A bespoke gene expression (GE) based small molecule screen in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) was conducted with the aim of identifying drug targets and pathways with the potential to reverse heart failure (HF) pathologic GE and the resultant decompensated HF phenotype. The screen utilized a composite human-murine HF gene expression signature, a target annotated compound set, and a HF gene expression reversal scoring algorithm to identify small molecules and their associated targets as potential modulators of HF pathologic GE. Following hit triage, a lead optimization program, and compound characterization in preclinical rodent models of HF and human iPSC-CM contractility assays, KEAP1 kelch domain blockers were identified as potent efficacious agents in the restoration of contractile function in the setting of oxidant stress and pressure overload induced cardiac dysfunction. ### Competing Interest Statement The authors have declared no competing interest. GlaxoSmithKline

Background The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. Methods and Results Consistent with its ability to inhibit prolyl‐tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

Transient receptor potential canonical channel 3 (TRPC3) is a nonselective cation channel that is associated with hypertension. Previous studies show enhanced basal and angiotensin II‐induced TRPC3 expression in the vasculature of SHR. We tested the hypothesis that TRPC3 plays an important role in the regulation of blood pressure by using a novel TRPC3 agonist and antagonist. In patch‐clamp studies of HEK293 cells over expressing TRPC3, GSK1702934A stimulated basal TRPC3 current (EC50 = 0.06 uM) and GSK2820986A inhibited GPCR‐activated TRPC3 current (IC50 = 0.005 uM). In conscious, chronically instrumented SD rats and SHR, GSK1702934A (1.0 mg/kg, i.v.) rapidly and transiently increased mean arterial pressure (MAP) by 33 ± 3 mmHg and 39 ± 3 mmHg, respectively. GSK2820986A (10, 50, 150 ug/kg/min, i.v.) dose‐dependently blocked the acute pressor response to GSK1702934A (IC50 = 0.009 uM). In telemetered SD and SHR, GSK2820986A (300 mg/kg/d, chow) administration for three days increased plasma GSK2820986A concentrations to 0.02 uM and 0.05 uM, respectively, and had no effect on MAP (100 ± 2 to 105 ± 2 mmHg in SD; 135 ± 2 to 138 ± 2 mmHg in SHR) or heart rate (373 ± 8 to 390 ± 8 bts/min in SD; 322 ± 2 to 330 ± 3 bts/min in SHR). These data suggest that in normotensive and essential hypertensive rats TRPC3 activation acutely increases blood pressure similarly, but TRPC3 has no major role in the maintenance of steady‐state blood pressure.

Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator G5K2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP HSFD increased MAP urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of G5K2181236A, decreased urine output, and improved survival. Conversely, the low dose of G5K2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did G5K2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both G5K2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible for the vascular dysfunction. In summary, G5K2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end-organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the oxidative state of sGC and might help direct the clinical development of these novel classes of therapeutic agents.

The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that highly expressed in renal tubules. Co-localization of TRPV4 with sodium transporters suggests that TRPV4 might have a role in the regulation of sodium and water homeostasis. To investigate this potential interaction, we determined the blood pressure and renal responses to chronic TRPV4 blockade (GSK2193874A, 30 mg/kg/d) in rats in the presence and absence of Na, K,2Cl− cotransporter, Na, Cl cotransporter, or arginine vasopressin V2 receptor (AVP-V2) inhibition. Under normal conditions, TRPV4 blockade had no effect on mean arterial pressure, renal excretory function, free water clearance, or plasma sodium concentrations. The diuretic and natriuretic responses to Na, K,2Cl− cotransporter inhibition (furosemide, 30 mg/kg) or Na, Cl cotransporter inhibition (hydrochlorothiazide, 30 mg/kg) were unaltered after 5 days of TRPV4 blockade. In contrast, GSK2193874A increased the diuretic, natriuretic, and plasma sodium responses to AVP-V2 inhibition (tolvaptan, 10 mg/kg). Further investigation showed that GSK2193874A increased the plasma concentration of tolvaptan which accounted for the enhanced renal response. In conclusion, TRPV4 has no major effect on blood pressure or sodium and water homeostasis in normal rats either directly or through modulation of sodium transporter or AVP-V2 function in the kidney.

Purpose— This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. Methods and Results— Apolipoprotein E −/− mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group ( P <0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis ( P <0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group ( P <0.05). Conclusion— An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

Background: Experimental studies have demonstrated the ability of bone marrow-derived stem cells to enhance cardiac repair and regeneration post-myocardial infarction (MI). However, clinical trials...

Purpose: To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model. Materials andMethods: Osmotic pumps were implanted subcutaneously in apoE(-/-) mice for continuous infusion of Angiotensin II (Ang-II). Weekly bright-blood gradient echo scans were performed on the suprarenal abdominal aorta to evaluate aneurysm development. Once an AAA was detected, animals were administered 1000 mu mol/kg of the USPIO contrast agent ferumoxtran-10 (Combidex (R)) followed by in vivo scanning 24 h post-USPIO administration. After in vivo imaging, aortas were harvested for ex vivo imaging, histology, iron quantification, and gene expression analysis.Results: Reduced signal intensity was evident in the post-USPIO transverse images of the abdominal aorta. The areas of reduced signal were primarily along the aneurysm shoulder and outer perianeurysm areas and corresponded to regions of macrophage infiltration and colocalized USPIO determination by mew-is of histological staining. The absolute iron content measured significantly correlated to the area of signal reduction in the ex vivo images (r = 0.9; P < 0.01). In the AAA tissue, the macrophage-driven cytokine gene expression was up-regulated along with a matrix metalloproteinase known to mediate extracellular matrix breakdown in this disease model.Conclusion: These results demonstrate the feasibility of using an USPIO contrast agent as a surrogate for detecting the acute inflammatory process involved in the development of abdominal aneurysms.

Background— Hyperlipidimic mice administered angiotensin II have been used for the study of abdominal aortic aneurysms (AAAs). The purpose of this study was to examine the use of MRI for studying AAA development and for examining the effects of pharmacological intervention on AAA development in the apolipoprotein E–deficient mouse. Methods and Results— Suprarenal aortic aneurysms were generated in apolipoprotein E–deficient mice administered angiotensin II (1000 ng/kg per min) for up to 28 days. In vivo MRI was performed serially (once weekly) to assess AAA development and rupture. Comparison of AAA size as measured by in vivo and ex vivo MRI resulted in excellent agreement ( r =0.96, P <0.0001). In addition, MRI correlated with histology-derived AAA area assessment (in vivo versus histology: r =0.84, P <0.0001; ex vivo versus histology: r =0.89, P <0.0001). In a separate study, angiotensin II–administered apolipoprotein E–deficient mice were treated with doxycycline (broad-based matrix metalloproteinase inhibitor; 30 mg/kg per day for 28 days). MRI was able to noninvasively assess a reduced rate of AAA development (46% versus 71%, P <0.05), a decreased AAA area (2.56 versus 4.02 mm 2 , P <0.01), and decreased incidence of rupture (43% versus 100%) in treated versus control animals. Inhibition of aorta matrix metalloproteinase 2/9 activity was observed in the treated animals. Conclusions— These results demonstrate the use of MRI to noninvasively and temporally assess AAA development on pharmacological intervention in this preclinical cardiovascular disease model.

Objective— Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation. The purpose of this study was to noninvasively evaluate USPIO uptake in aorta of apoE −/− mice and to determine the effects of Angiotensin II (Ang II) infusion and chronic antiinflammatory treatment with a p38 MAPK inhibitor on this uptake. Methods and Results— ApoE −/− mice were administered saline or Ang II (1.44 mg/kg/d) for 21 days. In vivo MRI assessment of USPIO uptake in the aortic arch was observed in all animals. However, although the Ang II group had significantly higher absolute iron content (↑103%, P <0.001) in the aortic arch compared with the saline group, the p38 MAPK inhibitor (SB-239063, 150 mg/kg/d) treatment group did not (↑6%, NS). The in vivo MRI signal intensity was significantly correlated to the absolute iron content in the aortic arch. Histological evaluation of the aortic root lesion area showed colocalization of USPIO with macrophages and a reduction in USPIO but not macrophage content with SB-239063 treatment. Conclusion— The present study demonstrates that noninvasive assessment of USPIO uptake, as a marker for inflammation in murine atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the uptake of USPIO in aorta of Ang II–infused apoE −/− mice.

The purpose of this study was to investigate the role of Sgk2 in the regulation of salt and water homeostasis. Kidney function and expression of Sgk1, Sgk2, Sgk3, and αENaC were determined during normal and 0% salt intake in Sgk1−/−,Sgk2−/−, and Sgk1−/−,Sgk2−/− double KO mice (DKO). During normal salt intake, renal function of Sgk1−/−, Sgk2−/− and DKO mice were similar to wild type mice. The renal response to salt deprivation was impaired in Sgk1−/− but not in Sgk2−/− mice. DKO mice on 0% salt diet gained less body weight, had higher urine flow, sodium and chloride excretions, and similar glomerular filtration rate than Sgk1+/+,Sgk2+/+ mice. Plasma concentrations of sodium, chloride and potassium were increased, renal cortical Sgk3 expression was increased, and there was no difference in renal αENaC expression in DKO compared to wild type mice. DKO mice had an impaired ability to reabsorb water, sodium, chloride, and potassium during salt deprivation compared to Sgk1−/−or Sgk2−/− mice. In conclusion, Sgk2 has a minimal role in the regulation of salt and water homeostasis during normal or reduced salt intake in vivo. However, Sgk2 significantly contributes to water and electrolyte homeostasis during salt deprivation in the absence of Sgk1, suggesting that Sgk2 can compensate for functional changes in Sgk1.

-/mice (Fig 2) while treatment with SB239063 decreased normalized signal loss in the apoE -/mouse arch. However, while the Ang II group had significantly higher absolute iron content (↑103%, P<0.001) in the arch compared with the saline group, the Ang II+SB-239063 group did not (↑6%, NS). There was a significant positive correlation between the absolute iron content of the aortic arch and the normalized in vivo MRI signal intensity loss present in the aortic arch (r=0.79, P<0.001). Immunohistochemical localization of iron (Perl’s) and macrophages (Cat-S) within the atherosclerotic lesion can be seen in Fig. 3. Perl’s staining demonstrated that iron accumulation is mainly associated with the macrophages. Conclusion The present study demonstrates that non-invasive assessment of USPIO uptake, as a marker for inflammation in mouse atherosclerotic plaque, is feasible and that p38 MAPK inhibition attenuates the pro-inflammatory uptake of USPIO in the aorta of Ang II infused apoE -/mice. Fig 1 Fig 2 Fig 3