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.

The lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid enhance survival during hypoxia in neonatal rat cardiac myocytes.

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) stimulate cellular proliferation and affect numerous cellular functions by signaling through G protein-coupled endothelial differentiation gene-encoded (Edg) receptors. S1P and LPA also act as survival factors in many cell types, but have not previously been studied in cardiac myocytes. We incubated neonatal rat cardiac myocytes either in room air/1% CO2 (normoxia) or in an atmosphere of 99% N2/1%CO2 (hypoxia) at 37 degrees C for 18-20 h in the absence of glucose. Cell viability was measured using a calcein ester green fluorescence assay. Under normoxic conditions 88.7+/-1.0% of the cells were viable after 18-20 h. Severe hypoxia reduced viability to 61.3+/-4.3% (n=6, P<0.05). In myocytes preincubated with either 10 microM S1P or 1 microM LPA for 2 h, the effects of severe hypoxia on cell viability were prevented resulting in survival equivalent to normoxia. Neither the protein kinase C inhibitor chelethyrine (1 microM) nor the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoic acid, (5-HD, 100 microM) had any effect on myocyte survival during severe hypoxia, but both agents completely abolished the ability of S1P to rescue cardiac myocytes from hypoxic cell death. We also tested the effects of dimethylsphingosine (DMS), which inhibits sphingosine kinase synthesis of S1P. Incubation of neonatal rat cardiac myocytes with 10 microM DMS for 2 h in the presence of serum resulted in 25-30% cell death during 18-20 h of normoxia. DMS-induced cell death was prevented by concurrent preincubation with either S1P or GM-1, a ganglioside that activates sphingosine kinase to increase intracellular levels of S1P. We conclude that both S1P and LPA are cardioprotective for hypoxic neonatal rat ventricular myocytes. S1P acts through cellular membrane receptors by signaling mechanisms involving protein kinase C and mitochondrial K(ATP) channels. Both endogenous and exogenously applied S1P are effective in preventing cell death induced by inhibition of sphingosine kinase.

Neonatal mouse cardiac myocytes exhibit cardioprotection induced by hypoxic and pharmacologic preconditioning and by transgenic overexpression of human Cu/Zn superoxide dismutase.

Although mouse models have been increasingly used for studies of cardiac pathophysiology, there is little information regarding cultured murine cardiac myocytes. Accordingly, we have developed a cell culture model of neonatal mouse cardiac myocytes by modifying a protocol used to prepare neonatal rat myocytes. The principal change is the substitution of cytosine arabinoside for bromodeoxyuridine to prevent fibroblast proliferation. Neonatal murine myocytes exhibited persistent spontaneous contraction and were viable for up to 14 days in culture. By flow cytometry 85% of the cells were cardiac myocytes. In sparse cultures (average cell density 259 cells/mm(2)), both hypoxic preconditioning (n=5) and phenylephrine pretreatment (n=8) produced significant protection of cardiac myocytes from cell death during a prolonged period of severe hypoxia (<0.5% O(2)for 18-20 h, both P<0.05). The phenylephrine effect was inhibited by the alpha(1)-adrenoceptor antagonist prazosin (n=4, P<0.05) and by an xi PKC peptide antagonist (xi V1-2) coupled to a TAT peptide (n=5, P<0. 05). Interestingly, the mixed alpha(1)- and beta -adrenoceptor agonist norepinephrine, which stimulates hypertrophy as measured by(14)[C]phenylalanine incorporation in neonatal rat cardiac myocytes, did not cause hypertrophy in mouse myocytes, suggesting that the signaling pathways for myocardial protection and hypertrophy are likely to be both divergent and species specific. In cardiac myocytes prepared from transgenic mice either homozygous or heterozygous for human Cu/Zn superoxide dismutase, there was protection from cell death (n=3) and restoration of(14)[C]phenyl- alanine uptake (n=4) during prolonged hypoxia (1% O(2)for 3 days, both P<0.05). We conclude that this cellular model, which is relatively simple to prepare, can be used for in-vitro examination of cardiac protection induced by preconditioning agents, various transgenes, and potentially by targeted gene deletions.

Gelsolin binding and cellular presentation of lysophosphatidic acid.

Lysophosphatidic acid (LPA) in biological fluids binds to serum albumin and other proteins that enhance its effects on cellular functions. The actin-severing protein gelsolin binds LPA with an affinity (K(d) = 6 nm) similar to that of the G protein-coupled LPA receptors encoded by endothelial differentiation genes 2, 4, and 7 (Edg-2, -4, and -7 receptors) and greater than that of serum albumin (K(d) = 360 nm). At concentrations of 10% or less of that in plasma, which are observed in fluids of injured tissues, purified and recombinant gelsolin augment LPA stimulation of nuclear signals and protein synthesis in rat cardiac myocytes (RCMs) that express Edg-2 and -4 receptors. At concentrations of 20% or more of that in plasma, gelsolin suppresses LPA stimulation of RCMs. The lack of effect of gelsolin on RCM responses to monoclonal anti-Edg-4 receptor antibody plus a phorbol ester without LPA attests to its specificity for LPA delivery and the absence of post-receptor effects. Inhibition of gelsolin binding and cellular delivery of LPA by l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP2) and peptides constituting the two PIP2 binding domains of gelsolin suggests competition between LPA and PIP2 for the same sites. Thus, delivery of LPA to RCMs is affinity-coupled to Edg receptors by gelsolin in a PIP2-regulated process.

Mechanisms of lysolipid phosphate effects on cellular survival and proliferation.

The specificity of cellular effects of lysolipid phosphate (LLP) growth factors is determined by binding to endothelial differentiation gene-encoded G protein-coupled receptors (EDG Rs), which transduce diverse proliferative and effector signals. The primary determinants of cellular responses to LLPs are the generative and biodegradative events, which establish steady-state concentrations of each LLP at cell surfaces, and the relative frequency of expression of each EDG R. There are major differences among types of cells in the net effective generation of the LLPs, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), and in their profile of expression of EDG Rs. The less well characterized secondary determinants of cellular specificity of LLPs are high-affinity binding proteins with carrier and cell-presentation functions, cell-selective regulators of expression of EDG Rs, and cellular factors that govern coupling of EDG Rs to G protein transductional pathways. The roles of components of the LLP-EDG R system in normal physiology and disease processes will be definitively elucidated only after development of animal models with biologically meaningful alterations in genes encoding EDG Rs and the discovery of potent and selective pharmacological probes.

Endothelin-1 stimulates cardiac fibroblast proliferation through activation of protein kinase C.

After myocardial ischemia, circulating levels of the mitogen endothelin-1 (ET-1) increase. The effects of ET-1 on cardiac fibroblasts are poorly characterized. Therefore we examined the influence of ET-1 on cardiac fibroblast proliferation with a view to elucidating the signal transduction mechanisms underlying this effect. ET-1 (10 n m) stimulated [(3)H]thymidine incorporation and cell proliferation in cultured neonatal rat cardiac fibroblasts, consistent with its activity as a mitogen. We examined the role of protein kinase C (PKC) on this function. Inhibition of PKC activation with either chelerythrine (1 microm) or staurosporine (1 n m) attenuated ET-1-induced increases in DNA synthesis and cell number. Downregulation of PKC by chronic pretreatment with 10 n m phorbol 12-myristate 13-acetate (PMA) also prevented ET-1-induced mitogenesis. In contrast to previous reports that cardiac fibroblast proliferation stimulated by angiotensin II acts independently of PKC, the ET-1 mediated mitogenic effect requires activation of PKC in these cells. Findings in adult rat cardiac fibroblasts were identical. In addition, we noted that concurrent treatment with the pro-inflammatory cytokine interleukin 1 beta which, like ET-1, is released after myocardial ischemia, attenuated the ET-1-induced increases in DNA synthesis and cell number. This effect was not mediated through a nitric oxide synthase pathway.

Hypoxia differentially regulates stress proteins in cultured cardiomyocytes: role of the p38 stress-activated kinase signaling cascade, and relation to cytoprotection.

OBJECTIVE: Stress proteins (heat shock proteins, HSPs) are molecular chaperones that have been shown to enhance the survival of cells exposed to environmental stress. We sought to investigate the effects of hypoxia on the levels of HSP27 and heme oxygenase-1 (HO-1 or HSP32) in an established model of rat neonatal cardiac myocytes in culture. METHODS: Myocytes were subjected to hypoxia (<0.5% O(2) for 16 h). Studies of cell viability and nuclear morphology showed no evidence of cell death under these conditions. RESULTS: Messenger RNA analysis demonstrated constitutive expression of HSP27 and low levels of HO-1. Hypoxia strongly induced HO-1 mRNA without affecting HSP27 mRNA. In parallel to mRNA levels, hypoxia increased HO-1 protein level without affecting HSP27. To further assess the signaling pathways implicated in HO-1 induction, we used inhibition experiments. The tyrosine kinase inhibitor tyrphostin and the mitogen-activated protein kinase inhibitor PD98059 did not prevent HO-1 induction, while the protein kinase C inhibitor chelerythrine partially blocked this response. The p38 stress-activated kinase inhibitor SB203580 was the most potent in suppressing hypoxia-induced HO-1. In vitro kinase assays, cell labeling and immunoprecipitation showed activation of signaling pathways downstream of p38 stress-activated kinase as revealed by an increase in phosphorylation of MAPKAPK-2/3 kinases and HSP27. CONCLUSIONS: These data show a differential pattern of hypoxia-induced HSP expression and implicate the stress kinase in HO-1 induction. Thus, selective regulation of HSP levels may play a role in the cardioprotective mechanisms that participate in the adaptive response to hypoxia-induced stress.

Altered gene expression during hypoxia and reoxygenation of the heart.

The heart is exposed to alterations in oxygen tension under different pathophysiological conditions. In order to maintain function, changes in the pattern of cardiac gene expression arise. Through the activity of multiple transcription factors, which include activating protein-1, hypoxia-inducible factor-1, and nuclear factor kappaB, there is up-regulation of mRNA encoding factors that enable the cardiomyocyte to adapt to the new environment. In the case of hypoxia or anoxia, there is an increased expression of growth factors, glucose transporters, enzymes associated with anaerobic glycolysis, and stress proteins. When the cardiomyocyte is reoxygenated after hypoxia, there is a rapid increase in antioxidants, pro-inflammatory cytokines, and stress proteins.

The green fluorescent protein is an efficient biological marker for cardiac myocytes.

There is a need for a non-toxic marker for cardiac myocytes in studies of cardiac development and in experimentally induced pathophysiologic states in adult animals. We investigated the possibility of using the enhanced green fluorescent protein (EGFP) gene as such a biological marker for cardiac myocytes in both whole animal and cell culture systems. Several lines of transgenic mice were constructed harboring an EGFP gene directed by a 2.38-kb promoter fragment of the hamster beta -myosin heavy chain gene. The transgene was preferentially expressed in the cardiac progenitor cells of embryos at E7.5, a developmental stage that precedes the formation of the cardiomyotube. It was specifically expressed in the cardiomyotube and myotomes along the somites of embryos at E8.5. The EGFP transgene expression continued in the heart throughout gestation and became very intense at birth. When neonatal cardiac cells were fractionated into myocytes and non-myocytes by a differential plating procedure, only myocytes from the transgenic mice showed specific green fluorescence of the transgene product that can be used as a marker for flow cytometry analysis. Although the expression levels were heterogeneous, EGFP expression persisted in the hearts of postnatal animals. In addition to the heart, some skeletal and smooth muscles from transgenic animals also expressed the transgene. The transgenic mice were healthy and had a normal life span, identical to their non-transgenic littermates. These results demonstrate that EGFP is an efficient non-toxic biological marker for cardiac myocytes.

Expression and regulation of adhesion molecules in cardiac cells by cytokines: response to acute hypoxia.

Adhesion molecules mediate inflammatory myocardial injury after ischemia/reperfusion. Cytokine release and hypoxia are features of acute ischemia that may influence expression of these molecules. Accordingly, we studied intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) responses to cytokines and acute hypoxia in cultured myocardial cells. Northern blot analysis and immunoassay showed that the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha stimulated concentration-dependent increases in ICAM and VCAM mRNA and protein. In both cardiac myocytes and fibroblasts, pretreatment with a specific inhibitor of nuclear transcription factor-kappaB (NF-kappaB) prevented cytokine induction of both molecules. We also found that inhibition of tyrosine kinase and p38/RK (stress-activated protein kinase) pathways prevented IL-1beta-induced ICAM and VCAM protein synthesis, whereas extracellular signal-regulated protein kinase (ERK1/ERK2) inhibition did not. Neither hypoxia (0% O2 for 6 hours) alone nor hypoxia/reoxygenation had any significant effect on ICAM and VCAM mRNA. However, hypoxia did enhance IL-1beta-induced ICAM mRNA expression in myocytes. As a possible mechanism of this synergistic action on CAM expression, hypoxia induced a time-dependent increase in the DNA binding activity of both NF-kappaB and activator protein-1 (AP-1), two transcription factors important for cell adhesion molecule expression. In contrast to the enhanced ICAM mRNA induced by IL-1beta during hypoxia, however, protein levels for this adhesion molecule were unchanged beyond IL-1beta-stimulated levels, suggesting posttranscriptional and/or posttranslational control mechanisms. We conclude that cytokines regulate ICAM and VCAM mRNA and protein in both cardiac myocytes and fibroblasts. Furthermore, adhesion molecule induction requires translocation of at least two transcription factors, NF-kappaB and AP-1.

Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from fibroblasts.

OBJECTIVE: We sought to determine whether angiotensin II (Ang II) promotes hypertrophy of cardiac directly or via paracrine mechanisms mediated by cardiac fibroblasts. METHODS: We studied neonatal rat cardiac myocytes and fibroblasts in culture as a model system. Paracrine effects of Ang II were identified using conditioned medium and co-culture experiments. RESULTS: Ang II type 1 (AT1) receptors responsible for myocyte growth localized to fibroblasts in radioligand binding, emulsion autoradiography, Western analysis, and immunofluorescence staining experiments. The bulk of AT1 receptor binding in myocyte cultures (1343 +/- 472 sites/cell) was to Ang II receptors on contaminating fibroblasts (9747 +/- 2126 sites/cell). Ang II induced significant paracrine trophic effects on myocytes in conditioned medium (40% increase in protein synthesis over control) and co-culture (4-fold increase over control) experiments. TGF-beta 1 and endothelin-1 were paracrine mediators of hypertrophy in neutralization experiments. CONCLUSIONS: Ang II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from cardiac fibroblasts in a neonatal rat cell culture model.

Chronic hypoxia modulates the interleukin-1beta-stimulated inducible nitric oxide synthase pathway in cardiac myocytes.

BACKGROUND: We wished to determine whether the cytokine-inducible nitric oxide synthase (iNOS) pathway is modulated by chronic hypoxia in vitro. METHODS AND RESULTS: We investigated the effects of the proinflammatory cytokine interleukin (IL)-1beta on expression of iNOS mRNA, iNOS protein, and NO production in cultured neonatal rat cardiomyocytes subjected to 1% O2 for 48 hours. Among several cytokines tested, IL-1beta was the most effective in stimulating NO production, which was maximum at 48 hours. In parallel, IL-1beta induced expression of both iNOS mRNA and protein. Hypoxia alone had no effect on NO production, iNOS gene expression, or protein induction. However, chronic hypoxia decreased IL-1beta-stimulated NO production, mRNA expression, and protein level in cardiac myocytes. Radioligand binding and electrophoretic mobility shift assays showed that during chronic hypoxia, IL-1 receptor density and activity of the transcription factor NF-kappaB induced by IL-1beta were decreased, which may account at least in part for the decrease in iNOS expression. CONCLUSIONS: These data indicate that IL-1beta induces iNOS gene expression, de novo synthesis of iNOS protein, and NO generation in neonatal rat cardiomyocytes and that chronic hypoxia appears to be a potent negative regulator of iNOS expression in these cells.

Cross talk between angiotensin AT1 and alpha 1-adrenergic receptors: angiotensin II downregulates alpha 1a-adrenergic receptor subtype mRNA and density in neonatal rat cardiac myocytes.

Signaling mediated by the angiotensin (Ang) II and alpha1-adrenergic receptor (alpha1-AR) pathways is important for cardiovascular homeostasis. However, it is unknown whether Ang II has any direct effect on alpha1-AR expression and signaling in cardiac myocytes. In the present study, we determined alpha1-AR subtype mRNA levels by RNase protection; receptor density by competition binding with 5-methylurapidil; and alpha1-AR-mediated c-fos expression by Northern blot analysis. We found that Ang II had no effect on alpha1b- and alpha1d-AR mRNA levels but decreased the alpha1a-AR mRNA level in a time- and dose-dependent manner. The maximal effect occurred at 6 hours with 100 nmol/L Ang II (40.0+/-8.2% reduction, n=4, P<.01). The decrease in alpha1a-AR mRNA level induced by Ang II is mediated by the Ang II AT1 receptor subtype and is associated with decreased stability of alpha1a-AR mRNA. Corresponding to the changes in the alpha1a-AR mRNA level, Ang II (100 nmol/L, 24 hours) reduced the density of high-affinity sites for 5-methylurapidil (alpha1A-AR) by 29% (56.5+/-6.4 versus 79.0+/-11.6 fmol/mg protein, n=4, P<.05). Alpha1-AR-stimulated c-fos induction, which could be blocked by 5-methylurapidil but not by chloroethylclonidine, was attenuated by Ang II preincubation (100 nmol/L, 24 hours). We conclude that there is previously undescribed cross talk between AT1 receptors and alpha1-ARs. Ang II selectively downregulates alpha1a-AR subtype mRNA and its corresponding receptor as well as alpha1a-AR-mediated expression of the immediate-early gene c-fos in cardiac myocytes.

A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death.

Protein kinase C activation is thought to protect cardiac tissue from subsequent ischemic injury by a process termed preconditioning. The protein kinase C isozyme that mediates preconditioning has not yet been identified. Using a cell culture model of hypoxic preconditioning, we found that cardiac myocyte viability after 9 h of hypoxia was increased by more than 50% over control. Preconditioning activated protein kinase C isozymes as evidenced by translocation from one cell compartment to another as follows: there was a 2.1-fold increase in epsilon-protein kinase C activation, a 2. 8-fold increase in delta-protein kinase C activation, and no increase in betaI-protein kinase C activation. 4beta-Phorbol 12-myristate 13-acetate mimicked hypoxic preconditioning, increasing myocyte survival after prolonged hypoxia by 34% compared with control. We previously identified an epsilon-protein kinase C-selective antagonist, epsilonV1-2 peptide, that inhibits epsilon-protein kinase C translocation and function in cardiac myocytes (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). epsilonV1-2 peptide abolished hypoxic preconditioning and phorbol ester-mediated cardiac protection. Therefore, preconditioning can be induced in this culture model, and activation of epsilon-protein kinase C is critical for cardiac myocyte protection.

Chronic hypoxia increases beta 1-adrenergic receptor mRNA and density but not signaling in neonatal rat cardiac myocytes.

BACKGROUND: It is well recognized that the beta-adrenergic receptor-adenylylcyclase system is altered during myocardial ischemia/hypoxia. However, there are no data regarding either regulation of beta-adrenergic receptors, particularly at the mRNA level, or adenylylcyclase activity in isolated cardiac myocytes exposed to chronic hypoxia. METHODS AND RESULTS: In a chronic hypoxia model in which neonatal rat ventricular myocytes were exposed to a 1% O2 environment for 72 hours, we investigated (1) beta 1-mRNA and receptor expression and adenylylcyclase activity and (2) beta 1-mRNA and receptor downregulation and adenylylcyclase desensitization induced by prolonged norepinephrine incubation. We found that hypoxia for 72 hours increased myocardial membrane beta 1-adrenergic receptor density by 44%. This increase was not associated with a corresponding decrease in cytosolic beta 1-adrenergic receptors. RNase protection assays demonstrated that hypoxia increased the steady-state levels of beta 1-mRNA by 109%. Adenylylcyclase activity stimulated by isoproterenol, sodium fluoride, guanyl-5'-imidodiphosphate, and forskolin in hypoxic membranes was not altered compared with normoxic controls. Hypoxia for 72 hours also did not affect norepinephrine-induced beta 1-mRNA and receptor downregulation and adenylylcyclase desensitization in response to isoproterenol, guanyl-5'-imidodiphosphate, or forskolin. CONCLUSIONS: In neonatal rat cardiac myocytes, chronic hypoxia (1) increases beta 1-mRNA and receptor expression but does not alter adenylylcyclase activity stimulated at either the receptor or the postreceptor level and (2) does not affect agonist-induced beta 1-mRNA and receptor downregulation and desensitization of the adenylylcyclase response.

An improved permeabilization protocol for the introduction of peptides into cardiac myocytes. Application to protein kinase C research.

We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C epsilon fragment into the cells. The intracellular molar concentration of the introduced peptide was approximately 10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester-modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [-14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester-induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.

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.

Chronic hypoxia differentially regulates alpha 1-adrenergic receptor subtype mRNAs and inhibits alpha 1-adrenergic receptor-stimulated cardiac hypertrophy and signaling.

BACKGROUND: After myocardial ischemia and/or infarction, surviving cardiac myocytes in and around the injured zone develop hypertrophy to compensate for the loss of contractile units due to myocyte injury and death. One of the factors that may be involved in the development of hypertrophy after ischemic injury is norepinephrine (NE), an agent that induces hypertrophy of cardiac myocytes through the alpha 1-adrenergic receptor (AR). It is not known, however, whether hypoxia, a major component of ischemia, has any direct effect on NE-stimulated hypertrophy. Therefore, we sought to determine whether chronic hypoxia could alter NE-stimulated hypertrophy and if so, whether this alteration was related to alpha 1-AR-mediated signaling and alpha 1-AR changes at both the protein and mRNA levels. METHODS AND RESULTS: We developed a model of chronic hypoxia in cultured neonatal rat cardiac myocytes in which myocytes were exposed to 1% oxygen for 72 hours. Initially, we observed that chronic hypoxia inhibited NE-stimulated hypertrophy, as reflected by decreases in both new protein synthesis and total protein content during chronic hypoxia. Then we found that chronic hypoxia also inhibited alpha 1-AR-transduced phosphatidylinositol hydrolysis, as indicated by a reduction in alpha 1-AR-stimulated inositol phosphate production in hypoxic cells. These observations suggested that the inhibition of NE-stimulated hypertrophy seen during chronic hypoxia was due to impairment of alpha 1-AR-mediated signaling and could result from changes in alpha 1-AR numbers and/or subtype distribution. To address this issue, we determined alpha 1-AR density and subtype distribution by radioligand binding and alpha 1-AR subtype mRNAs, including alpha 1A/D-, alpha 1B-, and alpha 1C-ARs, by RNase protection assays. We found that chronic hypoxia differentially regulated both the pharmacologically defined alpha 1-AR subtypes and the mRNAs for the alpha 1-AR subtypes. Thus, hypoxia for 72 hours coordinately downregulated both the pharmacologically defined alpha 1A-AR density and the alpha 1C-AR mRNA level. During normoxia, NE increased the pharmacologically defined alpha 1A-AR density and the alpha 1C-AR mRNA level, but hypoxia for 72 hours prevented these NE-mediated changes. CONCLUSIONS: Chronic hypoxia (1) inhibits alpha 1-AR-mediated hypertrophy of cardiac myocytes and alpha 1-AR-transduced phosphatidylinositol hydrolysis and (2) downregulates both the pharmacologically defined alpha 1A-AR density and the alpha 1C-AR mRNA level.

Prolonged incubation with neuropeptide Y upregulates beta-adrenoceptors yet does not cause supersensitivity of beta-adrenoceptor signaling.

In neonatal rat ventricular myocytes prolonged incubation with 1 microM neuropeptide Y (2 h-48 h) increased beta-adrenoceptor density 16-24% (n = 4--8, all p < 0.05), an effect prevented by pertussis toxin pretreatment. Prolonged incubation with neuropeptide Y had no effect on adenylycylclase activity stimulated by 5'-guanylylimidodiphosphate or (-)-isoprenaline, probably because of a neuropeptide Y-induced decrease in affinity of the beta-adrenoceptor for agonist. Thus, chronic incubation with an inhibitory agonist does not inevitably lead to supersensitivity of the adenylylcyclase pathway.