Most cells can dynamically shift their relative reliance on glycolytic versus oxidative metabolism in response to nutrient availability, during development, and in disease. Studies in model systems have shown that re-directing energy metabolism from respiration to glycolysis can suppress oxidative damage and cell death in ischemic injury. At present we have a limited set of drugs that safely toggle energy metabolism in humans. Here, we introduce a quantitative, nutrient sensitized screening strategy that can identify such compounds based on their ability to selectively impair growth and viability of cells grown in galactose versus glucose. We identify several FDA approved agents never before linked to energy metabolism, including meclizine, which blunts cellular respiration via a mechanism distinct from canonical inhibitors. We further show that meclizine pretreatment confers cardioprotection and neuroprotection against ischemia-reperfusion injury in murine models. Nutrient-sensitized screening may offer a useful framework for understanding gene function and drug action within the context of energy metabolism.

A major challenge in drug discovery is to distinguish the molecular targets of a bioactive compound from the hundreds to thousands of additional gene products that respond indirectly to changes in the activity of the targets. Here, we present an integrated computational-experimental approach for computing the likelihood that gene products and associated pathways are targets of a compound. This is achieved by filtering the mRNA expression profile of compound-exposed cells using a reverse-engineered model of the cell's gene regulatory network. We apply the method to a set of 515 whole-genome yeast expression profiles resulting from a variety of treatments (compounds, knockouts and induced expression), and correctly enrich for the known targets and associated pathways in the majority of compounds examined. We demonstrate our approach with PTSB, a growth inhibitory compound with a previously unknown mode of action, by predicting and validating thioredoxin and thioredoxin reductase as its target.

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2؉ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2؉ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2؉ /CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Key Words: phosphodiesterase Ⅲ cGMP Ⅲ cardiomyocyte Ⅲ cardiac hypertrophy C a 2ϩ /calmodulin (CaM)-dependent signaling has been implicated in promoting pathological gene expression involved in hypertrophy and heart failure through the activation of Ca 2ϩ /CaM-dependent kinases, phosphatases, and ion channels. 1 Recently, a number of intrinsic negative regulators of cardiac growth have been identified which activate cGMPdependent signaling. 2 Stimulation of cGMP synthesis through genetic upregulation of natriuretic peptide receptor (guanylyl cyclase-A) prevents neurohumoral or pressure overload induced hypertrophy, 3 whereas disruption of cGMP synthesis results in enhanced hypertrophy and deteriorated cardiac function. 4 Likewise, chronic inhibition of cGMP metabolism by a cyclic nucleotide phosphodiesterase (PDE)5 inhibitor prevents and reverses pressure overload induced cardiac hypertrophy. 5 PDEs, by degrading cAMP and/or cGMP, regulate the amplitude, duration, and compartmentation of intracellular cyclic nucleotide signaling. PDEs constitute a superfamily of enzymes grouped into 11 broad families based on their distinct kinetic, regulatory, and inhibitory properties. PDE family members are also differentially expressed in various tissues and present within distinct subcellular domains. Together, these properties enable PDE enzymes to regulate the spatiotemporal, intracellular cAMP and cGMP gradients in response to various external stimuli. At least 5 PDE families, PDE1 to -5, have been reported in the heart, of which PDE1 and PDE5 are most likely responsible for cGMP hydrolysis. Logically, alteration of cGMP-hydrolyzing PDE expression/ activity...

Ischemic preconditioning (IPC) affords cardioprotection against ischemia-reperfusion (IR) injury, and while the molecular mechanisms of IPC are debated, the mitochondrial ATP-sensitive K(+) channel (mK(ATP)) has emerged as a candidate effector for several IPC signaling pathways. The molecular identity of this channel is unknown, but significant pharmacologic overlap exists between mK(ATP) and mitochondrial respiratory complex II (succinate dehydrogenase). In this investigation, we utilized isolated cardiac mitochondria, Langendorff perfused hearts, and a variety of biochemical methods, to make the following observations: (i) The competitive complex II inhibitor malonate is formed in mitochondria under conditions resembling IPC. (ii) IPC leads to a reversible inhibition of complex II that has likely been missed in previous investigations due to the use of saturating concentrations of succinate. (iii) Malonate opens mK(ATP) channels even when mitochondria are respiring on complex I-linked substrates, suggesting an effect of this inhibitor on the mK(ATP) channel independent of complex II inhibition. Together, these observations suggest that complex II inhibition by endogenously formed malonate may represent an important activation pathway for mK(ATP) channels during IPC.

Reactive Oxygen Species (ROS) are known to cause oxidative damage to DNA, proteins and lipids. In addition, recent evidence suggests that ROS can also initiate signaling cascades that respond to stress and modify specific redox-sensitive moieties as a regulatory mechanism. This suggests that ROS are physiologically-relevant signaling molecules. However, these sensor/effector molecules are not uniformly distributed throughout the cell. Moreover, localized ROS damage may elicit site-specific compensatory measures. Thus, the impact of ROS can be likened to that of calcium, a ubiquitous second messenger, leading to the prediction that their effects are exquisitely dependent upon their location, quantity and even the timing of generation. Despite this prediction, ROS signaling is most commonly intuited through the global administration of chemicals that produce ROS or by ROS quenching through global application of antioxidants. Optogenetics, which uses light to control the activity of genetically-encoded effector proteins, provides a means of circumventing this limitation. Photo-inducible genetically-encoded ROS-generating proteins (RGPs) were originally employed for their phototoxic effects and cell ablation. However, reducing irradiance and/or fluence can achieve sub-lethal levels of ROS that may mediate subtle signaling effects. Hence, transgenic expression of RGPs as fusions to native proteins gives researchers a new tool to exert spatial and temporal control over ROS production. This review will focus on the new frontier defined by the experimental use of RGPs to study ROS signaling.