Overexpression of adenylyl cyclase type 5 (AC5) confers a proarrhythmic substrate to the heart.

Inhibition of ß-adrenergic receptor (ß-AR) signaling is one of the most common therapeutic approaches for patients with arrhythmias. Adenylyl cyclase (AC) is the key enzyme responsible for transducing ß-AR stimulation to increases in cAMP. The two major AC isoforms in the heart are types 5 and 6. Therefore, it is surprising that prior studies on overexpression of AC5 and AC6 in transgenic (Tg) mice have not examined mediation of arrhythmogenesis. Our goal was to examine the proarrhythmic substrate in AC5Tg hearts. Intracellular calcium ion (Ca(2+) i) was imaged in fluo-4 AM-loaded ventricular myocytes. The sarcoplasmic reticulum (SR) Ca(2+) content, fractional Ca(2+) release, and twitch Ca(2+) transient were significantly higher in the AC5Tg vs. wild-type (WT) myocytes, indicating Ca(2+) overload in AC5Tg myocytes. Action potential (AP) duration was significantly longer in AC5Tg than in WT myocytes. Additionally, AC5Tg myocytes developed spontaneous Ca(2+) waves in a larger fraction compared with WT myocytes, particularly when cells were exposed to isoproterenol. The Ca(2+) waves further induced afterdepolarizations and triggered APs. AC5Tg hearts had increased level of SERCA2a, oxidized Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and phosphorylation of ryanodine receptors (RyR) at the CaMKII site, especially after isoproterenol treatment. This was consistent with higher reactive oxygen species production in AC5Tg myocytes after isoproterenol treatment. Isoproterenol induced more severe arrhythmias in AC5Tg than in WT mice. We conclude that AC5 overexpression promotes arrhythmogenesis, by inducing SR Ca(2+) overload and hyperactivation of RyR (phosphorylation by CaMKII), which in turn induces spontaneous Ca(2+) waves and afterdepolarizations.

H11 kinase/heat shock protein 22 deletion impairs both nuclear and mitochondrial functions of STAT3 and accelerates the transition into heart failure on cardiac overload.

BACKGROUND: Cardiac overload, a major cause of heart failure, induces the expression of the heat shock protein H11 kinase/Hsp22 (Hsp22). METHODS AND RESULTS: To determine the specific function of Hsp22 in that context, a knockout mouse model of Hsp22 deletion was generated. Although comparable to wild-type mice in basal conditions, knockout mice exposed to pressure overload developed less hypertrophy and showed ventricular dilation, impaired contractile function, increased myocyte length and accumulation of interstitial collagen, faster transition into heart failure, and increased mortality. Microarrays revealed that hearts from knockout mice failed to transactivate genes regulated by the transcription factor STAT3. Accordingly, nuclear STAT3 tyrosine phosphorylation was decreased in knockout mice. Silencing and overexpression experiments in isolated neonatal rat cardiomyocytes showed that Hsp22 activates STAT3 via production of interleukin-6 by the transcription factor nuclear factor-κB. In addition to its transcriptional function, STAT3 translocates to the mitochondria where it increases oxidative phosphorylation. Both mitochondrial STAT3 translocation and respiration were also significantly decreased in knockout mice. CONCLUSIONS: This study found that Hsp22 represents a previously undescribed activator of both nuclear and mitochondrial functions of STAT3, and its deletion in the context of pressure overload in vivo accelerates the transition into heart failure and increases mortality.

Preemptive conditioning of the swine heart by H11 kinase/Hsp22 provides cardiac protection through inducible nitric oxide synthase.

The second window of ischemic preconditioning (SWOP) provides maximal protection against ischemia through regulation of the inducible nitric oxide synthase (iNOS), yet its application is limited by the inconvenience of the preliminary ischemic stimulus required for prophylaxis. Overexpression of H11 kinase/Hsp22 (Hsp22) in a transgenic mouse model provides cardioprotection against ischemia that is equivalent to that conferred by SWOP. We hypothesized that short-term, prophylactic overexpression of Hsp22 would offer an alternative to SWOP in reducing ischemic damage through a nitric oxide (NO)-dependent mechanism. Adeno-mediated overexpression of Hsp22 was achieved in the area at risk of the left circumflex (Cx) coronary artery in chronically instrumented swine and compared with LacZ controls (n = 5/group). Hsp22-injected myocardium showed an average fourfold increase in Hsp22 protein expression compared with controls and a doubling in iNOS expression (both P < 0.05). Four days after ischemia-reperfusion, regional wall thickening was reduced by 58 ± 2% in the Hsp22 group vs. 82 ± 7% in the LacZ group, and Hsp22 reduced infarct size by 40% (both P < 0.05 vs. LacZ). Treatment with the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) before ischemia suppressed the protection induced by Hsp22. In isolated cardiomyocytes, Hsp22 increased iNOS expression through the transcription factors NF-κB and STAT, the same effectors activated by SWOP, and reduced by 60% H(2)O(2)-mediated apoptosis, which was also abolished by NOS inhibitors. Therefore, short-term, prophylactic conditioning by Hsp22 provides NO-dependent cardioprotection that reproduces the signaling of SWOP, placing Hsp22 as a potential alternative for preemptive treatment of myocardial ischemia.

Activation of the bone morphogenetic protein receptor by H11kinase/Hsp22 promotes cardiac cell growth and survival.

H11 kinase/Hsp22 (H11K) is a chaperone promoting cardiac cell growth and survival through the activation of Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K). In this study, we tested whether H11K-induced activation of the PI3K/Akt pathway is mediated by the bone morphogenetic protein (BMP) signaling, both in a transgenic mouse model with cardiac-specific overexpression of H11K and in isolated cardiac myocytes. Microarrays in hearts from transgenic compared to wild-type mice showed an upregulation of the BMP receptors Alk3 and BMPR-II, and of their ligand BMP4 (P<0.01 versus wild type). Activation of the BMP pathway in transgenic mice was confirmed by increased phosphorylation of the "canonical" BMP effectors Smad 1/5/8 (P<0.01 versus wild type). In isolated myocytes, adenovirus-mediated overexpression of H11K was accompanied by a significant (P<0.01) increase in PI3K activity, phospho-Akt, Smad 1/5/8 phosphorylation and [(3)H]phenylalanine incorporation, and by a 70% reduction in H(2)O(2)-mediated apoptosis. All these effects were abolished by the BMP antagonist noggin. In presence of BMP4, Smad 1/5/8 phosphorylation was enhanced by 5-fold on H11K overexpression but decreased by 3-fold on H11K knockdown (P<0.01 versus control), showing that H11K potentiates the BMP signaling. In pull-down experiments, H11K increased both the association of Alk3 and BMPR-II together, and their interaction with the transforming growth factor-beta-activated kinase (TAK)1, a "noncanonical" mediator of the BMP receptor signaling. TAK1 inhibition prevented H11K-mediated activation of Akt. Therefore, potentiation of the BMP receptor by H11K promotes an activation of the PI3K/Akt pathway mediated by TAK1, which dictates the physiological effects of H11K on cardiac cell growth and survival.

Proteasome activation during cardiac hypertrophy by the chaperone H11 Kinase/Hsp22.

AIMS: The regulation of protein degradation by the proteasome during cardiac hypertrophy remains largely unknown. Also, the proteasome translocates to the nuclear periphery in response to cellular stress in yeast, which remains unexplored in mammals. The purpose of this study was to determine the quantitative and qualitative adaptation of the proteasome during stable cardiac hypertrophy. METHODS AND RESULTS: We measured proteasome activity, expression and sub-cellular distribution in a model of chronic cardiac hypertrophy induced by the stress-response chaperone H11 Kinase/Hsp22 (Hsp22). Over-expression of Hsp22 in a transgenic (TG) mouse leads to a 30% increase in myocyte cross-sectional area compared to wild-type (WT) mice (P < 0.01). Characterization of the proteasome in hearts from TG mice vs. WT revealed an increased expression of both 19S and 20S subunits (P < 0.05), a doubling in 20S catalytic activity (P < 0.01), a redistribution of both subunits from the cytosol to the nuclear periphery, and a four-fold increase in nuclear-associated 20S catalytic activity (P < 0.001). The perinuclear proteasome co-localized and interacted with Hsp22. Inhibition of proteasome activity by epoxomicin reduced hypertrophy in TG by 50% (P < 0.05). Adeno-mediated over-expression of Hsp22 in isolated cardiac myocytes increased both cell growth and proteasome activity, and both were prevented upon inhibition of the proteasome. Similarly, stimulation of cardiac cell growth by pro-hypertrophic stimuli increased Hsp22 expression and proteasome activity, and proteasome inhibition in that setting prevented hypertrophy. Proteasome inhibitors also prevented the increase in rate of protein synthesis observed after over-expression of Hsp22 or upon addition of pro-hypertrophic stimuli. CONCLUSIONS: Hsp22-mediated cardiac hypertrophy promotes an increased expression and activity, and a subcellular redistribution of the proteasome. Inhibition of the proteasome reverses cardiac hypertrophy upon Hsp22 over-expression or upon stimulation by pro-hypertrophic hormones, and also blocks the stimulation of protein synthesis in these conditions.

H11 kinase prevents myocardial infarction by preemptive preconditioning of the heart.

Ischemic preconditioning confers powerful protection against myocardial infarction through pre-emptive activation of survival signaling pathways, but it remains difficult to apply to patients with ischemic heart disease, and its effects are transient. Promoting a sustained activation of preconditioning mechanisms in vivo would represent a novel approach of cardioprotection. We tested the role of the protein H11 kinase (H11K), which accumulates by 4- to 6-fold in myocardium of patients with chronic ischemic heart disease and in experimental models of ischemia. This increased expression was quantitatively reproduced in cardiac myocytes using a transgenic (TG) mouse model. After 45 minutes of coronary artery occlusion and reperfusion, hearts from TG mice showed an 82+/-5% reduction in infarct size compared with wild-type (WT), which was similar to the 84+/-4% reduction of infarct size observed in WT after a protocol of ischemic preconditioning. Hearts from TG mice showed significant activation of survival kinases participating in preconditioning, including Akt and the 5'AMP-activated protein kinase (AMPK). H11K directly binds to both Akt and AMPK and promotes their nuclear translocation and their association in a multiprotein complex, which results in a stimulation of survival mechanisms in cytosol and nucleus, including inhibition of proapoptotic effectors (glycogen synthase kinase-3beta, Bad, and Foxo), activation of antiapoptotic effectors (protein kinase Cepsilon, endothelial and inducible NO synthase isoforms, and heat shock protein 70), increased expression of the hypoxia-inducible factor-1alpha, and genomic switch to glucose utilization. Therefore, activation of survival pathways by H11K preemptively triggers the antiapoptotic and metabolic response to ischemia and is sufficient to confer cardioprotection in vivo equally potent to preconditioning.

The valosin-containing protein promotes cardiac survival through the inducible isoform of nitric oxide synthase.

AIMS: Expression of the heat shock protein 22 (Hsp22) in the heart stimulates cardiac cell survival through activation of the Akt pathway and expression of the inducible nitric oxide (NO) synthase (iNOS), the mediator of ischaemic preconditioning and the most powerful prophylaxis against cardiac cell death. The goal of the present study was to elucidate the downstream effector by which Hsp22 and Akt increase iNOS expression. We tested both in vivo and in vitro the hypothesis that such an effector is the valosin-containing protein (VCP), an Akt substrate, which activates the transcription factor NF-κB, using a transgenic mouse with cardiac-specific over-expression of Hsp22, as well as isolated rat cardiac myocytes. METHODS AND RESULTS: Using two-dimensional gel electrophoresis and mass spectrometry combined with immunoprecipitation, we found that Hsp22 and Akt co-localize and interact together with VCP. Adeno-mediated over-expression of VCP in isolated cardiac myocytes activated NF-κB and dose-dependently increased the expression of iNOS, which was abolished upon NF-κB inhibition. Over-expression of a dominant-negative (DN) mutant of VCP did not increase iNOS expression. VCP, but not its DN mutant, protected against chelerythrine-induced apoptosis, which was suppressed by inhibition of either NF-κB or iNOS. VCP-mediated activation of the NF-κB/iNOS pathway was also prevented upon inhibition of Akt. CONCLUSION: We conclude that the Akt substrate, VCP, mediates the increased expression of iNOS downstream from Hsp22 through an NF-κB-dependent mechanism.

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.