Hypertrophic cardiac growth is a major compensatory response of the heart to an increased mechanical (hemodynamic) load in the form of either pressure or volume overload. Although this response is initially compensatory, a transition from this state to failure occurs when further growth of the heart is not sufficient to normalize the wall stress and maintain contractile function (1). Therefore, a major research interest in cardiovascular disease is to understand how the increase in hemodynamic load is transmitted intracellularly for mediating hypertrophic growth. Although the mechanical load appears to directly regulate the hypertrophic growth initiation, the signaling mechanism that connects load to such growth is not well understood.A major cellular event during cardiac hypertrophy is increased protein synthesis (1-5). Enhanced protein synthesis can occur via accelerated protein translation, increased biogenesis of translational components, or both. A significant amount of mRNA of vertebrate cells possesses a unique 5Ј-terminal oligopyrimidine (5Ј-TOP) 1 sequence in the 5Ј-untranslated region (5Ј-UTR), and these mRNA species generally code for specific ribosomal proteins (6, 7). Their translation is largely controlled via phosphorylation of the 40 S ribosomal S6 protein (S6 protein) at its C terminus (8) by p70/85 S6 kinase (S6K1) (9 -12). There are two isoforms of S6K1: the 70-kDa isoform was first isolated from mouse 3T3 cells (13), and the 85-kDa isoform of this kinase was then identified (14). The p85 isoform is expressed from the same transcript as the p70 isoform through an alternative translational initiation start site, which adds a 23-amino acid nuclear localization signal to the N terminus (15,16). Therefore, the 85-kDa isoform is predominantly in the nucleus, whereas the 70-kDa isoform is present mostly in the cytoplasm. Both the S6K isoforms are collectively called p70/85S6K, p70S6K, or S6K1 and have been shown to phosphorylate the S6 protein and mediate the biogenesis of the translational components, including several of the ribosomal proteins and elongation factors (12). The p85 isoform has been shown to have additional roles in translational control, G 1 to S phase transition, and increased DNA synthesis (17). Recent studies using S6K1 knockout mice (18) demonstrate no appreciable change in S6 protein phosphorylation, 5Ј-TOP mRNA translation, or cell growth, although these mice exhibited a small mouse phenotype. These studies (18) and other independent studies (19 -21) resulted in the discovery of another S6K (S6K2), which possesses 70% homology with the p70 isoform of

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.

SummaryStpA is a paralogue of the nucleoid-associated protein H-NS that is conserved in a range of enteric bacteria and had no known function in Salmonella Typhimurium. We show that 5% of the Salmonella genome is regulated by StpA, which contrasts with the situation in Escherichia coli where deletion of stpA only had minor effects on gene expression. The StpA-dependent genes of S. Typhimurium are a specific subset of the H-NS regulon that are predominantly under the positive control of s 38 (RpoS), CRP-cAMP and PhoP. Regulation by StpA varied with growth phase; StpA controlled s 38 levels at mid-exponential phase by preventing inappropriate activation of s 38 during rapid bacterial growth. In contrast, StpA only activated the CRP-cAMP regulon during late exponential phase. ChIP-chip analysis revealed that StpA binds to PhoP-dependent genes but not to most genes of the CRP-cAMP and s 38 regulons. In fact, StpA indirectly regulates s 38 -dependent genes by enhancing s 38 turnover by repressing the anti-adaptor protein rssC. We discovered that StpA is essential for the dynamic regulation of s 38 in response to increased glucose levels. Our findings identify StpA as a novel growth phasespecific regulator that plays an important physiological role by linking s 38 levels to nutrient availability.

Left ventricular (LV) pressure (PO) or volume (VO) overload is accompanied by myocardial remodeling, but mechanisms that contribute to this progressive remodeling process remain unclear. The matrix metalloproteinases (MMPs) contribute to tissue remodeling in a number of disease states. This study tested the hypothesis that increased MMP expression and activity occur after the induction of an LV overload, which is accompanied by a loss of endogenous MMP inhibitory control. LV MMP zymographic activity and species abundance were measured in dogs under the following conditions: acute PO induced by ascending aortic balloon inflation (6 h, n = 9), prolonged PO by aortic banding (10 days, n = 5), acute VO through mitral regurgitation secondary to chordal rupture (6 h, n = 6), prolonged VO due to mitral regurgitation (14 days, n = 7), and sham controls (n = 11). MMP zymographic activity in the 92-kDa region, indicative of MMP-9 activity, increased over threefold in acute PO and VO and fell to control levels in prolonged PO and VO. The MMP-9 activity-to-abundance ratio increased by over fourfold with acute VO and twofold in acute PO, suggesting a loss of inhibitory control. Endogenous MMP inhibitor content was unchanged with either PO or VO. Interstitial collagenase (MMP-1) content decreased by 50% with acute VO but not with acute PO. Stromelysin (MMP-3) levels increased by 40% with acute VO and increased by 80% with prolonged PO. Although changes in LV myocardial MMP activity and inhibitory control occurred in both acute and prolonged PO and VO states, these changes were not identical. These results suggest that the type of overload stimulus may selectively influence myocardial MMP activity and expression, which in turn would affect the overall LV myocardial remodeling process in LV overload.

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.

Because we find persistent increases both in microtubules and in their biosynthetic precursors in pressure-hypertrophied myocardium, the mechanisms for this cytoskeletal abnormality must be sought through studies of the control both of microtubule stability and of tubulin synthesis.

Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease.

This report identifies a rapid increase in the expression of cardiac Na(+)-Ca2+ exchanger mRNA in response to an acute pressure overload. This enhanced exchanger expression appeared within 1 h after the onset of right ventricular pressure overload in the cat and was sustained during cardiac overloading for at least 4 h. Maintenance of this right ventricular pressure overload for 48 h evoked an increase in the production of exchanger protein. Because of our previous finding that load imposition on the heart initiates cell growth and our hypothesis that this is in response to the enhanced entry of cellular cations, we then examined the effect of Na+ influx into cultured adult cardiac myocytes, or cardiocytes, in terms of early anabolic responses. Pressure overload of the heart and cardiocyte Na+ influx were found to produce a common, rapid result in terms of both enhanced Na(+)-Ca2+ exchanger expression and accelerated synthesis of general and contractile proteins, the hallmarks of cardiac hypertrophy.