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Recent studies have documented various roles of adrenal corticosteroid signaling in cardiac physiology and pathophysiology. It is known that glucocorticoids and aldosterone are able to bind glucocorticoid receptor (GR) and mineralocorticoid receptor, and these ligand-receptor interactions are redundant. It, therefore, has been impossible to delineate how these nuclear receptors couple with corticosteroid ligands and differentially regulate gene expression for operation of their distinct functions in the heart. Here, to particularly define the role of GR in cardiac muscle cells, we applied a ligand-based approach involving the GR-specific agonist cortivazol (CVZ) and the GR antagonist RU-486 and performed microarray analysis using rat neonatal cardiomyocytes. We indicated that glucocorticoids appear to be a major determinant of GR-mediated gene expression when compared with aldosterone. Moreover, expression profiles of these genes highlighted numerous roles of glucocorticoids in various aspects of cardiac physiology. At first, we identified that glucocorticoids, via GR, induce mRNA and protein expression of a transcription factor Kruppel-like factor 15 and its downstream target genes, including branched-chain aminotransferase 2, a key enzyme for amino acid catabolism in the muscle. CVZ treatment or overexpression of KLF15 decreased cellular branched-chain amino acid concentrations and introduction of small-interfering RNA against KLF15 cancelled these CVZ actions in cardiomyocytes. Second, glucocorticoid-GR signaling promoted gene expression of the enzymes involved in the prostaglandin biosynthesis, including cyclooxygenase-2 and phospholipase A2 in cardiomyocytes. Together, we may conclude that GR signaling should have distinct roles for maintenance of cardiac function, for example, in amino acid catabolism and prostaglandin biosynthesis in the heart. endocrinology; cardiovascular system; KLF15; cylooxygenase-2; phospholipase A2

The long QT syndrome genes human ether-a-go-go-related gene (HERG1) and voltage-gated [K.sup.+] channel, KQT-like subfamily, member 1, gene (KCNQ1), encoding [K.sup.+] channels critical to the repolarization rate and repolarization reserve in cardiac cells, and thereby the likelihood of arrhythmias, are both composed of two isoforms: HERG1a and HERG1b and KCNQ1a and KCNQ1b, respectively. Expression of these genes is dynamic, depending on the differentiation status and disease states. We identified their core promoter regions and transcription start sites. Our data suggest that HERG1a and HERG1b, and KCNQ1a and KCNQ1b, represent independent transcripts instead of being alternatively spliced variants of the same gene, for they each have their own transcription start sites and their own promoter regions. We obtained data pointing to the potential role of stimulating protein 1 (Sp1) in the transactivation of these genes. We compared expression profiling of these genes across a variety of human tissues. Consistent with the general lack of cis elements for cardiac-specific transcription factors and the presence of multiple sites for ubiquitous Sp1 sites in the core promoter regions of HERG1a/HERG1b and KCNQ1a/KCNQ1b genes, the transcripts demonstrated widespread distribution across a variety of human tissues. We further revealed that the mRNA levels of all HERG1 and KCNQ1 isoforms were asymmetrically distributed within the heart, being more abundant in the right atria and ventricles relative to the left atria and ventricles. These findings open up an opportunity for studying interventricular gradients of slow and rapid delayed rectifier [K.sup.+] current and of cardiac repolarization as well. Our study might help us understand the molecular mechanisms for arrhythmias since heterogeneity of ion channel activities is an important substrate for arrhythmogenesis. potassium channels; promoter; transcription; human ether-a-go-go-related gene; voltage-gated potassium channel, KQT-like subfamily, member 1; stimulating protein 1

TNF-[alpha] is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-[kappa]B family of transcription factors are essential regulators of myocardial TNF-[alpha] gene expression, recent developments in our understanding of the modulation of NF-[kappa]B activity through posttranslational modification of NF-[kappa]B subunits suggest that the present view of NF-[kappa]B-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-[alpha], production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-[alpha] production is accompanied by a 12-fold increase in phosphorylation of p65 at [Ser.sup.536], a modification associated with enhancement of p65 trans-activation potential. Pharmacological inhibition of IKK-[beta] reduced LPS-induced TNF-[alpha] production 38-fold, TNF-[alpha] mRNA levels 6-fold, and I[kappa]B-[alpha] phosphorylation 5-fold and degraded [kappa]B-[alpha] 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-[alpha] production 3.5-fold, whereas overexpression of dominant-negative IKK-[beta] reduced LPS-induced TNF-[alpha] production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-[alpha] had no effect on p65 phosphorylation or TNF-[alpha] production, revealing that IKK-[alpha], not IKK-[beta], plays a central role in regulation of p65 phosphorylation at [Ser.sup.536] and TNF-[alpha] production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of [Ser.sup.536]-phosphorylated p65 to the TNF-[alpha] gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-[kappa]B signaling in TNF-[alpha] gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction. signal transuction; gene expression; heart; cytokines

Serum response factor (SRF) is an important transcription factor that may have a role in the maintenance of cardiac structure and function. The level of SRF mRNA expression increases ~16% in the hearts of mice during adult aging. To model the effect of mild SRF elevation in the aging heart, transgenic mice with low levels of SRF overexpression were generated. By 6 mo of age, the transgenic mice had a 19% increase of heart-to-body weight ratio compared with nontransgenic mice. In addition, they had a 12% increase in myocyte size, a 6.7% increase in collagen deposition, and altered gene expression of a number of muscle-specific and cardiac genes. Doppler echocardiography revealed that these transgenic mice had increased left ventricular wall thickness and decreased left ventricular (LV) volumes, increased LV stiffness with 20% reduction in early diastolic LV filling (peak E), and 35% decline in peak E-to-peak A (late diastolic filling) ratio. The observed changes, especially those in the E/A ratio, are similar to those seen clinically in late life as a part of human adult myocardial aging. transcription factor; transgenic; cardiac structure and function

We previously reported that S100B, a 20-kDa [Ca.sup.2+]-binding homodimer, inhibited the postinfarct myocardial hypertrophic response mediated by [[alpha].sub.1]-adrenergic stimulation through the protein kinase C (PKC) signaling pathway. In the present study, we examined whether the same pathway induced the S100B gene, supporting the hypothesis that S100B is a feedback negative regulator of this pathway. We transfected cultured neonatal rat cardiac myocytes with a luciferase reporter gene driven by the maximal human S100B promoter and progressively shorter segments of this promoter sequentially deleted from the 5' end. We identified a basic promoter essential for transcription spanning 162 bp upstream of the transcription initiation site and positive (at -782/-162 and -6,689/ -4,463) and negative (at -4,463/-782) myocyte-selective regulatory elements. We showed that the basic and maximal S100B promoters were activated specifically by [[alpha].sub.1]-adrenergic agonists through the [[alpha].sub.1A]-adrenergic receptor, but not by any other trophic hormonal stimuli. The activation of the S100B promoter was mediated through the PKC signaling pathway. Transcription enhancer factor-1 (TEF-1) and related to TEF-1 (RTEF-1) influenced transcription from the maximal, but not the basic, promoter implicating active MCAT elements upstream from the basic promoter. Acting in opposing fashions, TEF-1 transrepressed the S100B promoter and RTEF-1 transactivated the promoter. Our results suggest that [[alpha].sub.1]-adrenergic stimulation induces the S100B gene after myocardial infarction through the PKC signaling pathway and that this induction is modulated by TEF-1 and RTEF-1. norepinephrine; TEF-1

The purpose of the study was to investigate interactions between myocardial nitric oxide synthase (NOS) and myocardial fibrosis, both of which determine left ventricular (LV) preload reserve in patients with nonischemic dilated cardiomyopathy (DCM). In previous animal experiments, chronic inhibition of NOS induced myocardial fibrosis and limited LV preload reserve. Twenty-eight DCM patients underwent LV catheterization, balloon caval occlusions (BCO; n = 8), intracoronary substance P infusion (n = 8), and procurement of LV endomyocardial biopsies for determinations of collagen volume fraction (CVF), of gene expression of NOS2, NOS3, heme oxygenase (HO)-1, and TNF-[alpha], and of NOS2 protein. CVF was unrelated to the intensity of NOS2, NOS3, HO-1, or TNF-[alpha] gene expression or of NOS2 protein expression. Preload recruitable LV stroke work (PR-LVSW) correlated directly with NOS2 gene expression (P = 0.001) and inversely with CVF (P = 0.04). High CVF (>10%) reduced baseline LVSW and PR-LVSW at each level of NOS2 gene expression. In DCM, myocardial fibrosis is unrelated to the intensity of myocardial gene expression of NOS, antioxidative enzymes (HO-l), or cytokines (TNF-[alpha]) and blunts NOS2-related recruitment of LV preload reserve. collagen; diastole; myocardial contraction

We have previously shown that ischemic preconditioning (IP) improves cardiac performance and sarcoplasmic reticulum (SR) function in hearts subjected to ischemia-reperfusion (I/R). In this study, we examined the effect of IP on I/R-induced changes in gene expression for SR proteins such as the Ca2+ release channel, [Ca.sup.2+] pump ATPase, phospholamban, and calsequestrin in the isolated rat heart. Normal isolated rat hearts exposed to three brief cycles of IP (5-min ischemia and 5-min reperfusion) exhibited a significant decrease in the transcript levels of SR genes. Nonpreconditioned I/R hearts when subjected to 30-min ischemia and 30-min reperfusion showed a marked decrease in mRNA levels for the SR proteins compared with normal hearts; this decrease was attenuated by preconditioning. Although hearts subjected to [Ca.sup.2+] paradox (CP) have been shown to exhibit intracellular [Ca.sup.2+] overload and SR dysfunction like those in I/R hearts, virtually nothing is known regarding the effect of CP on cardiac SR gene expression. Accordingly, CP (5-min [Ca.sup.2+]-free perfusion and 30-min reperfusion with normal medium) was observed to produce dramatic changes in SR gene expression, and the heart failed to contract; these alterations were attenuated by IP. Our results show that 1) both I/R and CP depress SR gene expression in the normal heart, 2) IP attenuates I/R- and CP-induced depression in cardiac function and SR gene expression, and 3) intracellular [Ca.sup.2+] overload may play a role in depressing SR gene expression in both I/R and CP hearts. ischemic preconditioning; ischemia-reperfusion; cardiac mRNA levels; [Ca.sup.2+] paradoxic heart

Research was conducted to investigate the influence of nitric oxide (NO) produced by cardiac inducible NO synthase on myocardial injury in rats after oxidative stress. S-nitroso-N-acetyl-penicillamine was utilized to study the effects of an exogenous NO releasing agent on cardiac myocytes while NO production by endogenous cardiac myocyte iNOS was performed using nitrite ion concentration measurements. Results showed interleukin-1-beta influenced cardiac myocytes to produce NO in the presence of L-arginine.

A recombinant megalovirus promoter was utilized for gene transfer in the pericardial sac of doxycycline-treated hearts to determine the efficiency of the technique as an alternative to intravascular gene transfer. Gene transfer in canine hearts was accomplished by utilizing the human adenovirus serotype-2 recombinant vector which is injected in the pericardial sac. The technique produced extensive transfection of the visceral and parietal pericardium of canine hearts.

The aim of this study was to examine two methods of [sup.31]P NMR quantitation of phosphocreatine (PCr), ATP, and Pi in rat heart and skeletal muscle in vivo. The first method employed an external standard of phenylphosphonic acid (PPA; 10 mM), and the second method used an enzymatic measurement of tissue ATP equated to the area under the [Beta]ATP peak. With the use of the external standard, the concentrations of ATP, PCr, and [P.sub.i] in the rat heart were 4.48 [+ or -] 0.33, 9.21 [+ or -] 0.65, and 2.25 [+ or -] 0.16 [micro]mol/g wet wt, respectively. With the use of the internal ATP standard, measured on the same tissue, the contents (means [+ or -] SE) were 4.78 [+ or -] 0.19, 9.83 [+ or -] 0.18, and 2.51 [+ or -] 0.33 [micro]mol/g wet wt, respectively (n = 7). In skeletal muscle, ATP, PCr, and Pi were 6.09 _[+ or -] 0.19, 23.44 _[+ or -] 0.88, and 1.81 _[+ or -] 0.18 [micro]mol/g wet wt using the PPA standard and 6.03 [+ or -] 0.19, 23.30 [+ or -] 1.30, and 1.82 [+ or -] 0.19 [micro]mol/g wet wt using the internal ATP standard (n = 6). There was no significant difference for each metabolite as measured by the two methods of quantification in heart or skeletal muscle. The results validate the use of an external reference positioned symmetrically above the coil and imply that each has similar NMR sensitivities (similar signal amplitude per mole of [sup.31]P between PPA and tissue phosphorus compounds). We conclude that PCr, ATP, and [P.sub.i] are nearly 100% visible in the normoxic heart and nonworking skeletal muscle given the errors of measurement. heart; ATP; ADP; [P.sub.i]

In vivo regulation of the [Beta]-myosin heavy chain gene in hypertensive rodent heart. Am J Physiol Cell Physiol 280: C1262-C1276, 2001.--The main goal of this study was to examine the transcriptional activity of different-length [Beta]-myosin heavy chain ([Beta]-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by ~45% relative to control. Results show that [Beta]-MHC promoter activity of all tested wild-type constructs, i.e., -3500, -408, -299, -215, -171, and -71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the -71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting [Beta]e1, [Beta]e2, GATA, and [Beta]e3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the [Beta]-MHC gene. Collectively, these results indicate that three separate regions on the [Beta]-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between -408 and -3500 bp, 2) a proximal region between -299 and -215 bp, and 3) a basal region within -71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate [Beta]-MHC transcriptional regulation in response to pressure overload. hypertension; transcription; dual luciferase; in vivo gene transfer

A study was conducted to determine the effects of electrogenic sodium/calcium exchange current, calcium-activated chloride conductance, and calcium-activated nonselective cation conductance on delayed afterdepolarizations in the midmyocardial cells of dogs. Patch electrodes were utilized to dialyzed endogenous calcium buffers from cells. Results indicated no proof for calcium-activaTed nonselective cation channels in a subset of cells within the midmyocardium.