Joel S. Karliner

Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-β1 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.

Pharmacologic and Hemodynamic Influences on the Rate of Isovolumic Left Ventricular Relaxation in the Normal Conscious Dog

We studied the effects of acute pharmacologic and hemodynamic interventions on isovolumic left ventricular relaxation in 19 conscious dogs using micromanometer tip catheters. Isoproterenol (11 studies) augmented peak rate of rise of left ventricular pressure [(+) dP/dt] by 1,275+/-227 (SE) mm Hg/s (P < 0.001) and dP/dt at an isopressure point of 35 mm Hg during isovolumic relaxation [(-) dP/dt(35)] by 435+/-80 mm Hg/s (P < 0.001). Peak (-) dP/dt decreased by 467+/-89 mm Hg/s (P < 0.002). The time constant, T, derived from the logarithmic fall of pressure during isovolumic relaxation, shortened from 20+/-2.8 to 14.9+/-1.8 ms (P < 0.003). Calcium (11 studies) increased peak (+) dP/dt and (-) dP/dt(35) (both P < 0.0001); peak (-) dP/dt was unchanged. T shortened from 20.4+/-1.8 to 17.3+/-1.5 ms (P < 0.002). Volume (13 studies) did not affect either dP/dt or T. Phenylephrine (13 studies) augmented peak (-) dP/dt, but reduced (-) dP/dt(35) (both P < 0.01); T lengthened from 22.1+/-1.5 to 32.5+/-1.5 ms (P < 0.01). In 15 studies, rapid atrial pacing increased peak (+) dP/dt and (-) dP/dt(35) (both P < 0.01). In the first post-pacing beat, peak (-) dP/dt and (-) dP/dt(35) decreased (both P < 0.01), although peak (+) dP/dt increased further. T paralleled values of (-) dP/dt(35). In five dogs, beta adrenergic blockade had no significant effect on any variable after calcium, volume, or phenylephrine infusion or during or after atrial pacing when the pre-and post-propranolol states were compared. We conclude that positive inotropic interventions augment both left ventricular contraction and relaxation. The changes in isovolumic relaxation are independent of alterations in sympathetic tone produced by beta-adrenergic blockade. Peak (-) dP/dt may not be a valid measure of left ventricular relaxation rate during acute alterations in inotropic state or afterload.

A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers.

Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.

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.

Sphingosine Kinase Activation Mediates Ischemic Preconditioning in Murine Heart

Background—Phosphorylation of sphingosine by sphingosine kinase (SK) is the rate-limiting step in the cellular synthesis of sphingosine 1-phosphate (S1P). The monoganglioside GM1, which stimulates SK, is cardioprotective in part through increased generation of S1P that protects myocytes by diverse mechanisms. Because protein kinase C (PKC)&egr; activation is necessary for myocardial ischemic preconditioning (IPC) and PKC activators increase SK activity, we tested the hypothesis that SK may be a central mediator of IPC. Methods and Results—In adult murine hearts, IPC sufficient to reduce infarct size significantly increased cardiac SK activity, induced translocation of SK protein from the cytosol to membranes, and enhanced cardiac myocyte survival. IPC did not increase SK activity in PKC&egr;-null mice. The SK antagonist N,N-dimethylsphingosine inhibited PKC&egr; activation and directly abolished the protective effects of IPC and the enhanced SK activity induced by IPC. Conclusions—These findings demonstrate that PKC&egr; is thus recruited by IPC and induces activation of SK that then mediates IPC-induced cardioprotection in murine heart.

-Adrenergic Receptor Subtype mRNAs Are Differentially Regulated by -Adrenergic and Other Hypertrophic Stimuli in Cardiac Myocytes in Culture and In Vivo REPRESSION OF α AND α BUT INDUCTION OF α

The three cloned α-adrenergic receptor (AR) subtypes, α, α, and α, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three α-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 α-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12-72 h). α and α mRNA levels were repressed by NE, whereas α mRNA was induced. Changes in mRNA levels were mediated by an α-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [3H]prazosin binding. α-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2α, and phorbol 12-myristate 13-acetate, also induced α mRNA and repressed α mRNA. In myocytes from hearts with pressure overload hypertrophy, α mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among α-AR subtypes expressed natively in the same cell. Transcriptional induction of the α-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.

Verapamil competitively inhibits alpha 1-adrenergic and muscarinic but not beta-adrenergic receptors in rat myocardium.

Recent studies indicate that antagonism of calcium channels may not be the only mechanism whereby drugs such as verapamil alter myocardial function. We have examined the effect of verapamil on the binding of [3H]prazosin (alpha 1-adrenergic), [3H]quinuclidinyl benzilate (QNB, muscarinic) and [3H]dihydroalprenolol (DHA, beta-adrenergic) to membranes prepared from rat heart. Verapamil competed for the binding of these radioligands in the following rank order: [3H]prazosin greater than [3H]QNB greater than [3H]DHA (Ki for verapamil = 0.6 microM, 7 microM, and 72 microM, respectively). Verapamil (10 microM) competitively inhibited [3H]prazosin binding to rat ventricular membranes; the apparent dissociation constant (KD) of [3H]prazosin increased from 0.13 +/- 0.02 to 1.5 +/- 0.6 nM (SD) without change in maximal binding capacity (Bmax). The effect of verapamil on the affinity of [3H]prazosin was completely reversed by washing the membranes. The verapamil derivative D-600 also inhibited [3H]prazosin binding (Ki = 1.1 microM). Verapamil (30 microM) competitively inhibited [3H]QNB binding in both atria and ventricles and increased the apparent KD of [3H]QNB fivefold (from 0.07 nM to 0.32 nM) without decreasing Bmax. Verapamil was a less potent inhibitor of [3H]DHA binding and its effect was noncompetitive: the KD for DHA was unaltered by 100 microM verapamil while the Bmax decreased severalfold. We conclude that verapamil, at concentrations clinically achieved in the myocardium (approximately 1 microM), competitively inhibits binding to alpha 1-adrenergic and muscarinic receptors and that this inhibition may play a role in the effects of verapamil on the heart.

Alpha1-adrenergic receptors in guinea pig myocardium: Identification by binding of a new radioligand, (3H)-prazosin

Abstract Binding of (3H)-prazosin to adrenoceptors in guinea pig myocardial membranes was rapid, readily reversible, stereospecific and saturable. By Scatchard analysis (n = 6) Bmax was 58 fmol of (3H)-prazosin bound/mg protein and the KD was 0.58 nm. The Hill number was 1.05. Adrenergic agonists competed with (3H)-prazosin as follows: (−)adrenaline > (−)noradrenaline > (−)phenylephrine ⪢ ( + )isoprenaline > (+)noradrenaline; antagonists competed in the order: non-radioactive prazosin > phentolamine ⪢ piperoxan > yohimbine > sulpiride > propranolol. The KD for beta-adrenoceptors assessed by (−3H)-dihydroalprenolol was 0.86 nM and the Bmax (96 fmol/mg protein) was almost twice that of alpha-adrenoceptors. (3H)-prazosin appears to be a useful radioligand for the study of post-synaptic (alpha1) adrenoceptors in myocardial tissue.

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.

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.