Increased length-dependent activation of human engineered heart tissue after chronic α1A-adrenergic agonist treatment: testing a novel heart failure therapy.

Chronic stimulation of cardiac α1A-adrenergic receptors (α1A-ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. Human engineered heart tissues (EHTs) provide a means of quantifying the effects of chronic α1A-AR stimulation on human cardiomyocyte physiology. EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. With a paired experimental design, EHTs were cultured for 3 wk, mechanically tested, cultured again for 2 wk with α1A-AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24 h. Separate control experiments determined the effects of EHT age (3-5 wk) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared with vehicle treatment (n = 7/group, P = 0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased by 35% relative to baseline testing (n = 7/group, P = 0.022), suggesting EHT maturation. Control experiments suggested that increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed that the α1A-AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways known to be activated by α1A-ARs, including the MAP kinase signaling pathway. In conclusion, increased LDA in human EHT after chronic A61603 treatment raises the possibility that chronic stimulation of the α1A-AR might be beneficial for increasing LDA in human myocardium and might be beneficial for treating human heart failure by restoring LDA.NEW & NOTEWORTHY Chronic stimulation of α1A-adrenergic receptors (α1A-ARs) is known to mediate therapeutic effects in animal heart failure models. To investigate the effects of chronic α1A-AR stimulation in human cardiomyocytes, we tested engineered heart tissue (EHT) created with iPSC-derived cardiomyocytes. RNA-seq analysis confirmed human EHT expressed α1A-ARs. Chronic (2 wk) α1A-AR stimulation with A61603 (10 nM) increased length-dependent activation (LDA) of contraction. Chronic α1A-AR stimulation might be beneficial for treating human heart failure by restoring LDA.

The cardiac beta-myosin heavy chain isogene is induced selectively in alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes.

Cardiac hypertrophy produced in vivo by pressure overload is characterized by selective up-regulation of the fetal/neonatal beta-cardiac myosin heavy chain (MHC) isogene. However, a molecular signal for beta-MHC isogene induction has not been identified. We examined cardiac MHC isogene expression in a cell culture model for hypertrophy. alpha-MHC and beta-MHC iso-protein and iso-mRNA levels in cultured cardiac myocytes were quantified during hypertrophy stimulated by the alpha 1-adrenergic agonist, norepinephrine (NE). beta-MHC iso-protein content was increased 3.2-fold vs. control (P less than 0.001), whereas alpha-MHC isoprotein content was not changed significantly (1.4-fold vs. control, P = NS). MHC iso-mRNA levels were quantified by nuclease S1 analysis, using a single oligonucleotide probe. NE increased beta-MHC iso-mRNA content by 3.9-fold vs. control (P less than 0.001), but there was no change in alpha-MHC iso-mRNA (1.1-fold vs. control, P = NS). The NE-stimulated increase in beta-MHC iso-mRNA preceded in time the increase in beta-MHC isoprotein accumulation. The EC50 for NE induction of beta-MHC was 40 nM, and pharmacologic experiments indicated alpha 1-adrenergic receptor specificity. alpha-MHC isogene expression was predominant in control myocytes (68% alpha-isoprotein and 60% alpha-iso-mRNA). In contrast, beta-MHC expression was equal to alpha-MHC or predominant after treatment with NE (51% beta-isoprotein and 69% beta-iso-mRNA). Thus, alpha 1-adrenergic receptor stimulation increases the cellular contents of beta-MHC iso-mRNA and beta-MHC isoprotein during hypertrophy of cultured neonatal rat cardiac myocytes, but does not change the levels of alpha-MHC iso-mRNA or isoprotein. The effect on beta-MHC is mediated primarily at the level of mRNA steady-state level (pretranslational). Activation of the alpha 1-adrenergic receptor is the first identified molecular signal for increased beta-MHC isogene expression in a model of cardiac hypertrophy.

Induction of the skeletal alpha-actin gene in alpha 1-adrenoceptor-mediated hypertrophy of rat cardiac myocytes.

Myocardial hypertrophy in vivo is associated with reexpression of contractile protein isogenes characteristic of fetal and neonatal development. The molecular signals for hypertrophy and isogene switching are unknown. We studied alpha (sarcomeric)-actin messenger RNA (mRNA) expression in cultured cardiac myocytes from the neonatal rat. In the cultured cells, as in the adult heart in vivo, expression of cardiac alpha-actin (cACT) predominated over that of skeletal alpha-actin (sACT) mRNA, the fetal/neonatal isoform. alpha 1-Adrenergic receptor stimulation induced hypertrophy of these cells, increasing total RNA and cytoskeletal actin mRNA by 1.8-fold over control, and total alpha-actin mRNA by 4.3 fold. This disproportionate increase in total alpha-actin mRNA was produced by a preferential induction of sACT mRNA, which increased by 10.6-fold over control versus only 2.6-fold for cACT mRNA. The alpha 1-adrenoceptor is the first identified molecular mediator of early developmental isogene reexpression in cardiac myocyte hypertrophy.

Alpha 1-adrenergic receptor stimulation of sarcomeric actin isogene transcription in hypertrophy of cultured rat heart muscle cells.

During pressure-load hypertrophy of the adult heart in vivo, there is up-regulation of the mRNA encoding skeletal alpha-actin, the sarcomeric actin iso-mRNA characteristic of mature skeletal muscle and the fetal/neonatal heart. We have shown previously that during alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes, the induction of skeletal alpha-actin mRNA is greater than that of the mRNA encoding cardiac alpha-actin, the sarcomeric actin iso-mRNA characteristic of the adult heart. To determine if this actin iso-mRNA switch during cardiac hypertrophy reflects changes in the transcriptional status of the myocyte nucleus, we quantified the rate of transcription of actin mRNAs and total RNA, using an in vitro run-on transcription assay with nuclei isolated from the cultured myocytes after stimulation with norepinephrine (NE). Transcription of skeletal alpha-actin was increased at 3 h after NE, reached a maximum 6.1-fold increase at 12 h, and returned to the control level at 24 h. The EC50 for NE was 200 nM, and pharmacologic studies indicated alpha 1-receptor specificity. Transcription of cardiac alpha-actin was also increased rapidly by NE (maximum 4.6-fold vs. control at 3 h). However, cardiac alpha-actin transcription had returned to the control level at 6 h, when NE-stimulated skeletal alpha-actin transcription was still increasing. Transcription of the cytoskeletal (beta) actin gene was not changed significantly by NE treatment. Total RNA transcription was not increased until 6 h after NE (1.5-fold vs. control) and remained elevated through 24 h. Inhibition of protein synthesis did not attenuate NE-stimulated actin gene transcription. Thus the alpha 1-adrenoceptor mediates a rapid, transient, and selective increase in transcription of the sarcomeric actin isogenes during cardiac myocyte hypertrophy. Skeletal alpha-actin, the fetal/neonatal isogene, is induced preferentially to cardiac alpha-actin, the adult isogene. The different kinetics of actin isogene and total RNA transcription and the independence of transcription from protein synthesis suggest that transcriptional induction via the alpha 1 receptor is complex and may involve preexisting regulatory factors. These results are the first to demonstrate that the alpha 1-adrenergic receptor is a molecular mediator of transcriptional changes underlying an isogene switch that is known to be associated with cardiac myocyte hypertrophy.

Thyroid hormone improves function and Ca2+ handling in pressure overload hypertrophy. Association with increased sarcoplasmic reticulum Ca2+-ATPase and alpha-myosin heavy chain in rat hearts.

We asked whether thyroid hormone (T4) would improve heart function in left ventricular hypertrophy (LVH) induced by pressure overload (aortic banding). After banding for 10-22 wk, rats were treated with T4 or saline for 10-14 d. Isovolumic LV pressure and cytosolic [Ca2+] (indo-1) were assessed in perfused hearts. Sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, and alpha- and beta-myosin heavy chain (MHC) proteins were assayed in homogenates of myocytes isolated from the same hearts. Of 14 banded hearts treated with saline, 8 had compensated LVH with normal function (LVHcomp), whereas 6 had abnormal contraction, relaxation, and calcium handling (LVHdecomp). In contrast, banded animals treated with T4 had no myocardial dysfunction; these hearts had increased contractility, and faster relaxation and cytosolic [Ca2+] decline compared with LVHcomp and LVHdecomp. Myocytes from banded hearts treated with T4 were hypertrophied but had increased concentrations of alpha-MHC and SERCA proteins, similar to physiological hypertrophy induced by exercise. Thus thyroid hormone improves LV function and calcium handling in pressure overload hypertrophy, and these beneficial effects are related to changes in myocyte gene expression. Induction of physiological hypertrophy by thyroid hormone-like signaling might be a therapeutic strategy for treating cardiac dysfunction in pathological hypertrophy and heart failure.

Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes. Comparison with rat.

Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a beta-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [(3)H]phenylalanine-labeled protein content. T3 and NE also increased alpha-myosin heavy chain (MyHC) mRNA and reduced beta-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including alpha(1)-adrenergic agonists, endothelin-1, prostaglandin F(2alpha), interleukin 1beta, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or beta-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal-regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and beta-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C-coupled agonists do not.

Regulation of thyroid hormone receptor isoforms in physiological and pathological cardiac hypertrophy.

Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of thyroid hormone (TH)-responsive genes, including alpha- and beta-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) beta1, alpha1, and alpha2 in pathological and physiological rat cardiac hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH target genes, alpha- and beta-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TRbeta1 was upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and promoters. In addition, TR cotransfection and treatment with the TRbeta1-selective agonist GC-1 suggested different functional coupling of the TR isoforms, TRbeta1 to transcription of beta-MyHC, SERCA, and TRbeta1, and TRalpha1 to alpha-MyHC transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in physiological and pathological cardiac hypertrophy.

Tumor cell splice variants of the transcription factor TEF-1 induced by SV40 T-antigen transformation.

The large tumor antigen (TAg) of simian virus 40 is able to transform cells through interactions with cellular proteins, notably p53 and Rb. Among the other proteins that form complexes with TAg is TEF-1, a transcription factor utilized by the viral enhancer to activate expression of the early gene which encodes TAg. We show that fibroblasts contain several alternately spliced TEF-1 mRNAs, the most abundant of which encodes a protein with an additional four amino acid exon compared to the database entry for Hela cell TEF-1. Transformation by TAg induces alternate splicing, producing a more abundant form lacking this exon and matching the published sequence. Splicing variants lacking this exon were detected in mouse pancreatic tumors and in cell lines derived from human pancreatic cancers, in contrast to a single isoform with the exon in normal mouse pancreas. A total of eight splice variants were identified, with the loss of the four amino acid exon typical of transformed cells. These and other data presented suggest that TAg 're-models' host cell transcription factors that are used early in viral infection, and thereby mimics an event that naturally occurs during transformation. The data indicate that TEF-1 alterations may be a hallmark feature of tumorigenesis.

Mechanisms and time course of beta 1 adrenoceptor desensitisation in mammalian cardiac myocytes.

To study the mechanisms and time course of beta1 adrenoceptor desensitisation in mammalian heart tissue neonatal rat cardiac myocytes (greater than 90% pure) were cultured in serum free medium. Cells were exposed to 1 mumol X litre-1 (-)-isoprenaline for 30 min, 4 h, and 16 h. In myocyte membranes mean(SEM)) 125I-iodocyanopindolol binding was 167(46) pmol X litre-1 (n = 5) and did not differ at 30 min, 4 h, or 16 h in control compared with (-)-isoprenaline treated cells. The maximum number of binding sites was 84(32) fmol X mg protein-1 and was unchanged at 30 min, but (-)-isoprenaline stimulated adenylate cyclase activity significantly decreased from 221(62) to 103(37) pmol X mg protein-1 30 min-1. (-)-Isoprenaline competition curves at 30 min showed a significant increase in the proportion of low affinity binding sites from 46% to 62% (n = 5). By 4 h the maximum number of binding sites was significantly decreased by 54%, adenylate cyclase activity remained depressed, and agonist affinity decreased threefold in the (-)-isoprenaline treated cells. At 16 h (-)-isoprenaline treated cells showed alterations similar to the 4 h values in the maximum number of binding sites, adenylate cyclase activity, and affinity for (-)-isoprenaline. (-)-Isoprenaline stimulated adenylate cyclase activity took 72 h to recover after desensitisation. Overnight ultracentrifugation of the cytosol showed a significant 40% increase in beta adrenoceptor density in cells exposed to (-)-isoprenaline for 4 h (n = 5), suggesting receptor internalisation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of proto-oncogenes in myocardial hypertrophy.

A question of major clinical significance in cardiology is the nature of the signals that initiate and maintain the various types of myocardial hypertrophy, either in response to hemodynamic loading or in the absence of altered load. This review suggests that the proto-oncogene model, a concept derived from the study of cancer, can be very useful in identifying these signals. The proto-oncogene model conceives of cell growth regulation in terms of a limited number of classes of critical regulatory proteins: growth factors, growth factor receptors, intracellular transducing proteins and ribonucleic acid (RNA) transcription factors. Growth of all cells has dissociable components: hypertrophy (growth in size), deoxyribonucleic acid synthesis, mitosis and cytokinesis. Hypertrophy may be the end result of activation of RNA transcription. The various types of hypertrophy could reflect transcription of specific myocyte genes in response to different growth factors. At least 1 member of each functional class of proto-oncogenes has been detected in the myocardium or myocytes, or both. The alpha 1-adrenergic receptor has been shown to be a growth factor receptor and to regulate RNA transcription. Continued work on proto-oncogenes in myocytes may open the way to manipulate the growth of these cells.

Trophic factors for cardiac myocytes.

During early neonatal myocardial growth, cardiac myocytes undergo a terminal differentiation stage after which cellular proliferation no longer occurs. Subsequent growth occurs by means of enlargement/hypertrophy of the existing cells. In an effort to dissect the molecular mechanisms underlying myocardial hypertrophy, a cell culture model of cardiac hypertrophy was developed in our laboratory. In this model, neonatal rat heart muscle cells respond to alpha 1-adrenergic receptor stimulation with an increase in cell size, total protein and the cellular contents of several messenger (m)RNA encoding fetal/neonatal contractile protein isoforms, and also that encoding the proto-oncogene c-myc. Similar changes in gene expression are seen in pressure-load hypertrophy in vivo. Recent observations on the production of peptide growth factors by the myocardium suggest an additional role of cell-cell interaction in cardiac growth and development. Some of the growth factors which have been found in normal myocardium include platelet-derived growth factor B chain, fibroblast growth factor (both acidic and basic), transforming growth factor beta, insulin-like growth factor-1 and insulin-like growth factor-2, and nerve growth factor. We recently identified a heparin-binding growth factor produced by cardiac non-myocytes in culture, which acts in a dose-dependent fashion to produce hypertrophy of cardiac muscle cells in culture. Preliminary work suggests that this is dissimilar from previously identified growth factors. Furthermore, the quantitative response of the cardiac myocytes to our growth factor exceeds that seen for other factors. Further work is necessary to ascertain how these factors and the alpha 1-adrenergic system interact to produce the different clinical forms of myocardial hypertrophy.

Alpha1A/B-knockout mice explain the native alpha1D-adrenoceptor's role in vasoconstriction and show that its location is independent of the other alpha1-subtypes.

BACKGROUND AND PURPOSE: Theoretically, three alpha(1)-adrenoceptor subtypes can interact at the signalling level to alter vascular contraction or at the molecular level to alter each other's cellular location. The alpha(1A/B)-adrenoceptor knockout mouse (alpha(1A/B)-KO) was used to study the isolated alpha(1D)-adrenoceptor to consider these potential interactions in native tissue. EXPERIMENTAL APPROACH: Pharmacological analysis of carotid and mesenteric arteries employed wire myography and fluorescent ligand binding (alpha(1)-adrenoceptor ligand BODIPY FL-prazosin, QAPB). KEY RESULTS: alpha(1A/B)-KO carotid had clear alpha(1D)-adrenoceptor-induced contractions. In WT carotid alpha(1D)-adrenoceptor dominated but all three alpha(1)-subtypes participated. alpha(1A/B)-KO mesenteric had alpha(1D)-adrenoceptor responses with high sensitivity and small maximum, explaining how alpha(1D)-adrenoceptor could determine agonist sensitivity in WT. In both arteries alpha(1A/B)-KO fluorescence levels were reduced but pharmacologically more consistent with 'pure'alpha(1D)-adrenoceptors. alpha(1D)-Adrenoceptor binding in alpha(1A/B)-KO was observed on the cell surface and intracellularly and was present in a high proportion of smooth-muscle cells in both strains, regardless of artery type. CONCLUSIONS AND IMPLICATIONS: 'Pure'alpha(1D)-adrenoceptor pharmacology in alpha(1A/B)-KO provides a quantitative standard. Functionally, the alpha(1D)- and alpha(1A)-adrenoceptors produce additive responses and do not significantly compensate for each other. alpha(1D)-Adrenoceptor contributes to sensitivity even in resistance arteries. In alpha(1A/B)-KO, the loss of alpha(1A)- and alpha(1B)-adrenoceptors is reflected by a general decrease in fluorescence, but similar binding distribution to WT indicates that the alpha(1D)-adrenoceptor location in native smooth-muscle cells is not influenced by other alpha(1)-adrenoceptors. Equivalent levels of receptors did not correspond to equivalent responses. In conclusion, alpha(1)-subtypes do not interact but provide independent alternative signals for vascular regulation.

Identification of alpha 1L-adrenoceptor in mice and its abolition by alpha 1A-adrenoceptor gene knockout.

BACKGROUND AND PURPOSE: The alpha(1L)-adrenoceptor has pharmacological properties that distinguish it from three classical alpha(1)-adrenoceptors (alpha(1A), alpha(1B) and alpha(1D)). The purpose of this was to identify alpha(1L)-adrenoceptors in mice and to examine their relationship to classical alpha(1)-adrenoceptors. EXPERIMENTAL APPROACH: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and alpha(1A)-, alpha(1B)- and alpha(1D)-adrenoceptor gene knockout (AKO, BKO and DKO) mice. KEY RESULTS: The radioligand [(3)H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of alpha(1A)-adrenoceptors and alpha(1L)-adrenoceptors. In membrane preparations of WT mouse cortex, however, [(3)H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of alpha(1A)-adrenoceptors alone. In contrast, [(3)H]-prazosin bound to two components having alpha(1A)-adrenoceptor and alpha(1B)-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked alpha(1A)-adrenoceptor profiles and BKO mice lacked alpha(1B)-adrenoceptor profiles. Noradrenaline produced contractions through alpha(1L)-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice. CONCLUSIONS AND IMPLICATIONS: alpha(1L)-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the alpha(1L)-adrenoceptor is one phenotype derived from the alpha(1A)-adrenoceptor gene.

Ischemic preconditioning depends on age and gender.

UNLABELLED: The goal of this study was to determine if an ischemic preconditioning (IPC) protocol improved post-ischemic functional recovery of female mouse hearts. A previous study found that IPC did not occur in hearts from 10-week-old females. We studied Langendorff-perfused hearts from both 10- and 18-week-old mice (males and females). Hearts were subjected to 45 min ischemia and 45 reperfusion (I/R); IPC involved pretreatment with 3 min ischemia. We measured hemodynamics, infarct size and levels of the phosphorylated prosurvival kinase Akt (p-Akt). Similar to a previous study, for 10- week-old mice we found that the IPC protocol appreciably improved recovery of LV developed pressure (LVDP) for hearts from males but not females. However, for 18-week-old mice we found that the IPC protocol doubled the recovery of LVDP for both males and females. For both ages, hearts from females had greater recovery of LVDP and higher levels of p-Akt compared to males. CONCLUSIONS: These findings are consistent with growing evidence that preconditioning induced by ischemia or other interventions can occur in hearts from females. However, for hearts from females, preconditioning depends on age. Moreover, consistent with previous studies, hearts from females have greater inherent resistance to ischemic injury, possibly involving increased signaling via p-Akt.

Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to alpha 1-adrenergic and phorbol ester stimulation.

Both alpha 1-adrenergic agonists (e.g. norepinephrine, NE*) and tumor-promoting phorbol esters (e.g. phorbol myristate acetate, PMA) are known to activate protein kinase C (PKC) (Abdel-Latif, 1986, Niedel and Blackshear, 1986). However, alpha 1 agonists and PMA produce very different effects on cardiac function (see Simpson, 1985; Benfey, 1987; Meidell et al., 1986; Leatherman et al., 1987; Yuan et al., 1987; for examples). PKC activation in heart cells has been studied only for PMA treated perfused heart (Yuan et al., 1987). Therefore, acute activation and chronic regulation of PKC by NE and PMA were compared in cultured neonatal rat heart myocytes. NE acutely and transiently activated PKC, as measured by translocation of PKC activity to the cell particulate fraction (Niedel and Blackshear, 1986). Particulate PKC activity peaked at 23% of total after NE for 30 s, as compared with 8% for control (P less than 0.001). By contrast, acute PKC activation by PMA was more pronounced and persistent, with particulate PKC activity 62% of total at 5 min (P less than 0.001). Calcium/lipid-independent kinase activity increased acutely with PMA, but not with NE. Chronic treatment with NE (24 to 48 h) increased total per cell PKC activity and 3H-phorbol dibutyrate (PDB) binding sites, an index of the number of PKC molecules (Niedel and Blackshear, 1986), by 30 to 60% over control (all P less than 0.05 to 0.01). In contrast with NE, chronic treatment with PMA down-regulated PKC, reducing total per cell PKC activity and 3H-PDB binding sites to 3% and 12% of control, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenoceptor and adenylate cyclase regulation in cardiac myocyte growth.

We studied the effect of growth on beta-adrenergic receptor properties of neonatal rat heart myocytes cultured in serum-free medium with transferrin and insulin. Growth was induced by addition of 1 microM (-)-norepinephrine for two days, 200 nM of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for two days, or 30 nM T3 for six days. The Kd values for beta-receptor binding (125I-ICYP) were unaffected by growth. The maximum number of beta-receptor binding sites calculated as sites/cell was increased 1.47-fold by T3 (p less than .005), but was decreased to 54% of control values by (-)-norepinephrine (p less than .005): TPA had no effect on either Kd or Bmax values. (-)-Isoproterenol-stimulated adenylate cyclase activity was augmented only in membranes from T3-treated cells and was reduced by 69% in membranes from (-)-norepinephrine treated cells. TPA had no effect on (-)-isoproterenol-stimulated adenylate cyclase activity. We conclude that the mechanisms controlling beta-adrenergic receptor number may be distinct from those controlling growth, since receptor number does not correlate with cell enlargement. Furthermore, in (-)-norepinephrine-stimulated growth, which we have shown previously is an alpha 1-adrenoceptor mediated response, beta-adrenergic signal transduction is modulated in a directionally opposite fashion.

scyllo-inositol and myo-inositol levels in tissues of the skate Raja erinacea.

1. Many organs of the skate Raja erinacea have been found to contain scyllo-inositol levels that are much higher than myo-inositol, the opposite to that found in mammals. 2. Both inositols were found in all skate organs studied. 3. myo-Inosose-2 was found to accompany the inositols in many of these organs.

A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.

Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.

Effects of pertussis toxin on alpha 1-agonist-mediated phosphatidylinositide turnover and myocardial cell hypertrophy in neonatal rat ventricular myocytes.

In neonatal rat ventricular myocytes pretreatment with pertussis toxin did not affect 1 microM (-)-norepinephrine stimulation of inositol phosphates or myocardial cell hypertrophy as measured either by protein radiolabelling or by myocardial cell protein content. Thus guanine nucleotide protein(s) ADP-ribosylated by pertussis toxin do not play a role in two alpha 1-adrenoceptor-mediated processes, phosphatidylinositide turnover and induction of myocardial cell hypertrophy.