow-to-fast fiber transformation. Furthermore, HG increased fast myosin heavy chain (MHC) IId/x but did not change slow MHCI/β expression. Protein phosphatase 2A (PP2A) was shown to mediate the effects of HG on GAPDH and MHCIId/x. Carbohydrate response element-binding protein (ChREBP), a glucose-dependent transcription factor downstream of PP2A, partially mediated the effects of glucose on metabolic markers. The glucose-induced increase in PP2A activity was associated with an increase in p38 mitogen-activated protein kinase activity, which presumably mediates the increase in MHCIId/x promoter activity. Liver X receptor, another possible mediator of glucose effects, induced only an incomplete metabolic shift, mainly increasing the expression of the glycolytic marker. Taken together, HG induces a partial slow-to-fast transformation comprising metabolic enzymes together with an increased expression of MHCIId/x. This work demonstrates a functional role for ChREBP in determining the metabolic type of muscle fibers and highlights the importance of glucose as a signaling molecule in muscle.

The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor.

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromasomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is ∼45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, ∼190 kDa; human, ∼200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions. In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.