In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNF ␣ induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNF ␣ stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNF ␣ -mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNF ␣ . These findings suggest that the elevated TNF ␣ levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNF ␣ -induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors. (J. Clin. Invest. 1996. 98:2854-2865 )

Pharmacologic arm-selective unfolded protein response (UPR) signaling pathway activation is emerging as a promising strategy to ameliorate imbalances in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. The small molecule N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (147) was previously identified (Plate et al., 2016) to preferentially activate the ATF6 arm of the UPR, promoting protective remodeling of the ER proteostasis network. Here we show that 147-dependent ATF6 activation requires metabolic oxidation to form an electrophile that preferentially reacts with ER proteins. Proteins covalently modified by 147 include protein disulfide isomerases (PDIs), known to regulate ATF6 activation. Genetic depletion of PDIs perturbs 147-dependent induction of the ATF6-target gene, BiP, implicating covalent modifications of PDIs in the preferential activation of ATF6 afforded by treatment with 147. Thus, 147 is a pro-drug that preferentially activates ATF6 signaling through a mechanism involving localized metabolic activation and selective covalent modification of ER resident proteins that regulate ATF6 activity.

Key Words: Akt Ⅲ PHLPP Ⅲ phosphatase Ⅲ heart Ⅲ protection N umerous studies have demonstrated that activation of Akt contributes to the cardioprotective effects of receptors tyrosine kinases, 1,2 glycoprotein 130 -linked receptors, [3][4][5] and G protein-coupled receptors. 6,7 These receptors activate phosphatidylinositol 3-kinase (PI3K) and the resultant increase in phosphatidylinositol (3,4,5) triphosphate (PIP 3 ) levels drives Akt translocation to the plasma membrane. Akt is subsequently activated through phosphorylation at Thr308 by the upstream kinase phosphoinositide-dependent kinase 1 (PDK1) and phosphorylation at Ser473 by a mechanism that depends on both TORC2 and the intrinsic catalytic activity of Akt. 8,9 The lipid phosphatase PTEN, which dephosphorylates PIP 3 to PIP 2 , has been shown to limit Akt activation by decreasing PIP 3 . Deletion or mutation of PTEN is observed in many types of tumors and is accompanied by high Akt activity. 10 A recent study identified a PH domain-only protein, PHLDA3, that competes with the PH domain of Akt for binding of PIP 3 . 11 These molecules regulate the activation of Akt via various mechanisms but far less is known about mechanisms involved in terminating Akt activity by its dephosphorylation.Protein phosphatase (PP)2A has been shown to dephosphorylate Akt at Thr308 and/or Ser473 in noncardiac cells. 12,13 A pharmacological study also suggests that in retina PP2B (calcineurin) can dephosphorylate Akt at both sites. 14 A more specific Akt-directed novel PP2C family member protein phosphatase, PHLPP (PH domain leucine-rich repeat protein phosphatase), [15][16][17] In cardiac myocytes, overexpression of PTEN has been shown to be proapoptotic, whereas genetic deletion of PTEN rescues hearts from ischemia/reperfusion (I/R) injury. 18,19 These data support observations made in noncardiac cells which demonstrate that modulation of Akt activity regulates cell survival. A recent article showed that either PP2A or PP2B (calcineurin) can dephosphorylate Akt and thereby, regulate insulin signaling in cardiomyocytes. 20 It has been generally believed that phosphatases such as PP2A and PP2B have poor substrate selectivity, eliciting dephosphorylation of diverse target molecules. In contrast, PHLPP has been reported to be a selective Akt phosphatase. [15][16][17] In this study, we demonstrate a role for endogenous PHLPP-1 in regulation of cardiomyocyte Akt activity and survival in vitro and in vivo. MethodsPHLPP-1 knockout (KO) mice were generated in the C57BL/6 strain as described previously. 21 All mice used in the present study were male at 8 to 10 weeks of age. All procedures were performed in accordance with NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. To knockdown PHLPP-1, predesigned PHLPP-1 ONTARGETplus small interfering (si)RNA for rat and control siRNA were purchased from Thermo Scientific. NRVMs were transfected with siRNA using DharmaFECT-I transfection reagent (Thermo Scientific) based on ...