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.

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.

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.

LV systolic performance improves with development of hypertrophy after transverse aortic constriction in mice.

Transverse aortic constriction (TAC) is an effective technique for inducing left ventricular (LV) hypertrophy in mice. With the use of transthoracic echocardiography and Doppler measurements, we studied the effects of an acute increase in pressure overload on LV contractile performance and peak systolic wall stress index (WSI) at early time points after TAC and the time course of the development of LV hypertrophy in mice. The LV mass index was similar between TAC and sham-operated mice at postoperative day 1 but progressively increased in TAC mice by day 10. There was no further increase in the LV mass index between postoperative days 10 and 20. On day 1, whereas peak systolic WSI increased significantly, the LV ejection fraction (LVEF) and percent fractional shortening (%FS) decreased in TAC mice compared with sham-operated mice. By day 10, peak systolic WSI, LVEF, and %FS had recovered to baseline levels and were not significantly different between postoperative days 10 and 20. Thus LV systolic performance in mice declines immediately after TAC, associated with increased peak systolic WSI, but recovers to baseline levels with the development of compensatory LV hypertrophy over 10-20 days.

Cloning and characterization of the mouse alpha1C/A-adrenergic receptor gene and analysis of an alpha1C promoter in cardiac myocytes: role of an MCAT element that binds transcriptional enhancer factor-1 (TEF-1).

alpha1-Adrenergic receptor (AR) subtypes in the heart are expressed by myocytes but not by fibroblasts, a feature that distinguishes alpha1-ARs from beta-ARs. Here we studied myocyte-specific expression of alpha1-ARs, focusing on the subtype alpha1C (also called alpha1A), a subtype implicated in cardiac hypertrophic signaling in rat models. We first cloned the mouse alpha1C-AR gene, which consisted of two exons with an 18 kb intron, similar to the alpha1B-AR gene. The receptor coding sequence was >90% homologous to that of rat and human. alpha1C-AR transcription in mouse heart was initiated from a single Inr consensus sequence at -588 from the ATG; this and a putative polyadenylation sequence 8.5 kb 3' could account for the predominant 11 kb alpha1C mRNA in mouse heart. A 5'-nontranscribed fragment of 4.4 kb was active as a promoter in cardiac myocytes but not in fibroblasts. Promoter activity in myocytes required a single muscle CAT (MCAT) element, and this MCAT bound in vitro to recombinant and endogenous transcriptional enhancer factor-1. Thus, alpha1C-AR transcription in cardiac myocytes shares MCAT dependence with other cardiac-specific genes, including the alpha- and beta-myosin heavy chains, skeletal alpha-actin, and brain natriuretic peptide. However, the mouse alpha1C gene was not transcribed in the neonatal heart and was not activated by alpha1-AR and other hypertrophic agonists in rat myocytes, and thus differed from other MCAT-dependent genes and the rat alpha1C gene.

Abnormal contraction caused by expression of G(i)-coupled receptor in transgenic model of dilated cardiomyopathy.

Although increased G(i) signaling has been associated with dilated cardiomyopathy in humans, its role is not clear. Our goal was to determine the effects of chronically increased G(i) signaling on myocardial function. We studied transgenic mice that expressed a G(i)-coupled receptor (Ro1) that was targeted to the heart and regulated by a tetracycline-controlled expression system. Ro1 expression for 8 wk resulted in abnormal contractions of right ventricular muscle strips in vitro. Ro1 expression reduced myocardial force by >60% (from 35 +/- 3 to 13 +/- 2 mN/mm(2), P < 0.001). Nevertheless, sensitivity to extracellular Ca(2+) was enhanced. The extracellular [Ca(2+)] resulting in half-maximal force was lower with Ro1 expression compared with control (0.41 +/- 0.05 vs. 0.88 +/- 0.05 mM, P < 0.001). Ro1 expression slowed both contraction and relaxation kinetics, increasing the twitch time to peak (143 +/- 6 vs. 100 +/- 4 ms in control, P < 0.001) and the time to half relaxation (124 +/- 6 vs. 75 +/- 6 ms in control, P < 0.001). Increased pacing frequency increased contractile force threefold in control myocardium (P < 0.001) but caused no increase of force in Ro1-expressing myocardium. When stimulation was interrupted with rests, postrest force increased in control myocardium, but there was postrest decay of force in Ro1-expressing myocardium. These results suggest that defects in contractility mediated by G(i) signaling may contribute to the development of dilated cardiomyopathy.

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.

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.

Turn geometry for minimizing band broadening in microfabricated capillary electrophoresis channels.

Turns in microfabricated capillary electrophoresis channels generally result in degraded separation quality. To circumvent this limitation, channels were constructed with different types of turns to determine the design that minimizes turn-induced band broadening. In particular, tapered turns were created by narrowing the separation channel width before the start of a turn and widening the channel after the turn is complete. The radius of curvature of the turn, the length over which the channel is tapered, and the degree of tapering were explored. The column efficiencies were determined by examining the resolution of the 271/281 base pair doublet in the separation of a phiX174 HaeIII DNA sizing ladder. Tapered turns with the smallest radius of curvature (250 microm), the shortest tapering length between the separation and turn widths (55 microm), and the largest tapering ratio (4:1 separation channel width to turn channel width) produced the highest resolution separations. These results are discussed by comparison to theoretical predictions of the effect of tapers and turns on analyte band dispersion in capillary electrophoresis.

Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis.

The design, fabrication, and operation of a radial capillary array electrophoresis microplate and scanner for high-throughput DNA analysis is presented. The microplate consists of a central common anode reservoir coupled to 96 separate microfabricated separation channels connected to sample injectors on the perimeter of the 10-cm-diameter wafer. Detection is accomplished by a laser-excited rotary confocal scanner with four color detection channels. Loading of 96 samples in parallel is achieved using a pressurized capillary array system. High-quality separations of 96 pBR322 restriction digest samples are achieved in < 120 s with the microplate system. The practical utility and multicolor detection capability is demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel using a noncovalent 2-color staining method. This work establishes the feasibility of performing high-throughput genotyping separations with capillary array electrophoresis microplates.

Post-infarction heart failure in the rat is associated with distinct alterations in cardiac myocyte molecular phenotype.

The myocardial molecular and cellular responses to hemodynamic and other hypertrophic stimuli have been characterized extensively, but less is known of the alterations in gene expression during the evolution of heart failure following myocardial infarction, and specifically those affecting the cardiac myocytes. Therefore, the present study was undertaken to test the hypothesis that post-infarction heart failure and remodeling in the rat is associated with a distinct myocyte molecular phenotype. To address this question, hemodynamic measurements were performed in vivo; and myocytes isolated from the non-infarcted myocardium 1 day, 1 week, and 6 weeks post-coronary artery ligation in post-infarct rats and sham controls. Myocyte size, mRNA levels for immediate early genes, contractile proteins, and sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban were assayed by Northern analyses, and SERCA and phospholamban proteins were examined by Western blotting. Hemodynamic evidence of heart failure was present at all post-infarct time points. Myocyte size was increased significantly at 6 weeks. c-myc expression was increased at 1 day and 1 week in the infarcted rats, but returned to baseline by 6 weeks. Atrial natriuretic peptide and VEGF mRNAs were elevated at 1 and 6 weeks. Both beta-myosin heavy chain and skeletal alpha-actin expression were increased at all post-MI time points. In contrast, neither changes in the expression of the calcium-handling proteins (SERCA and phospholamban) were not observed, nor was there a change in TGFbeta1 or TGFbeta3. These results demonstrate that in rats with post-MI heart failure, there was an immediate induction of the fetal/embryonic transcriptional gene program which preceded myocyte hypertrophy and appeared to persist longer than in pressure-overload models. In further contrast to pressure-overload, expression of sarcoplasmic reticulum Ca2+-ATPase and phospholamban, was not altered despite a comparable degree of cellular hypertrophy and more severe hemodynamic decompensation. These findings suggest that there may be important differences in the regulatory mechanisms underlying these two forms of myocardial hypertrophy and heart failure.

Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure.

Growing evidence suggests that cardiac nonmyocyte cells may play an important regulatory role in the response to myocardial overload and injury via altered expression of paracrine products, such as cytokines and growth factors, but information concerning the cell-specific changes in the expression of these substances in heart-failure models is limited. Therefore, cardiac nonmyocytes were isolated from rats 1 day and 1 and 6 wk after left coronary artery ligation with resulting hemodynamic evidence of heart failure and in sham-operated control animals. mRNAs for tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, transforming growth factors (TGF)-beta1 and TGF-beta3, and type I and type III collagen were measured by Northern analyses. The temporal and quantitative relationships between the expression of these cytokines and collagen and myocyte hypertrophy were determined. mRNA expression of IL-1beta was increased by 1.3-fold at 1 day and 1 wk, and expression of TNF-alpha, IL-1beta, IL-6, TGF-beta1, and TGF-beta3 were increased by 1.4- to 2.1-fold at the 1-wk time point before returning toward baseline at 6 wk. There were significant correlations between the expression of these cytokines and the expression of types I and III collagen, which also peaked at 1 wk. Myocyte hypertrophy was seen first at 6 wk. These observations are consistent with a hypothesis that nonmyocyte cells play a regulatory role in the extracellular matrix changes during postinfarction remodeling and highlight the importance of examining cell-specific changes in gene expression and elucidating the role of cell-to-cell interactions within the myocardium.

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates.

Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal fluorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and fluorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

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.

Alpha1-adrenergic receptor subtype mRNAs are differentially regulated by alpha1-adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo. Repression of alpha1B and alpha1D but induction of alpha1C.

The three cloned alpha1-adrenergic receptor (AR) subtypes, alpha1B, alpha1C, and alpha1D, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three alpha1-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the alpha1-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12 72 h). Alpha1B and alpha1D mRNA levels were repressed by NE, whereas alpha1C mRNA was induced. Changes in mRNA levels were mediated by an alpha1-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [3H]prazosin binding. alpha1-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2alpha, and phorbol 12-myristate 13-acetate, also induced alpha1C mRNA and repressed alpha1B mRNA. In myocytes from hearts with pressure overload hypertrophy, alpha1 mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among alpha1-AR subtypes expressed natively in the same cell. Transcriptional induction of the alpha1C-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.

M-CAT, CArG, and Sp1 elements are required for alpha 1-adrenergic induction of the skeletal alpha-actin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth.

Induction of the fetal isogenes skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) is characteristic of cardiac growth in many models, suggesting a conserved signaling pathway. However, divergent regulation has also been observed. beta-Protein kinase C (PKC) and transcriptional enhancer factor-1 (TEF-1) are involved in induction of beta-MHC in alpha 1-adrenergic-stimulated hypertrophy of cultured cardiac myocytes (Kariya, K., Farrance, I.K. G., and Simpson, P.C. (1993) J. Biol. Chem. 268, 26658-26662; Kariya, K., Karns, L. R., and Simpson, P.C. (1994) J. Biol. Chem. 269, 3775-3782). In the present study, we asked whether the skACT promoter used the same mechanism. A mouse skACT promoter fragment (-113/-46) was induced by both alpha 1-adrenergic stimulation and co-transfection of activated beta-PKC, and contained three required DNA sequence elements: M-CAT, CArG, and Sp1. The skACT M-CAT element bound TEF-1 in cardiac myocytes. Thus the skACT and beta-MHC promoters both require a TEF-1 binding site for activation by alpha 1-adrenergic stimulation, but differ in that skACT also requires a CArG box. These results provide a potential molecular basis for divergent regulation of the fetal program, and also imply that PKC and TEF-1 are conserved transducers for this program during cardiac growth.

Cloning of the rat alpha 1C-adrenergic receptor from cardiac myocytes. alpha 1C, alpha 1B, and alpha 1D mRNAs are present in cardiac myocytes but not in cardiac fibroblasts.

alpha 1-Adrenergic receptor (AR) activation in cardiac muscle has several different physiological effects that might be mediated through different alpha 1-AR subtypes. Two alpha 1-AR subtypes have been cloned from the rat, the alpha 1B and the alpha 1D; both are present in adult rat heart. A third subtype, the alpha 1C, cloned from the cow and human, was reported to be absent in the rat. However, we recently found alpha 1C mRNA in adult rat heart by using a partial alpha 1C cDNA. Thus, all three cloned alpha 1-AR subtypes are present in the heart, but it is unknown whether each is expressed in cardiac myocytes or in cardiac fibroblasts. In the present study, the full-length rat alpha 1C-AR was cloned from cultured neonatal cardiac myocytes. alpha 1C mRNA transcripts of 3, 9.5, and 11 kb were present in adult rat heart by Northern blot analysis. alpha 1B-, alpha 1C-, and alpha 1D-subtype mRNAs were each present in isolated adult and neonatal cardiac myocytes by RNase protection assay. In addition, cultured neonatal cardiac myocytes expressed the three alpha 1-AR subtype mRNAs. In contrast, none of the alpha 1-AR mRNAs was detected in cultured neonatal cardiac fibroblasts. In addition, alpha 1-ARs were absent in fibroblasts by [3H]prazosin binding and norepinephrine-stimulated [3H]inositol phosphate production. The absence of alpha 1-ARs in cardiac fibroblasts differs from beta-adrenergic and angiotensin II receptors, which are present in both cardiac fibroblasts and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)