Activation of NF-κB is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes

The transcription factor NF-κB regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-κB in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-κB activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-κB and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-κB was inhibited by expression of a “supersuppressor” IκBα mutant that is resistant to stimulation-induced degradation and a dominant negative IκB kinase (IKKβ) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced IκBα degradation in an IKK-dependent manner, suggesting that NF-κB is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor IκBα mutant or the dominant negative IKKβ mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-κB itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-κB plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

In human patients, a wide range of mutations in keratin (K) 5 or K14 lead to the blistering skin disorder epidermolysis bullosa simplex. Given that K14 deficiency does not lead to the ablation of a basal cell cytoskeleton because of a compensatory role of K15, we have investigated the requirement for the keratin cytoskeleton in basal cells by inactivating the K5 gene in mice. We report that the K5−/− mice die shortly after birth, lack keratin filaments in the basal epidermis, and are more severely affected than K14−/− mice. In contrast to the K14−/− mice, we detected a strong induction of the wound-healing keratin K6 in the suprabasal epidermis of cytolyzed areas of postnatal K5−/− mice. In addition, K5 and K14 mice differed with respect to tongue lesions. Moreover, we show that in the absence of K5 and other type II keratins, residual K14 and K15 aggregated along hemidesmosomes, demonstrating that individual keratins without a partner are stable in vivo. Our data indicate that K5 may be the natural partner of K15 and K17. We suggest that K5 null mutations may be lethal in human epidermolysis bullosa simplex patients.

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.

The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor.

More than 30 years ago, Brambell published the hypothesis bearing his name [Brambell, F. W. R., Hemmings, W. A. & Morris, 1. C. (1964) Nature (London) 203, 1352-1355] that remains as the cornerstone for thinking on IgG catabolism. To explain the long survival of IgG relative to other plasma proteins and its pattern of increased fractional catabolism with high concentrations of IgG, Brambell postulated specific IgG "protection receptors" (FcRp) that would bind IgG in pinocytic vacuoles and redirect its transport to the circulation; when the FcRp was saturated, the excess unbound IgG then would pass to unrestricted lysosomal catabolism. Brambell subsequently postulated the neonatal gut transport receptor (FcRn) and showed its similar saturable character. FcRn was recently cloned but FcRp has not been identified. Using a genetic knockout that disrupts the FcRn and intestinal IgG transport, we show that this lesion also disrupts the IgG protection receptor, supporting the identity of these two receptors. IgG catabolism was 10-fold faster and IgG levels were correspondingly lower in mutant than in wild-type mice, whereas IgA was the same between groups, demonstrating the specific effects on the IgG system. Disruption of the FcRp in the mutant mice was also shown to abrogate the classical pattern of decreased IgG survival with higher IgC concentration. Finally, studies in normal mice with monomeric antigen-antibody complexes showed differential catabolism in which antigen dissociates in the endosome and passes to the lysosome, whereas the associated antibody is returned to circulation; in mutant mice, differential catabolism was lost and the whole complex cleared at the same accelerated rate as albumin, showing the central role of the FcRp to the differential catabolism mechanism. Thus, the same receptor protein that mediates the function of the FcRn transiently in the neonate is shown to have its functionally dominant expression as the FcRp throughout life, resolving a longstanding mystery of the identity of the receptor for the protection of IgG. This result also identifies an important new member of the class of recycling surface receptors and enables the design of protein adaptations to exploit this mechanism to improve survivals of other therapeutic proteins in vivo.

Axotomized neonatal motoneurons overexpressing the bcl2 proto-oncogene retain functional electrophysiological properties.

Bcl2 overexpression prevents axotomy-induced neuronal death of neonatal facial motoneurons, as defined by morphological criteria. However, the functional properties of these surviving lesioned transgenic neurons are unknown. Using transgenic mice overexpressing the protein Bcl2, we have investigated the bioelectrical properties of transgenic facial motoneurons from 7 to 20 days after neonatal unilateral axotomy using brain-stem slices and whole cell patch-clamp recording. Nonaxotomized facial motoneurons from wild-type and transgenic mice had similar properties; they had an input resistance of 38 +/- 6 M omega and fired repetitively after injection of positive current pulses. When cells were voltage-clamped at or near their resting membrane potential, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), or vasopressin generated sustained inward currents. In transgenic axotomized mice, facial motoneurons could be found located ipsilaterally to the lesion; they had an input resistance of 150 +/- 30 M omega, indicating that they were smaller in size, fired repetitively, and were also responsive to AMPA, NMDA, and vasopressin. Morphological measurements achieved 1 week after the lesion have shown that application of brain-derived neurotrophic factor prevented the reduction in size of axotomized transgenic motoneurons. These data indicate that Bcl2 not only prevents morphological apoptotic death of axotomized neonatal transgenic motoneurons but also permits motoneurons to conserve functional electrophysiological properties.

The estrogen receptor locus is associated with a major gene influencing litter size in pigs.

Identification of individual major genes affecting quantitative traits in livestock species has been limited to date. By using a candidate gene approach and a divergent breed cross involving the Chinese Meishan pig, we have shown that a specific allele of the estrogen receptor (ER) locus is associated with increased litter size. Female pigs from synthetic lines with a 50% Meishan background that were homozygous for this beneficial allele produced 2.3 more pigs in first parities and 1.5 more pigs averaged over all parities than females from the same synthetic lines and homozygous for the undesirable allele. This beneficial ER allele was also found in pigs with Large White breed ancestory. Analysis of females with Large White breed background showed an advantage for females homozygous for the beneficial allele as compared to females homozygous for the other allele of more than 1 total pig born. Analyses of growth performance test records detected no significant unfavorable associations of the beneficial allele with growth and developmental traits. Mapping of the ER gene demonstrated that the closest known genes or markers were 3 centimorgans from ER. To our knowledge, one of these, superoxide dismutase gene (SOD2), was mapped for the first time in the pig. Analysis of ER and these linked markers indicated that ER is the best predictor of litter size differences. Introgression of the beneficial allele into commercial pig breeding lines, in which the allele was not present, and marker-assisted selection for the beneficial allele in lines with Meishan and Large White background have begun.

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Genetic variation in vulnerability to the behavioral effects of neonatal hippocampal damage in rats.

We explored how two independent variables, one genetic (i.e., specific rat strains) and another environmental (i.e., a developmental excitotoxic hippocampal lesion), contribute to phenotypic variation. Sprague-Dawley (SD), Fischer 344 (F344), and Lewis rats underwent two grades of neonatal excitotoxic damage: small and large ventral hippocampal (SVH and LVH) lesions. Locomotion was tested before puberty [postnatal day 35 (P35)] and after puberty (P56) following exposure to a novel environment or administration of amphetamine. The behavioral effects were strain- and lesion-specific. As shown previously, SD rats with LVH lesions displayed enhanced spontaneous and amphetamine-induced locomotion as compared with controls at P56, but not at P35. SVH lesions in SD rats had no effect at any age. In F344 rats with LVH lesions, enhanced spontaneous and amphetamine-induced locomotion appeared early (P35) and was exaggerated at P56. SVH lesions in F344 rats resulted in a pattern of effects analogous to LVH lesions in SD rats--i.e., postpubertal onset of hyperlocomotion (P56). In Lewis rats, LVH lesions had no significant effect on novelty- or amphetamine-induced locomotion at any age. These data show that the degree of genetic predisposition and the extent of early induced hippocampal defect contribute to the particular pattern of behavioral outcome. These results may have implications for modeling interactions of genetic and environmental factors involved in schizophrenia, a disorder characterized by phenotypic heterogeneity, genetic predisposition, a developmental hippocampal abnormality, and vulnerability to environmental stress.

Aod1, the immunoregulatory locus controlling abrogation of tolerance in neonatal thymectomy-induced autoimmune ovarian dysgenesis, maps to mouse chromosome 16.

Mice thymectomized at three days of age (D3Tx) develop during adulthood a variety of organ-specific autoimmune diseases, including autoimmune ovarian dysgenesis (AOD). The phenotypic spectrum of AOD is characterized by the development of anti-ovarian autoantibodies, oophoritis, and atrophy. The D3Tx model of AOD is unique in that disease induction depends exclusively on perturbation of the normal developing immune system, is T-cell-mediated, and is strain specific. For example, D3Tx A/J mice are highly susceptible to AOD, whereas C57BL/6J mice are resistant. After D3Tx, self ovarian antigens, expressed at physiological levels, trigger an autoimmune response capable of eliciting disease. The D3Tx model provides, therefore, the opportunity to focus on the mechanisms of self-tolerance that are relevant to disease pathogenesis. Previous studies indicate that the principal mechanisms involved in AOD susceptibility are genetically controlled and govern developmental processes associated with the induction and maintenance of peripheral tolerance. We report here the mapping of the Aod1 locus to mouse chromosome 16 within a region encoding several loci of immunologic relevance, including scid, Igl1, VpreB, Igll, Igl1r, Mtv6 (Mls-3), Ly-7, Ifnar, and Ifgt.

Neonatal lethality associated with respiratory distress in mice lacking cytochrome P450 1A2.

Cytochrome P450 1A2 (CYP1A2) is a constitutively expressed hepatic enzyme that is highly conserved among mammals. This protein is primarily involved in oxidative metabolism of xenobiotics and is capable of metabolically activating numerous procarcinogens including aflatoxin B1, arylamines, heterocyclic amine food mutagens, and polycylic aromatic hydrocarbons. Expression of CYP1A2 is induced after exposure to certain aromatic hydrocarbons (i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin). Direct evidence for a role of CYP1A2 in any physiological or developmental pathway has not been documented. We now demonstrate that mice homozygous for a targeted mutation in the Cyp1a-2 gene are nonviable. Lethality occurs shortly after birth with symptoms of severe respiratory distress. Mutant neonates display impaired respiratory function associated with histological signs of lung immaturity, lack of air in alveoli at birth, and changes in expression of surfactant apoprotein in alveolar type II cells. The penetrance of the phenotype is not complete (19 mutants survived to adulthood out of 599 mice). Surviving animals, although lacking expression of CYP1A2, appear to be normal and are able to reproduce. These findings establish that CYP1A2 is critical for neonatal survival by influencing the physiology of respiration in neonates, thus offering etiological insights for neonatal respiratory distress syndrome.

Invariance and restriction toward a limited set of self-antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults.

Analysis of the reactivity of IgM with self-antigens in tissues by a quantitative immunoblotting technique showed striking invariance among newborns in the human and in the mouse. The self-reactive repertoire of IgM of adults was also markedly conserved; it comprised most anti-self reactivities that prevailed among neonates. Multivariate analysis confirmed the homogeneity of IgM repertoires of neonates toward self- and non-self-antigens. Multivariate analysis discriminated between newborn and adult repertoires for reactivity with two of five sources of self-proteins and with non-self-antigens. Our observations support the concept that naturally activated B lymphocytes are selected early in development and throughout life for reactivity with a restricted set of self-antigens.

Free, long-chain, polyunsaturated fatty acids reduce membrane electrical excitability in neonatal rat cardiac myocytes.

Because previous studies showed that polyunsaturated fatty acids can reduce the contraction rate of spontaneously beating heart cells and have antiarrhythmic effects, we examined the effects of the fatty acids on the electrophysiology of the cardiac cycle in isolated neonatal rat cardiac myocytes. Exposure of cardiomyocytes to 10 microM eicosapentaenoic acid for 2-5 min markedly increased the strength of the depolarizing current required to elicit an action potential (from 18.0 +/- 2.4 pA to 26.8 +/- 2.7 pA, P < 0.01) and the cycle length of excitability (from 525 ms to 1225 ms, delta = 700 +/- 212, P < 0.05). These changes were due to an increase in the threshold for action potential (from -52 mV to -43 mV, delta = 9 +/- 3, P < 0.05) and a more negative resting membrane potential (from -52 mV to -57 mV, delta = 5 +/- 1, P < 0.05). There was a progressive prolongation of intervals between spontaneous action potentials and a slowed rate of phase 4 depolarization. Other polyunsaturated fatty acids--including docosahexaenoic acid, linolenic acid, linoleic acid, arachidonic acid, and its nonmetabolizable analog eicosatetraynoic acid, but neither the monounsaturated oleic acid nor the saturated stearic acid--had similar effects. The effects of the fatty acids could be reversed by washing with fatty acid-free bovine serum albumin. These results show that free polyunsaturated fatty acids can reduce membrane electrical excitability of heart cells and provide an electrophysiological basis for the antiarrhythmic effects of these fatty acids.