The recent discovery that hydrogen sulfide (H2S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H2S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H2S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemiareperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H2S by cardiac-specific overexpression of cystathionine ␥-lyase (␣-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H2S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H2S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 M epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.

Gonadotropin-releasing hormone (GnRH) regulates the expression of all three gonadotropin genes, encoding the common ␣ subunit (␣GSU) and hormone-specific ␤ subunits, through the activation of several signal transduction pathways. We have shown that GnRH also upregulates calcineurin, and we hypothesized that calcineurin mediates the effects of GnRH on the transcription of the ␣GSU and follicle-stimulating hormone ␤ (FSH␤) genes through two of its targets: nuclear factor of activated T cells (NFAT) and CREB-regulated transcription coactivator (TORC). We show that calcineurin is essential for GnRH-induced expression of both genes but that NFAT and TORC1 play quite distinct roles in activating each gene. GnRH induces calcineurindependent nuclear import of NFAT3, which activates the ␣GSU promoter, while TORC1 also mediates GnRH activation of this promoter, but not through CREB. GnRH initially stimulates the degradation of TORC1 but protects the N terminus of the newly synthesized protein to enhance its activity. Calcineurin induces Nur77 expression, likely via NFAT3, and Nur77 interacts synergistically with TORC1 and CREB to increase FSH␤ promoter activity. Although TORC plays a role in the basal activity of the FSH␤ promoter, it does not interact with phosphorylated CREB and probably does not play a major role in direct GnRH signaling to this gene. TORC may be part of an alternatively regulated pathway, possibly involving cross talk with other stimulatory hormones.In the pituitary gonadotrope, the gonadotropin-releasing hormone (GnRH) plays a crucial role in activating the transcription of all three gonadotropin subunit genes: the common ␣ subunit (␣GSU) and the hormone-specific ␤ subunits encoding luteinizing hormone (LH␤) and follicle-stimulating hormone (FSH␤). Upon binding its G-protein-coupled receptor (GPCR), GnRH activates protein kinase C (PKC) and a number of well-characterized mitogen-activated protein kinase (MAPK) pathways, as well as elevating cyclic AMP (cAMP) levels to activate protein kinase A (PKA) (7,20,33,42,53). It also induces an increase in intracellular calcium levels by stimulating calcium release from intracellular stores as well as the influx of extracellular calcium through voltage-sensitive channels (7, 42). One of the effects of this increase in calcium levels is the activation of calmodulin and calmodulin-dependent kinases (CaMKs), which mediate various aspects of GnRH-induced signaling, including inhibition of histone deacetylases and regulation of extracellular signal-regulated kinase (ERK) activity (6,10,23,24,34,44,61). Calmodulin also activates the serine/threonine protein phosphatase calcineurin to regulate the expression of various genes (26). In the gonadotrope, calcineurin moderates the activity of the prolyl isomerase Pin1, facilitating its localization to the nucleus, where it plays an important role in GnRH signaling to several key transcription factors (36). Calcineurin also plays a role in the GnRH-induced derepression of FSH␤ gene expression in immature ␣T3-1 gonadotrope ...

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time-and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.

Circadian clocks are cell autonomous, transcriptionally-based, molecular mechanisms that confer the selective advantage of anticipation, enabling cells/organs to respond to environmental factors in a temporally appropriate manner. Critical to circadian clock function are two transcription factors, CLOCK and BMAL1. The purpose of the present study was to reveal novel physiologic functions of BMAL1 in the heart, as well as determine the pathologic consequences of chronic disruption of this circadian clock component. In order to address this goal, we generated cardiomyocyte-specific Bmal1 knockout (CBK) mice. Following validation of the CBK model, combined microarray and in silico analyses were performed, identifying 19 putative direct BMAL1 target genes, which included a number of metabolic (e.g., β-hydroxybutyrate dehydrogenase 1 [Bdh1]) and signaling (e.g., the p85α regulatory subunit of phosphatidylinositol 3-kinase [Pik3r1]) genes. Results from subsequent validation studies were consistent with regulation of Bdh1 and Pik3r1 by BMAL1, with predicted impairments in ketone body metabolism and signaling observed in CBK hearts. Furthermore, CBK hearts exhibited depressed glucose utilization, as well as a differential response to a physiologic metabolic stress (i.e., fasting). Consistent with BMAL1 influencing critical functions in the heart, echocardiographic, gravimetric, histologic, and molecular analyses revealed age-onset development of dilated cardiomyopathy in CBK mice, which was associated with a severe reduction in lifespan. Collectively, our studies reveal that BMAL1 influences metabolism, signaling, and contractile function of the heart.

Rationale: Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown. Objective: To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice. Methods and Results: Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Key Words: chronobiology Ⅲ ischemia/reperfusion Ⅲ myocardium N umerous aspects of cardiovascular physiology and pathophysiology demonstrate circadian rhythms. 1 In humans, heart rate, blood pressure, and cardiac output all increase in the early hours of the morning, as does the onset of adverse cardiac events, such as myocardial infarction. 2,3 These rhythms have been attributed primarily to time-of-day oscillations in neurohumoral influences, such as sympathetic or autonomic stimulation. 3,4 Although extracardiac factors undoubtedly play critical roles in modulation of cardiovascular function/dysfunction, increasing evidence suggests that intrinsic factors, such as cellautonomous circadian clocks, likely contribute. 1 Circadian clocks are transcriptionally based molecular mechanisms, composed of positive-and negative-feedback loops, with a free-running period of Ϸ24 hours. 5 This mechanism allows the cell to anticipate alterations in environmental stimuli, through time-of-day-dependent modulation of cellular responsiveness to extrinsic factors. 5 Circadian clocks have been identified/characterized in multiple cardiovascular-relevant cell types, including cardiomyocytes, vascular smooth muscle cells, and endothelial cells. 6 -8 Ubiquitous genetic ablation of circadian clock function markedly influences multiple cardiovascular parameters, including heart rate and blood pressure. 9 We have recently used a CCM (cardiomyocyte-specific circadian clock mutant) mouse to reveal regulation of myocardial gene expression, ␤-adrenergic responsiveness, metabolism, heart rate, and cardiac power by this mechanism. 10,11 Although circadian rhythms in myocardial infarction onset are well established, time-of-day oscillations in myocardial ischemia/reperfusion (I/R) tolerance have not been reported. Given that the cardiomyocyte circadian clock influences Original

The p38 and JNK stress-activated MAPK signal transduction pathways are activated by T cell receptor (TCR) signaling and are required for IFN-gamma production by TH1 effector cells. Here, we show that the expression of GADD45gamma is induced during T cell activation and that the level of expression is higher in TH1 cells than in TH2 cells. TH1 cells from GADD45gamma(-/-) mice are severely compromised in their abilities to activate p38 and JNK in response to TCR signaling, produce much less IFN-gamma upon restimulation, and are deficient in activation-induced cell death (AICD). Additionally, GADD45gamma deficiencies caused reduced contact hypersensitivity in mice. Thus, GADD45gamma mediates activation of the p38 and JNK pathways and effector function of TH1 cells.

Interleukin (IL)-6 is produced by professional antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been previously shown that APC-derived IL-6 promotes the differentiation of naive CD4+ T cells into effector T helper type 2 (Th2) cells. Here, we have studied the molecular mechanism for IL-6–mediated Th2 differentiation. During the activation of CD4+ T cells, IL-6 induces the production of IL-4, which promotes the differentiation of these cells into effector Th2 cells. Regulation of IL-4 gene expression by IL-6 is mediated by nuclear factor of activated T cells (NFAT), as inhibition of NFAT prevents IL-6–driven IL-4 production and Th2 differentiation. IL-6 upregulates NFAT transcriptional activity by increasing the levels of NFATc2. The ability of IL-6 to promote Th2 differentiation is impaired in CD4+ T cells that lack NFATc2, demonstrating that NFATc2 is required for regulation of IL-4 gene expression by IL-6. Regulation of NFATc2 expression and NFAT transcriptional activity represents a novel pathway by which IL-6 can modulate gene expression.