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.

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 (KO) mouse model of Hsp22 deletion was generated. Although comparable to wild type mice in basal conditions, KO 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 KO mice failed to transactivate genes regulated by the transcription factor STAT3. Accordingly, nuclear STAT3 tyrosine phosphorylation was decreased in KO. Silencing and over-expression experiments in isolated neonatal rat cardiomyocytes showed that Hsp22 activates STAT3 via production of interleukin-6 by the transcription factor NF-κB. In addition to its transcriptional function, STAT3 also translocates to the mitochondria where it increases oxidative phosphorylation. Both mitochondrial STAT3 translocation and respiration were significantly decreased in KO mice as well. Conclusions Hsp22 represents a previously undescribed activator of both nuclear and mitochondrial functions of STAT3, and its deletion in a context of pressure overload in vivo accelerates the transition into heart failure and increases mortality.

Abstract-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 82Ϯ5% reduction in infarct size compared with wild-type (WT), which was similar to the 84Ϯ4% 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 5ЈAMP-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-3␤, Bad, and Foxo), activation of antiapoptotic effectors (protein kinase C⑀, endothelial and inducible NO synthase isoforms, and heat shock protein 70), increased expression of the hypoxia-inducible factor-1␣, 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. (Circ Res. 2006;98:280-288.)

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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.

We conclude that the Akt substrate, VCP, mediates the increased expression of iNOS downstream from Hsp22 through an NF-κB-dependent mechanism.

Abstract-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 (PϽ0.01 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 (PϽ0.01 versus wild type). In isolated myocytes, adenovirus-mediated overexpression of H11K was accompanied by a significant (PϽ0.01) 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 (PϽ0.01 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-␤-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. Key Words: Akt Ⅲ bone morphogenetic protein Ⅲ heat shock protein Ⅲ phosphatidylinositol 3-kinase H 11 kinase/Hsp22 (H11K), which belongs to the crystallin family of heat shock proteins, is mainly expressed in heart and skeletal muscle. 1 An increase in H11K expression was found in a canine model of left ventricular hypertrophy, 2 in swine models of acute and repetitive myocardial ischemia, 3,4 and in patients with ischemic heart disease. 4 We generated a transgenic (TG) mouse with cardiac-specific overexpression of H11K to a level (3-to 5-fold) reproducing the increased expression found in the animal models and in patients. This TG model shows that H11K stimulates cardiac cell growth 2 and promotes cardiac cell survival to an extent reproducing the protection conferred by ischemic preconditioning. 5 We showed previously that these protective effects of H11K correlate with the activation of Akt, and of its downstream survival pathway. 2,5,6 The main activator of Akt is the phosphatidylinositol 3-kinase (PI3K), which phosphorylates Akt via the phosphatidylinositol-dependent kinase (PDK)1, and which is itself under the control of multiple growth factor receptors. 7 Our goal was to elucidate the mechanisms leading to H11K-mediated ...

Vatner SF, Depre C. Preemptive conditioning of the swine heart by H11 kinase/Hsp22 provides cardiac protection through inducible nitric oxide synthase. Am J Physiol Heart Circ Physiol 300: H1303-H1310, 2011. First published February 11, 2011 doi:10.1152/ajpheart.00979.2010.-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 ϭ 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 Ͻ 0.05). Four days after ischemia-reperfusion, regional wall thickening was reduced by 58 Ϯ 2% in the Hsp22 group vs. 82 Ϯ 7% in the LacZ group, and Hsp22 reduced infarct size by 40% (both P Ͻ 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-B and STAT, the same effectors activated by SWOP, and reduced by 60% H2O2-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. gene delivery; myocardial ischemia; preconditioning ALTHOUGH ANGIOPLASTY and thrombolytic therapy have reduced the mortality of acute ischemic heart disease, they do not prevent the deterioration of cardiac function after myocardial infarction (MI) that eventually leads to heart failure. Despite the progress in stem cell research, the only alternative approach to restore impaired cardiac function due to MI to date is cardiac transplantation, which is severely limited by donor availability. A promising concept is preemptive conditioning of the heart (32) [or prophylactic cardioprotection (6)], in which the activation of prosurvival pathways in patients at risk of future heart attack might prevent cell death if an episode of potentially lethal ischemia occurs. In this study, we tested the hypothesis that H11 kinase/Hsp22 (Hsp22) represents a candidate for such an approach.Hsp22, which belongs to the crystallin family of small heat shock proteins (24), shows an expression pattern that is restricted to a limited n...