ECE-1 influences prostate cancer cell invasion via ET-1-mediated FAK phosphorylation and ET-1-independent mechanisms.

Plasma concentrations of the mitogenic peptide endothelin-1 (ET-1) are significantly elevated in men with metastatic prostate cancer (PC). ET-1 also contributes to the transition of hormonally regulated androgen-dependent PC to androgen-independent disease. ET-1 is generated from big-ET-1 by endothelin-converting enzyme (ECE-1). ECE-1 is present in PC cell lines and primary tissue and is elevated in primary malignant stromal cells compared with benign. siRNA or shRNA-mediated knockdown of endogenous ECE-1 in either the epithelial or stromal compartment significantly reduced PC cell (PC-3) invasion and migration. The re-addition of ET-1 only partially recovered the effect, suggesting ET-1-dependent and -independent functions for ECE-1 in pPC. The ET-1-independent effect of ECE-1 on PC invasion may be due to modulation of downstream signalling events. Addition of an ECE-1 specific inhibitor to PC-3 cells reduced phosphorylation of focal adhesion kinase (FAK), a signalling molecule known to play a role in PC. siRNA-mediated knockdown of ECE-1 resulted in a significant reduction in FAK phosphorylation. Accordingly, transient ECE-1 overexpression in PNT1-a cells increased FAK phosphorylation. In conclusion, ECE-1 influences PC cell invasion via both ET-1-mediated FAK phosphorylation and ET-1 independent mechanisms.

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes.

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.

Modifications of eukaryotic initiation factor 4F (eIF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on eIF4F activity and total protein synthesis rates.

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F (eIF4F) activity are correlated with accelerated rates of total protein synthesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load,cardiocytes were electrically stimulated to contract (2 Hz,5 ms pulses). Non-stimulated cardiocytes were used as controls.Adenovirus-mediated overexpression of wild-type eIF4E increased the total eIF4E pool by 120-140% above endogenous levels after 24 h and produced a corresponding increase in eIF4F content.However, it did not accelerate total protein synthesis rates inquiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreased binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increased the quantity of eIF4F but the rate of total protein synthesis was decreased inquiescent and contracting cardiocytes. Overexpression of a mutant that blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity ofeIF4F without any significant effect on total protein synthesis rates in quiescent or contracting cardiocytes. Overexpression of the eIF4Ekinase Mnk-1 increased eIF4E phosphorylation without a corresponding increase in eIF4F complex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via adenoviral gene transfer; (2) the capbinding affinity of eIF4F is a rate-limiting determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4Falone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.

Transcriptional regulation of the rat eIF4E gene in cardiac muscle cells: the role of specific elements in the promoter region.

Eukaryotic initiation factor 4E (eIF4E) binds to the 7-methylguanosine cap of mRNA and facilitates binding of mRNA to the 40 S ribosome, a rate-limiting step in translation initiation. The expression of eIF4E mRNA and protein increases during growth of cardiac muscle cells (cardiocytes) in vitro. To examine transcriptional regulation of the rat eIF4E gene, 2.1 kB of the rat eIF4E promoter region was cloned and the contribution of specific elements in regulating transcription was determined in primary cultures of rat cardiocytes and in a murine C(2)C(12) myoblast cell line. Sequence analysis of the rat eIF4E promoter revealed 80% sequence similarity with human eIF4E. A putative distal E-box was found at -230 bp and a proximal E-box was located at -77 bp upstream of the transcription start site. Consensus AP-1 motifs were found at -839 and -901 bp and designated as the proximal AP-1 site and distal AP-1 site, respectively. Transfection of reporter gene constructs into cardiocytes showed that deletion of the region between -633 and -318 bp produced a 3-fold increase in basal transcription as compared to the 2.1 kB eIF4E promoter construct. Further deletion of the distal E-box region had no effect on transcription as compared with the 2.1 kB promoter, but deletion of both E-boxes eliminated transcriptional activity. Similar results were obtained in C(2)C(12) myoblasts. To further investigate transcriptional regulation, point mutations were made in the 2.1 kB eIF4E promoter. Mutation of either the distal or proximal E-box had minimal effects on activity in either cell type, but mutation of the distal AP-1 site significantly reduced eIF4E promoter activity by 66+/-4% in cardiocytes. In C(2)C(12) myoblasts, mutating the distal AP-1 site reduced activity by 30+/-4% We conclude that both E-boxes are required for maximal basal activity of the eIF4E promoter, and that the distal AP-1 motif may activate transcription.

Rutgers University case study: a guide to non-routine high dose rate procedures for biomedical facilities.

Non-routine potentially high dose rate procedures occasionally present themselves at biomedical facilities. Lacking significant operational experience, many biomedical facilities decide to contract out the work to consulting firms without seriously considering doing the work themselves. This study shares the University's experiences during a non-routine procedure to extract a 60Co irradiation source from an underground storage facility and relocate it to an above-ground shielded storage cask.

Differential effects of pressure or volume overload on myocardial MMP levels and inhibitory control.

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.

Translational mechanisms accelerate the rate of protein synthesis during canine pressure-overload hypertrophy.

This study examined how translational mechanisms regulate the rate of cardiac protein synthesis during canine pressure overload in vivo. Acute aortic stenosis (AS) was produced by inflating a balloon catheter in the ascending aorta for 6 h; sustained AS was created by controlled banding of the ascending aorta. AS caused significant hypertrophy as reflected by increased left ventricular (LV) mass after 5 and 10 days. To monitor LV protein synthesis in vivo, myosin heavy chain (MHC) synthesis was measured by continuous infusion of radiolabeled leucine. Acute AS accelerated the rate of myosin synthesis without a corresponding increase in ribosomal RNA, indicating an increase in translational efficiency. Total MHC synthesis (mg MHC/LV per day) was significantly increased at 5 and 10 days of sustained AS. Total MHC degradation was not significantly altered at 5 days of AS but increased at 10 days of AS in concordance with a new steady state with respect to growth. Translational capacity (mg total RNA/LV) was significantly increased after 5 and 10 days of AS and was preceded by an increase in the rate of ribosome formation. MHC mRNA levels remained unchanged during AS. These findings demonstrate that cardiac protein synthesis is accelerated in response to pressure overload by an initial increase in translational efficiency, followed by an adaptive increase in translational capacity during sustained hypertrophic growth.

Role of load in regulating eIF-4F complex formation in adult feline cardiocytes.

This study examined whether cardiocyte load increases eIF-4F complex formation. To increase load in vitro, adult feline cardiocytes were electrically stimulated to contract (1 Hz, 5-ms pulses). eIF-4F complex formation, measured by eIF-4G association with eIF-4E, increased 57 +/- 16% after 4 h of contraction compared with controls. eIF-4F complex formation did not increase on electrical stimulation with 2,3-butanedione monoxime (BDM), an inhibitor of active tension. Both insulin and phorbol ester increased eIF-4F complex formation, but these increases were unaffected by BDM. Insulin caused a shift of eIF-4E binding proteins (4E-BPs) into their hyperphosphorylated gamma-isoforms and dissociation of 4E-BPs from eIF-4E. Rapamycin inhibited 4E-BP phosphorylation in response to insulin but had no effect on eIF-4F complex formation. Electrically stimulated contraction caused a partial shift of 4E-BP1 and 4E-BP2 into the gamma-isoforms, but it had no effect on 4E-BP association with eIF-4E. Rapamycin blocked the increase in eIF-4F complex formation in electrically stimulated cardiocytes and depressed contractility. These data indicate that cardiocyte load causes a tension-dependent increase in eIF-4F complex formation that does not require dissociation of 4E-BPs from eIF-4E.

Mechanisms of cardiac hypertrophy in canine volume overload.

This study tested whether the modest hypertrophy that develops in dogs in response to mitral regurgitation is due to a relatively small change in the rate of protein synthesis or, alternatively, is due to a decreased rate of protein degradation. After 3 mo of severe experimental mitral regurgitation, the left ventricular (LV) mass-to-body weight ratio increased by 23% compared with baseline values. This increase in LV mass occurred with a small, but not statistically significant, increase in the fractional rate of myosin heavy chain (MHC) synthesis (Ks), as measured using continuous infusion with [3H]leucine in dogs at 2 wk, 4 wk, and 3 mo after creation of severe mitral regurgitation. Translational efficiency was unaffected by mitral regurgitation as measured by the distribution of MHC mRNA in polysome gradients. Furthermore, there was no detectable increase in translational capacity as measured by either total RNA content or the rate of ribosome formation. These data indicate that translational mechanisms that accelerate the rate of cardiac protein synthesis are not responsive to the stimulus of mitral regurgitation. Most of the growth after mitral regurgitation was accounted for by a decrease in the fractional rate of protein degradation, calculated by subtracting fractional rates of protein accumulation at each time point from the corresponding Ks values. We conclude that 1) volume overload produced by severe mitral regurgitation does not trigger substantial increases in the rate of protein synthesis and 2) the modest increase in LV mass results primarily from a decrease in the rate of protein degradation.

Increased expression of eukaryotic initiation factor 4E during growth of neonatal rat cardiocytes in vitro.

Eukaryotic initiation factor 4E (eIF-4E) is rate limiting for translational initiation. The purpose of this study was to determine whether eIF-4E levels are increased during cardiocyte growth produced by increased load in the form of electrically stimulated contraction. Neonatal rat cardiocytes were cultured on a matrix of aligned type I collagen. The cardiocytes aligned in parallel to the direction of the collagen fibrils and exhibited an elongated, rod-shaped morphology. Cardiocytes were electrically stimulated to contract at 3 Hz (alternating polarity, 5-ms pulse width). Nonstimulated cardiocytes were quiescent and used as controls. Electrically stimulated contraction produced hypertrophic growth as determined by the following criteria: 1) increased protein content, 2) increased RNA content, 3) accelerated rate of protein synthesis, and 4) threefold increase in promoter activity of the atrial natriuretic factor gene. Cardiocyte growth was associated with an increase in eIF-4E mRNA levels that reached 48 +/- 9% after 2 days of electrically stimulated contraction. eIF-4E protein levels were increased by more than twofold over the same time period. We conclude that an adaptive increase in eIF-4E is an important mechanism for maintaining translational efficiency during cardiocyte growth.

Synthesis and endectocidal activity of novel 1-(arylsulfonyl)-1-[(trifluoromethyl)sulfonyl]methane derivatives.

We have recently synthesized a series of novel disulfonylmethane compounds that have shown anthelmintic and insecticidal (endectocidal) activity. Several analogues have shown activity against the internal nematode Haemonchus contortus. In sheep studies, these analogues have shown 100% control of this internal parasite at a 10 mg/kg rate. In vitro activity against the biting flies, Stomoxys calcitrans and Haematobia irritans, has been observed at rates as low as 25 and 2.3 ppm, respectively. Only marginal activity against the liver fluke Fasciola hepatica and Trichostrongylus colubriformis was seen. Respiratory control index values on rat liver mitochondria for this series suggested uncoupling of oxidative phosphorylation as a mechanism of action. Compound 1 is considered to be a promising agent for treatment of parasitized sheep.

The 3'-untranslated region of the alpha2C-adrenergic receptor mRNA impedes translation of the receptor message.

We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha2C-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha2C-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating alpha2C-AR expression and associated signaling events.

Comparative effects of contraction and angiotensin II on growth of adult feline cardiocytes in primary culture.

The purposes of this study were 1) to determine whether angiotensin II causes growth of adult feline cardiocytes in long-term culture, 2) to compare the growth effects of angiotensin II with those resulting from electrically stimulated contraction, and 3) to determine whether the anabolic effects of contraction are exerted via the angiotensin type 1 receptor. Adult feline cardiocytes were cultured on laminin-coated trays in a serum-free medium. Cardiocytes were either electrically stimulated to contract (1 Hz, 5-ms pulse duration, alternating polarity) or were nonstimulated and quiescent. Quiescent cells were studied as controls and after treatment with angiotensin II (10(-8) M), losartan (10(-6) M; an angiotensin type 1-receptor antagonist), or angiotensin II plus losartan. Contracting cells were studied in the presence and absence of angiotensin II or losartan. In quiescent cardiocytes, angiotensin II treatment on day 7 significantly increased protein synthesis rates by 22% and protein content per cell by 17%. The effects of angiotensin II were completely blocked by losartan. Electrically stimulated contraction on days 4 and 7 in culture significantly increased protein synthesis rate by 18 and 38% and protein content per cell by 19 and 46%, respectively. Angiotensin II treatment did not further increase protein synthesis rate or protein content in contracting cardiocytes. Furthermore, losartan did not block the anabolic effects of contraction on protein synthesis rates or protein content. In conclusion, angiotensin II can exert a modest anabolic effect on adult feline cardiocytes in culture. In contracting feline cardiocytes, angiotensin II has no effect on growth. Growth caused by electrically stimulated contraction occurs more rapidly and is greater in magnitude than that caused by angiotensin II. Growth of contracting adult feline cardiocytes is not dependent on activation of the angiotensin receptor.

Passive load and angiotensin II evoke differential responses of gene expression and protein synthesis in cardiac myocytes.

This study introduced an improved model of loaded adult cardiocytes to address a proposed requirement for angiotensin II (Ang II) in the transduction pathway between load on the cardiac myocyte and its early anabolic responses of gene expression and acceleration of protein synthesis. The isolated cardiocytes were subjected to passive load by step increments of stretch and responded with proportional acceleration of protein synthesis in both adult and neonatal cardiocytes; this response was unaltered by 1 mumol/L [Sar1, Ile8]Ang II, an antagonist peptide to Ang II. Ang II from 1 nmol/L to 10 mumol/L did not increase protein synthesis after 4 hours in adult cardiocytes nor at 100 nmol/L in neonatal cardiocytes. However, 100 nmol/L Ang II did increase [3H]phenylalanine incorporation into neonatal cardiocyte protein over a 24-hour period by 10%, whereas passive load increased [3H]phenylalanine incorporation into protein by 30%, which was not blocked by [Sar1, Ile8]Ang II. Thus, the anabolic effect of load does not require ANG II to increase either 4-hour protein synthesis in both adult and neonatal cardiocytes or 24-hour [3H]phenylalanine incorporation into protein in neonatal cardiocytes. The genetic response of the cardiocyte to load was examined by assessing c-fos and Na+-Ca2+ exchanger mRNA levels, because there are rapidly expressed at the onset of cardiac pressure overload. The c-fos mRNA was increased fourfold within 1 hour after 100 nmol/L Ang II treatment of either adult or neonatal cardiocytes. This c-fos induction was blocked by [Sar1, Ile8]Ang II. One hour after loading of adult cardiocytes, induction of c-fos expression was increased threefold; this was also blocked by [Sar1, Ile8]Ang II. Thus, load-induced c-fos expression was Ang II dependent in adult cardiocytes. In contrast, exchanger mRNA levels were increased threefold 1 hour after loading of adult cardiocytes, but this increased expression was not blocked by [Sar1, Ile8]Ang II. For additional comparison, c-fos expression was induced by Ang II and phorbol myristate acetate, which did not induce exchanger expression; conversely, exchanger expression was induced by veratridine, which did not increase c-fos expression. Thus, separate c-fos and exchanger expression pathways can be differentiated in adult cardiocytes. This study demonstrated that Ang II is not required for load to initiate the anabolic processes of accelerated protein synthesis or enhanced Na+-Ca2+ exchanger expression pathways can be differentiated in adult cardiocytes. This study demonstrated that Ang II is not required for load to initiate the anabolic processes of accelerated protein synthesis or enhanced Na+-Ca2+ exchanger gene expression in cardiocytes; however, load induced c-fos expression is Ang II dependent.

Translational initiation factor eIF-4E. A link between cardiac load and protein synthesis.

To define the coupling mechanism between cardiac load and the rate of protein synthesis, changes in the extent of eIF-4E phosphorylation were measured after imposition of a load. Electrically stimulated contraction of adult feline cardiocytes increased eIF-4E phosphorylation to 34% after 4 h, as compared with 8% phosphorylation in quiescent controls. However, eIF-4E phosphorylation did not increase upon electrical stimulation in the presence of 7.5 mM 2,3-butanedione monoxime, an inhibitor of actin-myosin cross-bridge cycling and active tension development. Treatment of adult cardiocytes with either 0.1 microM insulin or 0.1 microM phorbol 12-myristate 13-acetate increased eIF-4E phosphorylation to 23 and 64%, respectively, but these increases were not blocked by 2,3-butanedione monoxime. In canine models of acute hemodynamic overload in vivo, eIF-4E phosphorylation increased to 23% in response to left ventricular pressure overload as compared with 7% phosphorylation in controls. Acute volume overload had no effect on eIF-4E phosphorylation. These changes in eIF-4E phosphorylation account for differences in anabolic responses to acute pressure versus acute volume overload. These data suggest that eIF-4E phosphorylation is a mechanism by which increased cardiac load is coupled to accelerated rates of protein synthesis.

Basis for increased microtubules in pressure-hypertrophied cardiocytes.

BACKGROUND: We have shown the levels of the sarcomere and the cardiocyte that a persistent increase in microtubule density accounts to a remarkable degree for the contractile dysfunction seen in pressure-overload right ventricular hypertrophy. In the present study, we have asked whether these linked phenotypic and contractile abnormalities are an immediate and direct effect of load input into the cardiocyte or instead a concomitant of hypertrophic growth in response to pressure overloading. METHODS AND RESULTS: The feline right ventricle was pressure-overloaded by pulmonary artery banding. The quantity of microtubules was estimated from immunoblots and immunofluorescent micrographs, and their mechanical effects were assessed by measuring sarcomere motion during microtubule depolymerization. The biogenesis of microtubules was estimated from Northern and Western blot analyses of tubulin mRNAs and proteins. These measurements were made in control cats and in operated cats during and after the completion of right ventricular hypertrophy; the left ventricle from each heart served as a normally loaded same-animal control. We have shown that the alterations in microtubule density and sarcomere mechanics are not an immediate consequence of pressure overloading but instead appear in parallel with the load-induced increase in cardiac mass. Of potential mechanistic importance, both these changes and increases in tubulin poly A+ mRNA and protein coexist indefinitely after a new, higher steady state of right ventricular mass is reached. CONCLUSIONS: 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.

Eukaryotic initiation factor 4E degradation during brain ischemia.

Suppression of protein synthesis in the brain following an ischemic insult has been thought to occur because of inhibition of translation initiation. All eukaryotic mRNAs, with the exception of heat-shock transcripts, require the activity of eukaryotic initiation factor (eIF) 4E for formation of the translation initiation complex, and eIF-4E availability is rate-limiting. The response of brain eIF-4E concentration and phosphorylation following decapitation ischemia was studied in rat brain homogenates after electrophoresis and western blotting with antibodies against eIF-4E and phosphoserine, respectively. There was no change in level of eIF-4E after 5 min of ischemia (p = 0.82 vs. time 0), but it had decreased 32 (p = 0.01) and 57% (p = 0.006) after 10 and 20 min of ischemia, respectively. There was no loss of serine phosphorylation on eIF-4E beyond signal loss observed due to degradation of the protein itself (p = 0.31). In vitro exposure of eIF-4E to activated mu-calpain resulted in a 50% loss in 10 min of eIF-4E on western blots. If active eIF-4E is required for translation of its own mRNA, degradation of this protein during ischemia, possibly by activated mu-calpain, could be a direct mechanism of irreversible neuronal injury, and the rate of proteolysis of eIF-4E could place an upper time limit on the maximal duration of global brain ischemia compatible with neurologic recovery.

Contraction accelerates myosin heavy chain synthesis rates in adult cardiocytes by an increase in the rate of translational initiation.

The purpose of this study was to determine the mechanism by which contraction acutely accelerates the synthesis rate of the contractile protein myosin heavy chain (MHC). Laminin-adherent adult feline cardiocytes were maintained in a serum-free medium and induced to contract at 1 Hz via electrical field stimulation. Electrical stimulation of contraction accelerated rates of MHC synthesis 28%, p < 0.05 by 4 h as determined by incorporation of [3H]phenylalanine into MHC. MHC mRNA expression as measured by RNase protection was unchanged after 4 h of electrical stimulation. MHC mRNA levels in messenger ribonucleoprotein complexes and translating polysomes were examined by sucrose gradient fractionation. The relative percentage of polysomebound MHC mRNA was equal at 47% in both electrically stimulated and control cardiocytes. However, electrical stimulation of contraction resulted in a reproducible shift of MHC mRNA from smaller polysomes into larger polysomes, indicating an increased rate of initiation. This shift resulted in significant increases in MHC mRNA levels in the fractions containing the larger polysomes of electrically stimulated cardiocytes as compared with nonstimulated controls. These data indicate that the rate of MHC synthesis is accelerated in contracting cardiocytes via an increase in translational efficiency.

Growth effects of electrically stimulated contraction on adult feline cardiocytes in primary culture.

The purpose of this study was to determine effects of long-term electrical stimulation of cardiocyte contraction on protein synthesis rates and total protein content. Adult feline cardiocytes were plated on laminin-coated culture trays and maintained in a serum-free medium consisting of M199 supplemented with ascorbate, bovine serum albumin, creatine, carnitine, taurine, and 10(-7) M recombinant insulin. Cardiocytes were electrically stimulated to contract with use of continuous electrical pulses of alternating polarity at a frequency of 1 Hz and pulse duration of 5 ms. Nonstimulated cardiocytes are normally quiescent and were used as the control group. In control quiescent cardiocytes, protein synthesis rate decreased by 14% between days 1 and 4 in culture and then remained stable through day 7. In electrically stimulated cardiocytes, protein synthesis rates increased by 19% between days 1 and 7. Protein synthesis rates were 18% higher on day 4 and 43% higher on day 7 in electrically stimulated than in quiescent cardiocytes. Protein content per cell was determined by measuring total fluorescence per cell by use of confocal microscopy of fluorescein isothiocyanate-stained cells. Electrical stimulation significantly increased cellular protein content by 52% after 7 days compared with controls. Quiescent and electrically stimulated cardiocytes remained rod shaped, retained their myofibrillar architecture, and were responsive to electrical stimulation over the 7-day period. These data demonstrated that electrically stimulated contraction of adult cardiocytes resulted in cell growth, as assessed by an increase in protein content per cell over 7 days in culture. This increase was due, at least in part, to an acceleration of steady-state protein synthesis rates.

Load effects on gene expression during cardiac hypertrophy.

Hemodynamic load is a primary regulator of cardiac mass. A potential proximal event in this regulatory pathway is thought to be the induction of immediate early genes, and markers of this process include the re-expression of genes for fetal sarcomeric proteins and the ventricular expression of atrial natriuretic factor (ANF). Previous in vivo models which have examined these questions have often neither quantified myocardial loading nor accounted for covariables which may affect gene expression such as the renin-angiotensin-aldosterone system, the sympathetic nervous system, or baroreceptors. Thus, whether load alone is sufficient to induce immediate early genes, which may ultimately result in cardiac hypertrophy, remains unknown. In the present study two models of right ventricular (RV) pressure overload were created by partially occluding the pulmonary artery (PA), either with a balloon catheter for 1 or 4 h, or with a surgically placed PA band for 12, 24, or 48 h. Serum catecholamine concentrations were determined in a subset of RV pressure overload cats at basal state, after 5 min of balloon inflation, and after 1 h of balloon inflation to examine the effects of this systemic trophic factor on IEG induction. Northern blot analysis for c-fos, egr-1, alpha-skeletal actin, and ANF from paired RV and left ventricular (LV) RNA allowed the effect of load (selectively increased in the RV) to be separated from other systemic variables (present in both ventricles). The relative signal intensities of the optical density of RV and LV mRNA autoradiograms were determined from northern blots, alternate lanes of which were loaded with 7.5 micrograms of total RNA from RV and LV tissue from the same cat. Partial PA occlusion caused RV systolic pressure to increase from a control value of 22 +/- 1 mmHg to 57 +/- 6 mmHg after 1 h, 59 +/- 5 mmHg after 4 h, and 58 +/- 5 mmHg after 48 h of RV pressure overload (RVPO). Serum norepinephrine and epinephrine levels at both 5 and 60 min of RVPO were not significantly different from basal levels. The RV/LV ratios of mRNA for both egr-1 and c-fos were equal in control and 48 h PA banded animals, but were increased in the 1 and 4 h balloon RVPO cats. The RV/LV ratio of mRNA for alpha-skeletal actin was equal in the basal state and did not increase after 12, 24, or 48 h of RVPO. After 48 h of RVPO, total RNA was increased in the RV compared with the LV (1.9 +/- 0.1 v 1.1 +/- 0.1 micrograms/g tissue, P < 0.05). ANF expression was present in the RV after 48 h of RVPO, but absent in same-animal LV and all control ventricles. Thus, while increased load alone did not alter the expression of alpha-skeletal actin, it was sufficient both to induce increased expression of two distinct classes of immediate early genes, as well as ANF, and to increase total RNA, indicating hypertrophic growth initiation.