Skeletal Alpha-actin mRNA Research Articles

Chronic Absence of Baroreceptors Induces Left Ventricular Dysfunction and Reduced Coronary Blood Flow in Spontaneously Hypertensive Rats

Hemodynamic and cardiac morphological variables were measured in normotensive Wistar (N), intacts hypertensive (H) and SHR submitted to sinoaortic denervation (DH) to evaluate the chronic baroreceptor dysfunction on left ventricular (LV) functions and coronary blood flow. Blood (BP) and end‐diastolic pressure (PD2) were obtained by direct quantification. The coronary blood flow was measured by the infusion of colored microspheres. The ratio of left ventricular weight to body weight, natriuretic atrial peptide (ANP), alpha‐skeletal actin (α‐skelectal), alpha and beta myosin heavy chain (α‐MHC, β‐MHC) genes mRNA expressions were evaluated by real time PCR to assess ventricular hypertrophy. The results were reported as means ± SEM. DH rats did not change BP values compared to H. PD2 of DH (9.91±1.78) was higher than H group (4.30±0.53) while an inverse result regarding coronary blood flow was observed (0.94±0.15 vs 1.80±0.25, respectively). The LV adaptations in DH were higher than in H (2.98 ±0.07 vs 2.15±0.05), as well as the expression of ANP (3.35±0.19 vs 2.39 ± 0.74), β‐MHC (3.36±0.18 vs 2.39 ± 0.37). The α‐MHC genes expression also were decreased (0.62±0.10 vs 0.79±0.10). Therefore, the chronic absence of baroreflex control by sinoaortic denervation induces left ventricular dysfunction and reduced coronary blood flow in SHR.Financial support: CAPES

Abstract P194: CCAAT/Enhancer-Binding Protein Beta Negatively Regulates Hemodynamic Stress-Induced Cardiac Hypertrophy

The CCAAT/enhancer-binding proteins (C/EBP) beta is a member of family of leucine-zipper transcription factors that regulate gene expression to control cellular proliferation, differentiation, inflammation and metabolism. Previous reports showed that the protein and mRNA levels of C/EBP beta were increased in rat ischemia-reperfused or human failing hearts. Recent study indicates an important role of C/EBP beta in physiological cardiac hypertrophic responses. In the present study, we generated cardiac-specific C/EBP beta-deficient mice (CKO) to elucidate its in vivo function in pathological cardiac hypertrophy. We crossed floxed C/EBP beta mice with mice expressing Cre recombinase in cardiac-specific manner. Echocardiographic and physiological analyses revealed that CKO showed no cardiac phenotypes under basal conditions at 10 weeks old, indicating that the C/EBP beta is not essential for mouse heart development. Then, we subjected CKO and control mice (CTL) to pressure overload by means of transverse aortic constriction (TAC). In wild-type mouse hearts, the expression level of C/EBP beta was increased after TAC. One week after TAC, CKO showed left ventricle (LV) hypertrophy (LV/body weight, CKO 4.64 ± 0.08 mg/g versus CTL 4.14 ± 0.06 mg/g, p<0.01, LV mass index, CKO 106.1 ± 1.7 versus CTL 98.5 ± 1.8, p<0.05), without showing contractile dysfunction. Cross-sectional area of cardiomyocytes was increased in CKO after TAC (CKO 373.6 ± 2.1 μm 2 versus CTL 300.5 ± 3.6 μm 2 , p<0.01). The increase in the atrial natriuretic factor (ANF) or alpha skeletal actin mRNA expression, molecular markers for cardiac remodeling, was observed in CKO hearts. Furthermore, overexpression of C/EBP beta in isolated neonatal rat cardiomyocytes inhibited an increase in 3 H-leucine uptake induced by phenylephrine stimulation. C/EBP beta is expressed as three distinct protein isoforms, namely FL, LAP, and LIP, which are encoded by a single gene, but transcribed from different initiation sites. Luciferase reporter gene assay showed that overexpression of LAP isoform attenuated the transcriptional activity of ANF induced by phenylephrine, but not LIP. Thus, we conclude that C/EBP beta attenuates the pathological cardiac hypertrophy induced by hemodynamic stresses.

Ligand-independent activation of the androgen receptor by insulin-like growth factor-I and the role of the MAPK pathway in skeletal muscle cells.

In this study, the roles of the p38 MAPK, ERK1/2 and JNK signaling pathway in IGF-I-induced AR induction and activation were examined. C2C12 cells were treated with IGF-I in the absence or presence of various inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK (SP600125). Inhibition of the MAPK pathway with SB203580, PD98059, or SP600125 significantly decreased IGF-I-induced AR phosphorylation and total AR protein expression. IGF-I-induced nuclear fraction of total AR and phosphorylated AR were significantly inhibited by SB203580, PD98059, or SP600125. Furthermore, IGF-I-induced AR mRNA and skeletal alpha-actin mRNA were blocked by those inhibitors in dose-dependent manner. Confocal images showed that IGF-I-induced AR nuclear translocation from cytosol was significantly blocked by SB203580, PD98059, or SP600125, suggesting that the MAPK pathway regulates IGF-I-induced AR nuclear localization in skeletal muscle cells. The present results suggest that the MAPK pathways are required for the ligand-independent activation of AR by IGF-I in C2C12 skeletal muscle cells.

Insulin-Like Growth Factor-I-Induced Androgen Receptor Activation Is Mediated by the PI3K/Akt Pathway in C2C12 Skeletal Muscle Cells

Although insulin-like growth factor-I (IGF-I) and androgen receptor (AR) are well known effectors of skeletal muscle, the molecular mechanism by which signaling pathways integrating AR and IGF-I in skeletal muscle cells has not been previously examined. In this study, the role of PI3K/Akt on IGF-I-induced gene expression and activation of AR in skeletal muscle cells was investigated. C2C12 cells were treated with IGF-I in the absence or presence of inhibitors of PI3K/Akt pathway (LY294002 and Wortmannin). Inhibition of the PI3K/Akt pathway with LY294002 or Wortmannin led to a significant decrease in IGF-I-induced AR phosphorylation and total AR protein expression. Furthermore, IGF-I-induced AR mRNA and skeletal alpha-actin mRNA were blocked by LY294002 or Wortmannin. Confocal images showed that IGF-I-induced AR translocation from cytosol to nucleus was inhibited significantly in response to treatment with LY294002 or Wortmannin. The present results suggest that modulating effect of IGF-I on AR gene expression and activation in C2C12 mouse skeletal muscle cells is mediated at least in part by the PI3K/Akt pathway.

ENOS Deficient Mice Develop Progressive Cardiac Hypertrophy with Altered Cytokine and Calcium Handling Protein Expression

Although studies have shown that endothelial nitric oxide synthase (eNOS) homozygous knockout mice (eNOS-/-) develop left ventricular (LV) hypertrophy, well compensated at least to 24 wks, uncertainty still exists as to the cardiac functional and molecular mechanistic consequences of eNOS deficiency at later time-points. To bridge the gap in existent data, we examined whole hearts from eNOS-/- and age-matched wild-type (WT) control mice ranging in age from 18 to 52 wks for macroscopic and microscopic histopathology, LV mRNA and protein expression using RNA Dot blots and Western blots, respectively, and LV function using isolated perfused work-performing heart preparations. Heart weight to body weight (HW/BW in mg/g) ratio increased significantly as eNOS-/- mice aged (82.2%, P < 0.001). Multi-focal replacement fibrosis and myocyte degeneration/death were first apparent in eNOS-/- mouse hearts at 40 wks. Progressive increases in LV atrial natriuretic factor (ANF) and alpha-skeletal actin mRNA levels both correlated significantly with increasing HW/BW ratio in aged eNOS-/- mice (r = 0.722 and r = 0.648, respectively; P < 0.001). At 52 wks eNOS-/- mouse hearts exhibited basal LV hypercontractility yet blunted beta adrenergic receptor (betaAR) responsiveness that coincided with a significant reduction in the LV ratio of phospholamban to sarcoplasmic reticulum Ca2+-ATPase-2a protein levels and was preceded by a significant upregulation in LV steady-state mRNA and protein levels of the 28 kDa membrane-bound form of tumor necrosis factor-alpha. We conclude that absence of eNOS in eNOS-/- mice results in a progressive concentric hypertrophic cardiac phenotype that is functionally compensated with decreased betaAR responsiveness, and is associated with a potential cytokine-mediated alteration of calcium handling protein expression.

Differential effects of muscle fibre length and insulin on muscle-specific mRNA content in isolated mature muscle fibres during long-term culture

The aims of this study were (1) to determine the relationship between muscle fibre cross-sectional area and cytoplasmic density of myonuclei in high- and low-oxidative Xenopus muscle fibres and (2) to test whether insulin and long-term high fibre length caused an increase in the number of myonuclei and in the expression of alpha-skeletal actin and of myogenic regulatory factors (myogenin and MyoD) in these muscle fibres. In high- and low-oxidative muscle fibres from freshly frozen iliofibularis muscles, the number of myonuclei per millimetre fibre length was proportional to muscle fibre cross-sectional area. The in vivo myonuclear density thus seemed to be strictly regulated, suggesting that the induction of hypertrophy required the activation of satellite cells. The effects of muscle fibre length and insulin on myonuclear density and myonuclear mRNA content were investigated on high-oxidative single muscle fibres cultured for 4-5 days. Muscle fibres were kept at a low length (~15% below passive slack length) in culture medium with a high insulin concentration (~6 nmol/l: "high insulin medium") or without insulin, and at a high length (~5% above passive slack length) in high insulin medium. High fibre length and high insulin medium did not change the myonuclear density of isolated muscle fibres during culture. High insulin increased the myonuclear alpha-skeletal actin mRNA content, whereas fibre length had no effect on alpha-skeletal actin mRNA content. After culture at high fibre length in high insulin medium, the myonuclear myogenin mRNA content was 2.5-fold higher than that of fibres cultured at low length in high insulin medium or in medium without insulin. Myonuclear MyoD mRNA content was not affected by fibre length or insulin. These in vitro experiments indicate that high muscle fibre length and insulin enhance muscle gene expression but that other critical factors are required to induce adaptation of muscle fibre size and performance.

Identification of PKCalpha isoform-specific effects in cardiac myocytes using antisense phosphorothioate oligonucleotides.

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in multiple cellular processes. In the heart, PKC signaling is involved in hypertrophic agonist-induced gene expression and hypertrophic growth. To investigate the specific function of PKC signaling in regulating cardiomyocyte growth, we used antisense oligonucleotides to inhibit PKC alpha, the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKCalpha oligonucleotides resulted in a marked reduction in both PKCalpha mRNA and protein levels. PKCalpha antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKCalpha. Antisense inhibition of PKCalpha led to reduction of PE-induced increase in skeletal alpha-actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had no significant effects on PE-induced beta-myosin heavy chain, ANP, or B-type natriuretic peptide (BNP) gene expression. On the other hand, antisense PKCalpha treatment attenuated endothelin-1-induced increase in ANP and BNP peptide secretion, whereas endothelin-1-induced gene expression of ANP and BNP remained unchanged. The hypertrophic agonist-induced growth of cardiomyocytes, characterized by increased [(3)H]leucine incorporation, was not affected with antisense PKCalpha treatment. Furthermore, we found that PE-induced increase in extracellular signal-regulated kinase (ERK) activity was partially inhibited by antisense PKCalpha treatment, implicating ERK as a downstream mediator for PKCalpha signaling. These results indicate that PKCalpha isozyme is involved in hypertrophic signaling in cardiomyocytes and provide novel strategies for future studies to identify other cellular targets controlled selectively by PKCalpha or other PKC isozymes.

Rescue of Contractile Parameters and Myocyte Hypertrophy in Calsequestrin Overexpressing Myocardium by Phospholamban Ablation

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

Differences in time course of myocardial mRNA expression in non-infarcted myocardium after myocardial infarction.

In non-infarcted myocardium after myocardial infarction, the change of cardiac phenotypic modulation of contractile protein, extracellular matrix and intracellular Ca2+ transport protein, such as sarcoplasmic reticulum Ca2+(SR-Ca2+)-ATPase, Na+-Ca2+ exchanger, have a important role during cardiac remodeling. However, the time course in this gene expression in the adjacent and remote left ventricular, or right ventricular myocardium after myocardial infarction has not been well examined. The purpose of this study was to examine the left ventricular function and regional cardiac gene expression after myocardial infarction. Myocardial infarction was produced in Wistar rats by the ligation of the left anterior descending coronary artery. After 3 weeks, 2 months and 4 months from myocardial infarction, we performed Doppler echocardiography and measured the systolic and diastolic function. Then, we analyzed the contractile protein, extracellular matrix and intracellular Ca2+ transport protein mRNAs of cardiac tissues in the adjacent and the remote noninfarcted myocardium, and right ventricular myocardium by Northern blot hybridization. Fractional shortening of infarcted heart progressively decreased. Peak early diastolic filling wave (E wave) velocity increased, and the deceleration rate of the E wave velocity was more rapid in myocardial infarction areas. Atrial filling wave (A wave) velocity decreased, resulting in a marked increase in the ratio of E wave to A wave velocity. Expression of myocardial alpha-skeletal actin, beta-MHC and ANP mRNA, or collagen I and III mRNA were higher at 3 weeks after myocardial infarction. SR Ca2+-ATPase mRNA in the adjacent non-infarcted myocardium was decreased at 2 months, and that in remote myocardium was decreased at 4 months after infarction. Na+-Ca2+ exchanger mRNA levels were increased at 3 weeks, but was decreased at 2 months in the adjacent non-infarcted myocardium and at 4 months in the remote myocardium. These findings suggest that the compensation for myocardial infarction by myocardial gene expression in non-infarcted myocardium may occur at an early phase after myocardial infarction, and myocardial dysfunction may begin from adjacent to remote non-infarcted myocardium during progressive cardiac remodeling.

Role and relation of p70 S6 and extracellular signal-regulated kinases in the phenotypic changes of hypertrophy of cardiac myocytes.

Cardiac hypertrophy is characterized by increased cardiomyocyte protein synthesis, increased cell volume, and a shift in cardiac-specific gene expression to fetal isoforms. Using neonatal rat cardiomyocytes stimulated with fetal calf serum (FCS) as a model for cardiac hypertrophy, the present study investigated the role of 2 signal transduction pathways, extracellular signal-regulated kinase (ERK) and p70S6 kinase (p70S6K), in the attendant phenotype changes. FCS evoked both ERK and p70S6K activity, peaking at 20-40min, and simultaneously increased cardiac myocyte protein synthesis (evaluated by [3H]leucine incorporation and total cellular protein content), cell size (evaluated by morphometry and fluorescence-activated cell sorter analysis) and expression of a fetal isoform of the muscle specific gene skeletal alpha-actin (SKA). Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR), which is an upstream signaling of p70S6K, completely inhibited FCS-induced cell size increases and protein synthesis, but had no effect on SKA mRNA expression. PD98059, which inhibited ERK activity, attenuated cardiac-specific gene expression in a dose-dependent manner, but had no influence on protein synthesis or cell size. These results indicate divergent roles for the ERK and p70S6K pathways in the phenotypic changes associated with cardiac hypertrophy.

A subpopulation of cardiomyocytes expressing alpha-skeletal actin is identified by a specific polyclonal antibody.

The NH(2)-terminal decapeptide of alpha-skeletal actin that contains a primary sequence specific for this isoform was used to raise a polyclonal antibody in rabbits. Using sequential affinity chromatography, we recovered from serum antibodies reacting exclusively with alpha-skeletal actin when tested by immunoblotting and immunofluorescence. Epitope mapping by means of competition assays with synthetic peptides indicated that the acetyl group and the first 9 amino acids are essential for specificity. The monospecific antibody was then used to investigate the distribution of alpha-skeletal actin in the myocardium of newborn and normal or hypertensive (with or without fibrotic areas) adult rats. Immunostaining of normal heart revealed that alpha-skeletal actin is diffusely distributed within practically all myocardial fibers of the newborn rat, whereas it is restricted to a small proportion of adult rat cardiomyocytes, which appear intensely stained. A correlation, albeit not complete, was found between the distribution of alpha-skeletal actin and beta-myosin heavy chain. During cardiac hypertrophy induced by aortic ligature between the renal arteries, the expressions of alpha-skeletal actin mRNA and protein were increased. The distribution of immunostaining had a focal pattern similar to that of normal adult rats, reactive fibers being more numerous and more intensely stained compared with normal myocardium. Positive fibers were particularly abundant at the periphery of fibrotic areas. Using this antibody, we have demonstrated for the first time the differential distribution of alpha-skeletal actin in heart tissues. Changes in the distribution of this isoform in hypertrophic heart provide new insight into the mechanisms by which the heart adapts to work overload. This antibody will prove useful in exploring the mechanisms of expression of alpha-skeletal actin and in defining its role in physiological and pathological situations.

Fibre type-specific gene expression activated by chronic electrical stimulation of adult mouse skeletal muscle fibres in culture.

1. Fast-twitch skeletal muscle fibres were enzymatically dissociated from adult mouse flexor digitorum brevis (FDB) muscles and maintained in culture without or with chronic low frequency stimulation (one 5 s train of 5 Hz pulses per minute) for up to 6 days. Single fibre reverse transcriptase-polymerase chain reaction was conducted to coamplify beta-myosin heavy chain (beta-MHC) and alpha-skeletal actin mRNA from the same fibre. 2. Chronic low frequency electrical stimulation of FDB fibres in culture increased the level of mRNA for beta-MHC. In unstimulated fibres there was a slight decline in the beta-MHC mRNA level. As an internal control there was no increase in the level of mRNA for alpha-actin in the identical individual stimulated or unstimulated fibres. 3. Neither the percentage of fibres exhibiting beta-MHC protein nor the Ca2+ transients recorded from individual fibres subjected to the same pattern of stimulation showed any difference between stimulated and unstimulated fibres over the period in culture. 4. This system provides a convenient in vitro model system for studying activity-dependent control of fibre type-specific gene expression in adult skeletal muscle fibres in culture.

Effects of IGF-I on cardiac growth and expression of mRNAs coding for cardiac proteins after induction of heart hypertrophy in the rat.

Adult rat cardiomyocytes in long-term culture reexpress several fetal cardiac proteins which also reappear during overload heart hypertrophy in vivo. IGF-I decreases reexpression of some of these proteins and stimulates myofibrillogenesis. IGF-I might therefore contribute to enhancing readaptation of the heart to overload. In order to test this hypothesis, hypertension was induced in male Wistar Kyoto rats by constriction of the left renal artery, and an infusion of 500 microg/day of recombinant human IGF-I (rhIGF-I) or vehicle was started after the operation via intraabdominally implanted osmotic minipumps. In the vehicle-treated hypertensive animals body weight gain was reduced after 3, 7 and 14 days, whereas rhIGF-I-treated hypertensive animals continued to gain weight like sham-operated animals. Left ventricular weight and the left, but not the right ventricle/body weight ratio increased more in rhIGF-I- than in vehicle-infused rats. Left ventricular IGF-I mRNA levels remained unchanged after renal clipping in both vehicle- and rhIGF-I-treated rats. However, beta-myosin heavy chain (MHC) mRNA in the left ventricle was 6- to 10-fold increased in clipped controls during the whole postoperative period, and rhIGF-I reduced this increase by more than 50% on days 7 and 14. On the first postoperative day, rhIGF-I prevented the decrease (50%) of alpha-MHC mRNA and the increase (2.5-fold of atrial natriuretic factor mRNA in the left ventricle. Renal clipping did not alter cardiac alpha-actin, but enhanced skeletal alpha-actin mRNA expression in the left ventricle up to 2.5-fold. However, both mRNAs were unaffected by rhIGF-I treatment. Restoration of body weight gain and stimulation of left ventricular cardiac weight by rhIGF-I as well as partial reversion of hypertension-induced changes in cardiac protein expression may reflect beneficial effects contributing to enhance readaptation of the heart to overload.

Activation of the skeletal alpha-actin promoter during muscle regeneration.

Little is known concerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5-10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal alpha-actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal alpha-actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal alpha-actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal alpha-actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by -424 skeletal alpha-actin and -99 skeletal alpha-actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal alpha-actin promoter activities were similar to control values. Thus, initial activation of the skeletal alpha-actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal alpha-actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal alpha-actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box.

Temporal change in skeletal muscle IGF-I mRNA abundance and nitrogen metabolism responses to abomasal casein infusion in steers.

Skeletal muscle IGF-I and alpha-actin mRNA responses to increased amino acid availability were investigated in young, rapidly growing steers. Four Holstein steers (208 kg BW) were surgically implanted with an abomasal cannula and jugular catheters and allowed 2 wk to recover. Steers were offered hourly a 43:57 forage:concentrate diet at 95% of ad libitum intake supplemented with continuous abomasal infusion of glucose (to replace 12.5% of metabolizable ad libitum energy intake) for 13 d before the start of abomasal infusion of 67 g of casein N/d. Biopsies of the liver and both semimembranosus muscles were removed and frozen in liquid N, and casein infusion was begun. Muscle biopsies were collected at 8, 16, 24, and 48 h, and on d 7 and 14. Nitrogen balance increased from 23.6 to 71.5 g/d (P < .001) within 24 h and remained elevated (mean = 58.4 g/d) during the 14 d of casein infusion. Plasma urea N increased from 4 to 9.5 mg/dL at 24 h and remained unchanged to d 14. Muscle IGF-I mRNA abundance increased to 215% of basal values at 16 h (P < .01), 244% of basal values at 24 h, and 222% of basal values at 48 h after initiation of casein infusion. Values reached a maximum of 274% of basal values on d 7 and then declined to near preinfusion levels on d 14. The IGF-I mRNA abundance was approximately 100 times higher in liver than in skeletal muscle and was not different on d 0 and 14. Although plasma IGF-I concentrations were numerically higher during the first 24 h of abomasal casein infusion, they were not significantly higher during the chronic phase of treatment. Plasma IGF binding protein (BP)-2 concentrations were higher at 16, 24, and 48 h after casein infusion was begun, but IGFBP-3 concentrations were not altered at these sampling times. Neither acute (first 24 h) nor chronic (daily) plasma insulin concentrations were altered by abomasal casein infusion. Plasma somatotropin concentrations were lower (P = .008) at 24 h of casein infusion and beyond. Results suggest that enhanced amino acid availability may modulate skeletal muscle protein synthesis and accretion through an autocrine or paracrine IGF-I influence.

SRF and TEF-1 control of chicken skeletal alpha-actin gene during slow-muscle hypertrophy.

The purpose of this study was to delineate the alpha-actin regulatory elements and transcription factors that are responsible for conferring stretch-overload responsiveness during hypertrophy of the anterior latissimus dorsi (ALD) muscle of young chickens by weighting one wing. Minimal promoter constructs were evaluated by direct injection into the ALD, which demonstrated that both serum response element 1 (SRE1) and the transcriptional enhancer factor 1 (TEF-1) elements were sufficient for increased expression during stretch overload. A mutated SRE1 prevented expression in both basal and stretched ALD muscles, whereas a mutated TEF-1 element reduced actin promoter function in both control and stretched muscles. The serum response factor (SRF)-SRE1 binding complex demonstrated faster migration in mobility shift assays from day 3-and day 6-stretched ALD nuclear extracts relative to their control. TEF-1 binding was qualitatively increased in stretched extracts at day 3 but not day 6 of stretch overload. Skeletal alpha-actin mRNA accumulated from day 3 to day 6 of stretch overload. These data demonstrate that SRE1 is necessary and sufficient for stretch-overload responsiveness from the skeletal alpha-actin promoter and that the SRF-SRE1 binding complex migrates faster in stretched nuclear extracts of hypertrophied relative to control extracts from intact ALD muscles of chickens.

1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.

1,25(OH)2 Vitamin D3 (VD3) and retinoic acid (RA) function as ligands for nuclear receptors which regulate transcription. Though the cardiovascular system is not thought to represent a classical target for these ligands, it is clear that both cardiac myocytes and vascular smooth muscle cells respond to these agents with changes in growth characteristics and gene expression. In this study we demonstrate that each of these ligands suppresses many of the phenotypic correlates of endothelin-induced hypertrophy in a cultured neonatal rat cardiac ventriculocyte model. Each of these agents reduced endothelin-stimulated ANP secretion in a dose-dependent fashion and the two in combination proved to be more effective than either agent used alone (VD3: 49%; RA:52%; VD3 + RA:80% inhibition). RA, at concentrations known to activate the retinoid X receptor, and, to a lesser extent, VD3 effected a reduction in atrial natriuretic peptide, brain natriuretic peptide, and alpha-skeletal actin mRNA levels. Similar inhibition (VD3:30%; RA:33%; VD3 + RA:59% inhibition) was demonstrated when cells transfected with reporter constructs harboring the relevant promoter sequences were treated with VD3 and/or RA for 48 h. These effects were not accompanied by alterations in endothelin-induced c-fos, c-jun, or c-myc gene expression, suggesting either that the inhibitory locus responsible for the reduction in the mRNA levels lies distal to the activation of the immediate early gene response or that the two are not mechanistically coupled. Both VD3 and RA also reduced [3H]leucine incorporation (VD3:30%; RA:33%; VD3 + RA:45% inhibition) in endothelin-stimulated ventriculocytes and, once again, the combination of the two was more effective than either agent used in isolation. Finally, 1,25(OH)2 vitamin D3 abrogated the increase in cell size seen after endothelin treatment. These findings suggest that the liganded vitamin D and retinoid receptors are capable of modulating the hypertrophic process in vitro and that agents acting through these or similar signaling pathways may be of value in probing the molecular mechanisms underlying hypertrophy.

Regulation of expression of contractile proteins with cardiac hypertrophy and failure.

Transitions in sarcomeric alpha-actin and cardiac myosin heavy chain (MHC) gene expression have been useful as molecular markers for the development of cardiac hypertrophy and failure. In simpler model systems, alpha-actin expression has been useful in delineating some of the molecular pathways responsible for its induction following growth stimulation in vitro. In this study, we report that the effects of adrenergic agonists on alpha-actin expression in neonatal cardiocytes is dependent upon the culture conditions. In cardiocytes plated at 5 x 10(4) cells/cm2, skeletal alpha-actin mRNA levels represent 47%, 37% or 42% of total sarcomeric alpha-actin accumulations following administrations of 4 microM norepinephrine (NE), isoproterenol (Iso), or phenylephrine (PE), respectively. Cultured cardiocytes treated with vehicle (ascorbate) only accumulated 19% skeletal alpha-actin. Under these tissue culture conditions, in contrast to data reported previously, skeletal alpha-actin expression is regulated by both alpha- and beta-adrenergic agonist stimulation. Furthermore, we present data showing that an endogenous anti-beta-MHC transcript is regulated by both pressure-overload- or thyroxine-induced cardiac hypertrophy. Although anti-beta-MHC transcripts do not play a major role in regulating beta-MHC gene expression, the presence of this antisense transcript is associated with a novel set of beta-MHC degradation products. In vitro studies, where oligonucleotides complementary to beta-MHC have been introduced into cardiomyocytes, show that the mRNA levels of beta-MHC are decreased by 14-21% within 72 h after addition of the oligonucleotides. This result together with the presence of beta-MHC degradation products suggest that endogenous anti-beta-MHC transcripts may be involved in a post-transcriptional regulatory mechanism affecting the steady-state levels of beta-MHC expression.

Endothelin-3 induces hypertrophy of cardiomyocytes by the endogenous endothelin-1-mediated mechanism.

We have recently reported that endothelin-1 (ET-1) mediates angiotensin II-induced hypertrophy of cardiomyocytes as an autocrine/paracrine factor. In the present study, we examined whether endothelin-3 (ET-3) induces hypertrophy of cultured neonatal rat cardiomyocytes and whether endogenous ET-1 mediates this effect. ET-3 (10(-7) M) increased the cell surface area of cardiomyocytes after 48 h. ET-3 dose dependently (10(-9)-10(-7) M) stimulated protein synthesis as evaluated by [3H]leucine incorporation; the maximum response was 1.4-fold increase over the control at 10(-7) M. Since the response of cardiac hypertrophy is characterized by enhanced expression of fetal isoforms of muscle specific genes, the effect of ET-3 on steady state levels of mRNA for skeletal alpha-actin was evaluated by Northern blot analysis. ET-3 (10(-9)-10(-7) M) increased mRNA level for skeletal alpha-actin with a maximum response after 6 h. ET-3-induced [3H]leucine incorporation, skeletal alpha-actin mRNA and cell surface area were inhibited by a synthetic ETB receptor antagonist (BQ788). Interestingly, ET-3-induced skeletal alpha-actin gene expression and [3H]leucine incorporation were inhibited by a synthetic ETA receptor antagonist (BQ123) as well as by antisense oligonucleotides against peproET-1 mRNA. ET-3 (10(-7) M) transiently increased mRNA levels for ET-1 peaking at 30 min and stimulated the release of immunoreactive ET-1 from cardiomyocytes. These results suggest that endogenous ET-1 locally generated and secreted by cardiomyocytes may contribute to ET-3-induced cardiac hypertrophy as an autocrine/paracrine factor.

Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart

Chronic infusion of isoproterenol (Iso) in rats results in cardiac hypertrophy via incompletely understood mechanisms. Our purpose was to determine whether Iso infusion would alter the expression of genes associated with hypertrophy. Male Wistar rats received either 2.4 mg Iso.kg-1.day-1, 9.9 mg propranolol (Prop).kg-1.day-1, both Iso and Prop, or vehicle (NaCl) via subcutaneously implanted osmotic pumps. In Iso-treated rats, the ventricular weight-to-body weight ratio was increased by 27% after 1 day and peaked on day 3 (+ 40%). Levels of atrial natriuretic factor (ANF) and fibronectin (FN) mRNA in the left ventricles were elevated 20-fold and 13-fold in Iso-treated rats, respectively, peaking at 3 days of infusion. The increase in FN mRNA accumulation was at least partially accounted for by elevated expression of extra type IIIA and IIIB (EIIIA and EIIIB) splicing variants. Levels of transforming growth factor (TGF)-beta 1 mRNA were elevated twofold after 3 days of Iso infusion. The abundance of skeletal alpha-actin (SK) mRNA increased fourfold after 1 day of Iso and declined thereafter. Iso infusion decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and preproenkephalin (PNK) gene expression by approximately 50% and induced a myosin heavy chain (MHC) isogene switch favoring beta-MHC. Prop partially inhibited the Iso-evoked increases in ANF and FN mRNA, completely prevented the Iso-induced changes in TGF-beta 1 and SERCA mRNA, but had no effect on the Iso-stimulated changes in SK and PNK gene expression. These results demonstrate that chronic Iso infusion elicits alterations in cardiac gene expression that are consistent with the development of myocyte hypertrophy and interstitial fibrosis and are directionally identical to those previously reported for pressure overload hypertrophy.