Diabetic retinopathy, a major cause of visual impairment, involves multiple pathological processes, including disruption of water homeostasis regulated by Müller glial cells. Aquaporin-4 (AQP4) is a water channel that mediates passive water transport driven by an osmotic gradient, whereas Na+/K+-ATPase (NKA) maintains the ionic balance underlying this process. We previously demonstrated that ouabain, an NKA inhibitor, preferentially induces neuronal cell loss in the inner retina. This study aimed to elucidate the role of AQP4 in ouabain-induced inner retinal injury using male Sprague-Dawley rats and rat retinal Müller cells (rMC-1). Immunohistochemical analysis revealed that AQP4 was localized to the endfeet of Müller cells along the deep retinal capillaries and was expressed throughout the cell body in rMC-1 cells. To assess the role of AQP4 in ouabain-induced retinal injury, we examined the effects of TGN020, an AQP4 inhibitor, using both in vitro and in vivo models. Ouabain induced acute cell swelling in rMC-1 cells, and this effect was significantly attenuated by TGN020 treatment. In vivo, rats were intravitreally injected with ouabain alone or in combination with TGN020, and retinal histological changes were evaluated 7 d after injection. Ouabain induced cell loss in both the ganglion cell layer (GCL) and inner nuclear layer (INL). Co-administration of TGN020 reduced cell loss in the INL, but not in the GCL. These findings suggest that the disruption of AQP4-mediated water transport in Müller cells may contribute to INL neurodegeneration associated with NKA dysfunction in rats.

Mitochondrial dysfunction in the retinal pigment epithelium (RPE) is a key pathological feature of age-related macular degeneration (AMD). However, mechanistically defined experimental models that recapitulate stress-mediated mitochondrial injury remain limited. Bcl-2-associated X (BAX), a key pro-apoptotic effector, serves as a critical upstream regulator of mitochondrial outer membrane permeabilization. In this study, we systematically characterized mitochondrial dysfunction induced by BAX trigger site activator 1 (BTSA1), a selective small-molecule BAX activator, in ARPE-19 cells. Treatment with BTSA1 (3-60 μM) for 24 and 48 h induced a concentration- and time-dependent reduction in cell viability, accompanied by caspase-3 activation. Mitochondrial membrane potential, assessed via tetramethylrhodamine ethyl ester staining, was markedly reduced in a BAX-dependent manner and associated with increased reactive oxygen species production following prolonged exposure or at high concentrations. BTSA1 profoundly altered mitochondrial dynamics by promoting DRP1-mediated fission while suppressing fusion through MFN2 downregulation and stress-associated OPA1 processing, resulting in pronounced mitochondrial fragmentation. Furthermore, BAX activation elicited a biphasic response in mitochondrial quality control pathways: mild stress induced impaired autophagic flux and compensatory mitochondrial biogenesis, whereas severe stress triggered mitophagy accompanied by failure of biogenic compensation. These coordinated alterations closely mirror mitochondrial pathologies observed in the degenerating RPE in AMD. Collectively, our findings demonstrate that BAX activation by BTSA1 is sufficient to induce a comprehensive cascade of mitochondrial dysfunction. This system represents a mechanistically defined experimental model for dissecting BAX-mediated mitochondrial pathology and evaluating therapeutic strategies to preserve mitochondrial integrity in AMD.

PURPOSE:Retinopathy of prematurity (ROP), a leading cause of visual impairment and blindness in preterm infants, is characterized by abnormal retinal vascular development, including tortuous arterioles and abnormally dense capillaries. Dysregulated production of vascular endothelial growth factor (VEGF) and disrupted interactions between glial and vascular cells contribute to its pathogenesis. This study investigated the effects of aflibercept, a clinically used anti-VEGF drug, and KRN633, a VEGF receptor tyrosine kinase inhibitor, on abnormal retinal vasculature and astrocyte distribution in a rat model of ROP. METHODS:ROP was induced in neonatal rats by subcutaneous injections of KRN633 on postnatal day (P) 7 and P8. Arteriolar tortuosity, capillary density, mammalian target of rapamycin complex 1 (mTORC1) activity (as indicated by phosphorylation of S6 protein [pS6]), and the distribution of glial fibrillary acidic protein (GFAP)-positive astrocytes were evaluated. RESULTS:In ROP model rats, tortuous arterioles and dense capillary plexuses were observed. At the vascular front, many vascular endothelial cells lacked GFAP-positive astrocyte coverage and exhibited strong pS6 immunoreactivity. Treatment with aflibercept or KRN633 significantly reduced capillary density and pS6-positive blood vessels at the vascular front. Following treatment, most vascular endothelial cells were covered by GFAP-positive astrocytes. However, neither aflibercept nor KRN633 ameliorated arteriolar tortuosity. CONCLUSION:These findings suggest that pathological angiogenesis in ROP is mediated through VEGF- and mTORC1-dependent mechanisms. Anti-VEGF therapies may help restore glial-vascular interactions by reducing abnormal blood vessels in the ROP retina.

AIMS:Heart failure is marked by suppression of fatty acid oxidation (FAO) and mitochondrial ATP production gene expression. While transcriptional downregulation via PGC-1 and PPARα/ERRα has been well documented, the involvement of the general transcriptional machinery remains insufficiently understood. Distinct from its salutary role in many cardiac conditions, endogenous Sirt1 negatively affects cardiac function during pressure overload (PO). This study investigates how Sirt1 modulates preinitiation complex (PIC) assembly and RNA polymerase II (Pol II) recruitment during pathological PO. METHODS AND RESULTS:Cardiac-specific Sirt1 knock-out (Sirt1 cKO) mice were subjected to PO. Interaction between Sirt1 and Sub1, a protein recruiting GTFs to the gene promoter, was assessed with co-immunoprecipitation, protein pull-down and molecular docking. Pol II recruitment was evaluated with ChIP-sequencing and -qPCR analyses. The binding affinity of Sirt1 and GTF to the Sub1 GTF binding domain was assessed by immunoprecipitation and protein pull-down assays. Unbiased ChIP-sequencing and -qPCR analyses showed that Pol II binding to metabolic gene promoters was downregulated during PO, which was reversed in Sirt1 cKO mice. PO upregulated Sirt1 in the heart and increased its binding to Sub1, which interacts PPARα and ERRα, but not NF-kB. The Sirt1 binding to Sub1 competitively displaced the interaction between Sub1 and GTFs, thereby inhibiting the PIC formation at the Sub1-PPARα/ERRα complex. CONCLUSIONS:Sirt1 impairs metabolic gene transcription during PO by competitively inhibiting Sub1-mediated recruitment of GTFs and PIC formation. This repression of general transcriptional machinery contributes to the metabolic disturbance and may represent a maladaptive component of the heart failure phenotype.

Na+/K+-ATPase (NKA) is an important transporter responsible for maintaining intracellular ion homeostasis. To identify retinal cell types most susceptible to NKA inhibition, we examined the effects of ouabain, an NKA inhibitor, on different retinal cell types in rats. Male Sprague-Dawley rats were used for this study. The effects of ouabain on the retina were assessed at 2, 7, and 14 days after intravitreal injection, using retinal histology, immunolabeling with cell-specific markers, and electroretinogram recordings. We observed a decrease in the number of cells in the ganglion cell layer (GCL) and inner nuclear layer (INL) after ouabain injection. Specifically, the number of NeuN- and Brn3a-positive cells in the GCL, as well as NeuN-, parvalbumin-, calretinin-, and choline acetyltransferase-, and secretagogin-positive cells in the INL, were significantly reduced in ouabain-injected eyes. The reduction in cell numbers in the INL occurred in the following order of susceptibility: parvalbumin-positive cells were most affected, followed by calretinin-positive cells, and then secretagogin-positive cells, and finally choline acetyltransferase- or NeuN-positive cells. Protein kinase Cα- and Sox9-positive cells showed modest changes, but calbindin-D-28k-positive cells remained unchanged. NKA α1 subunit was broadly expressed in the rat retina, whereas the ouabain-sensitive NKA α2 and/or α3 subunits were present in all retinal neuronal cells and in Müller cells. The electroretinogram showed significant reductions in both a- and b-wave amplitudes. These findings suggest that amacrine cells are particularly susceptible to NKA inhibition compared to other retinal cell types, and this differential susceptibility may contribute to cell type-specific damage in retinal neurodegenerative diseases.

The pharmacological treatment of pediatric heart failure (PHF) remains a long-standing unmet medical need. We previously reported that TRV027, a peptidyl angiotensin type 1 receptor (AT1R) agonist that selectively activates β-arrestin over G protein signaling and avoids G protein-mediated vasoconstriction, increased cardiac contractility in neonatal, but not adult, mice by activating CaV1.2 L-type Ca2 + channels via casein kinase 2 without detectable adverse effects. TRV027 also significantly increased twitch Ca2+ transients in human induced pluripotent stem cell-derived cardiomyocytes. To examine whether TRV027 ameliorates PHF, its effects were evaluated in preweaning cTnTΔK210 knock-in mice, a model of human pediatric dilated cardiomyopathy with overt heart failure, using candesartan (Cand) as a reference AT1R blocker. Saline, TRV027, or Cand was administered chronically by daily subcutaneous injection from postnatal day 1-30 to homozygous (HOMO) mice and wild-type littermates. TRV027, but not Cand, significantly improved the survival rate of HOMO mice at P35. Echocardiography showed that TRV027 significantly slowed the time-dependent decline in left ventricular ejection fraction and wall motion in HOMO mice, without arrhythmias or obvious toxicities in major organs. Conversely, Cand did not slow PHF progression in HOMO mice and further impaired postnatal metanephrogenesis in both genotypes. Our findings strongly support cardiac AT1R/β-arrestin signaling as a promising, safe, and effective therapeutic axis for PHF, addressing the long-standing unmet medical needs in pediatric cardiology.

Methylglyoxal (MGO), a highly reactive dicarbonyl compound produced via the glycolytic pathway, plays a key role in the pathogenesis of various diabetic complications, such as diabetic retinopathy. M & uuml;ller cells provide neurotrophic support and maintain retinal homeostasis, including the redox balance. This dysfunction leads to retinal disease. Yes-associated protein (YAP), a major downstream effector of the Hippo pathway, plays a crucial role in regulating cell survival. In this study, we investigated the roles of M & uuml;ller cell YAP during MGO-induced retinal injury using normal rats intravitreally injected with MGO and a rat M & uuml;ller cell line (rMC-1). Immunohistochemistry revealed that MGO injection increased the glial fibrillary acidic protein immunoreactivity in M & uuml;ller cells. The alignment of M & uuml;ller cell nuclei was disrupted in MGO-treated retinas. YAP increased and activated in M & uuml;ller cells two days after MGO injection. This increase in YAP levels was independent of the Hippo pathway and partially attributed to the upregulation of YAP mRNA levels. YAP inhibition by verteporfin exacerbated MGO-induced cell damage and decreased Bcl-xL levels in rMC-1 cells. Intravitreal verteporfin injection also enhanced MGO-induced retinal oxidative stress. Overall, our findings suggest that YAP activation in M & uuml;ller cells alleviates oxidative stress in the retina following MGO-induced retinal injury.

Empagliflozin (EMPA), a sodium-glucose cotransporter 2 inhibitor used in patients with type 2 diabetes, exerts various beneficial effects, including anti-inflammatory and antioxidant properties, in addition to its glucose-lowering effect. In this study, we examined whether EMPA protects against N-methyl-d-aspartic acid (NMDA)-induced retinal excitotoxicity and the mechanisms underlying its protective effects. Male Sprague-Dawley rats (7-8 weeks old) were used in this study. The number of cells in the ganglion cell layer (GCL) decreased 7 days after intravitreal injection of NMDA (50 nmol). Simultaneous intravitreal injection of EMPA (50 and 100 nmol) and NMDA reduced NMDA-induced cell loss in a dose-dependent manner. The protective effect of EMPA was significantly attenuated by the AMP-activated protein kinase (AMPK) inhibitor, compound C (10 nmol). NMDA increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the GCL 6 h after injection, and the response was significantly, but not completely, attenuated by EMPA. These results suggest that EMPA protects against NMDA-induced retinal excitotoxicity in rats. The protective effect of EMPA may be partly attributed to the activation of the AMPK pathway and inhibition of neuronal cell apoptosis.

Sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a) mediates Ca2+ reuptake into the SR in cardiomyocytes. The inactivation or downregulation of SERCA2a leads to reduced contractility in the failing heart. Here we show that SERCA2a is regulated by p22phox, a heterodimeric partner of NADPH oxidases. Endogenous p22phox was upregulated by pressure overload, but cardiac-specific p22phox knockout (cKO) in mice exacerbated heart failure, enhanced the downregulation of SERCA2a and increased oxidative stress in the SR. We show that p22phox interacts with SERCA2a, preventing its oxidation at Cys498 and subsequent degradation by the Smurf1 and Hrd1 E3 ubiquitin ligases. The exacerbation of SERCA2a downregulation and cardiac dysfunction following pressure overload in p22phox cKO mice was alleviated when these mice were crossed with SERCA2a-C498S knock-in mice, in which the oxidation-susceptible and degradation-promoting cysteine residue is mutated. Future molecular interventions to prevent the oxidation of SERCA2a at Cys498 may prevent its downregulation during heart failure. Nakada, Titus et al. show that p22phox, a heterodimeric partner of NADPH oxidases, prevents sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) oxidation at Cys498 and its downregulation. This study suggests that therapeutic interventions to protect this residue may sustain SERCA2a expression in heart failure.

β-Hydroxybutyrate, a metabolic substrate and signaling molecule, exerts neuroprotective effects against various neurodegenerative diseases, including retinal diseases. However, mechanisms underlying these neuroprotective effects are currently not fully understood. In this study, we examined the effects of β-hydroxybutyrate against N-methyl-D-aspartic acid (NMDA)-induced retinal injury in rats and the underlying mechanisms. The intravitreal injection of NMDA (200 nmol) resulted in a decrease in neuronal cells, an increase in apoptotic cells, and activation of microglial cells. Simultaneous intravitreal injection of β-hydroxybutyrate (50 nmol) and NMDA significantly attenuated these NMDA-induced responses. The protective effects of β-hydroxybutyrate against NMDA-induced retinal injury were suppressed by Compound C (an inhibitor of AMPK), but not AR-C155858 (an inhibitor of monocarboxylate transporters). These findings suggest that β-hydroxybutyrate exerts protective effects against NMDA-induced retinal excitotoxicity through inhibition of neuronal cell apoptosis and microglial cell activation. β-Hydroxybutyrate is transported across the plasma membrane via monocarboxylate transporters; however, the mechanisms associated with these transporters are unlikely to be involved in its protective effect. Instead, activation of AMPK pathway may contribute to β-hydroxybutyrate-mediated protection.

Diabetic retinopathy is a major ocular complication associated with diabetes mellitus. Pericyte loss is a hallmark of diabetic retinopathy. The platelet-derived growth factor (PDGF)-B-PDGF receptor-β (PDGFRβ) signaling pathway plays an important role in the proliferation and migration of pericytes. Imatinib, an antineoplastic drug primarily used to treat chronic myelogenous leukemia, inhibits the PDGFRβ tyrosine kinase. In this study, we aimed to determine the time-course of pathological changes in the retinal vasculature following pharmacological depletion of pericytes with imatinib. Rats were injected with imatinib once daily for 1, 2, or 4 days starting on postnatal day (P) 4. The distribution of endothelial cells and pericytes in the retina was assessed at P4, P5, P6, P8, and P11. Single and multiple injections of imatinib (100 mg/kg) significantly decreased the pericyte coverage within the retinal capillaries on the day after the completion of each injection protocol. After pericyte coverage decreased, endothelial cell degeneration and microaneurysm formation were initiated. Following the elimination of the inhibitory effect of imatinib on the PDGFRβ signaling pathway, the pericyte coverage returned to control levels but structural abnormalities of the retinal vasculature with microaneurysms and dense capillaries were observed. Vascular pathological features are similar to those of the early clinical manifestations of diabetic retinopathy. Therefore, these rats could serve as animal models to study the mechanisms underlying the pathological changes that occur after pericyte loss in diabetic retinopathy.

Activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway protects against Nmethyl-D-aspartic acid (NMDA)-induced excitotoxic retinal injury. AMPK activation enhances fatty acid metabolism and ketone body synthesis. Ketone bodies are transported into neurons by monocarboxylate transporters (MCTs) and exert neuroprotective effects. In this study, we examined the distribution and expression levels of MCT1 and MCT2 in the retina and analyzed the effects of pharmacological inhibition of MCTs on the protective effects of metformin and 5-aminoimidazole-4-carboxamide (AICAR), activators of AMPK, against NMDA-induced retinal injury in rats. MCT1 was expressed in the blood vessels, processes of astrocytes and M & uuml;ller cells, and inner segments of photoreceptors in the rat retina, whereas MCT2 was expressed in neuronal cells in the ganglion cell layer (GCL) and in astrocyte processes. The expression levels of MCT2, but not MCT1, decreased one day after intravitreal injection of NMDA (200 nmol). Intravitreal injection of NMDA decreased the number of cells in the GCL compared to the vehicle seven days after injection. Simultaneous injection of metformin (20 nmol) or AICAR (50 nmol) with NMDA attenuated NMDA-induced cell loss in the GCL, and these protective effects were attenuated by AR-C155858 (1 pmol), an inhibitor of MCTs. AR-C155858 alone had no significant effect on the retinal structure. These results suggest that AMPK-activating compounds protect against NMDA-induced excitotoxic retinal injury via mechanisms involving MCTs in rats. NMDA-induced neurotoxicity may be associated with retinal neurodegenerative changes in glaucoma and diabetic retinopathy. Therefore, AMPK-activating compounds may be effective in managing these retinal diseases.

Retinal neurodegeneration, characterized by retinal ganglion cell (RGC) death, is a leading cause of vision impairment and loss in blind diseases, such as glaucoma. Müller cells play crucial roles in maintaining retinal homeostasis. Thus, dysfunction of Müller cells has been implicated as one of the causes of retinal diseases. Yes-associated protein 1 (YAP), a nuclear effector of the Hippo pathway, regulates mammalian cell survival. In this study, we investigated the role of YAP in Müller cells during N-methyl-D-aspartic acid (NMDA)-induced excitotoxic RGC injury in rats. We found that YAP expression increased and was activated in Müller cells after NMDA-induced RGC injury. This YAP response was partly due to an increase in Yap mRNA levels, although it may be independent of the Hippo pathway and β-TrCP-mediated YAP degradation. Morphological analysis revealed that verteporfin, a selective YAP inhibitor, exacerbated NMDA-induced RGC degeneration, suggesting that YAP activation in Müller cells contributes to RGC survival in NMDA-treated retinas. Studies in the rat Müller cell line (rMC-1) demonstrated that overexpression of YAP increased the levels of Bcl-xL, while verteporfin decreased the levels of Bcl-xL and cell viability and increased the levels of cytochrome c released from mitochondria and cleaved caspase-3. Finally, we found that Bcl-xL expression increased slightly in NMDA-treated retinas, whereas intravitreal injection of verteporfin suppressed this increase. Our findings suggest that activated YAP in Müller cells protects against NMDA-induced RGC injury by upregulating Bcl-xL expression.

The heart utilizes glucose and its metabolites as both energy sources and building blocks for cardiac growth and survival under both physiological and pathophysiological conditions. YAP/TAZ, transcriptional co-activators of the Hippo pathway, are key regulators of cell proliferation, survival, and metabolism in many cell types. Increasing lines of evidence suggest that the Hippo-YAP/TAZ signaling pathway is involved in the regulation of both physiological and pathophysiological processes in the heart. In particular, YAP/TAZ play a critical role in mediating aerobic glycolysis, the Warburg effect, in cardiomyocytes. Here, we summarize what is currently known about YAP/TAZ signaling in the heart by focusing on the regulation of glucose metabolism and its functional significance.

Introduction: Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22 phox , a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22 phox in oxidative stress and SERCA2a levels during pressure overload (PO). Question: Does loss of p22 phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO? Methods/approach: Cardiac-specific p22 phox knockout ( p22 phox -cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22 phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling. Results: The p22 phox -cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p<0.05) and 50% lower total tissue H 2 O 2 levels compared to WT after 1-week TAC (p<0.01). Unexpectedly, p22 phox -cKO mice had higher mortality (+20%, p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22 phox directly interacted with SERCA2a. Lack of p22 phox led to oxidation of SERCA2a (50% reduction in BIAM labelling, p<0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and p22 phox -cKO mice. Furthermore, in the absence of p22 phox , SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22 phox knockdown restored SERCA2a to 70% of WT levels (ns and p<0.5 vs p22 phox knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice. Conclusion: The loss of p22 phox exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.

Resveratrol, a natural polyphenolic compound, reportedly possesses numerous biological activities, including anti-inflammatory and antioxidant effects. In the current study, we examined specialIntscript the dilator effects of resveratrol on retinal arterioles, specialIntscript the protective effects of resveratrol against excitotoxic retinal injury, and specialIntscript whether these effects are mediated by the AMP-activated kinase (AMPK)-dependent pathway in rats. Male Wistar rats (7 to 10 weeks old) were used in this study. The diameters of the retinal arterioles, mean arterial pressure, and heart rate were measured in vivo. The retinal injury was assessed by histological examination. Intravenous injection of resveratrol (3 mg/kg) increased the diameter of the retinal arterioles without affecting the mean arterial pressure and heart rate. The AMPK inhibitor, compound C (5 mg/kg, intravenously), significantly attenuated the retinal vasodilator response to resveratrol. Seven days after intravitreal injection of N-methyl-D-aspartic acid (NMDA; 25, 50, and 100 nmol/eye), the number of cells located in the ganglion cell layer (GCL) was reduced, along with thinning of the inner plexiform layer. Intravitreal resveratrol injection (100 nmol/eye) reduced the NMDA (25 and 50 nmol/eye)-induced cell loss in the GCL. The neuroprotective effect of resveratrol was significantly but not completely reversed by compound C (10 nmol/eye). These results suggest that resveratrol dilates retinal arte-rioles and protects against NMDA-induced retinal neurodegeneration via an AMPK-dependent pathway in rats. Resveratrol may have the potential to slow the onset and progression of diseases associated with retinal ischemia by improving impaired retinal circulation and protecting retinal neuronal cells.

Yes-associated protein 1 (YAP), a transcriptional co-activator, is known to regulate cell growth and organ size. We have shown previously that YAP is activated in response to acute pressure overload (PO), and that YAP cardiac-specific heterozygous knockout (YAPchKO) mice are suppressed adaptive cardiac hypertrophy with inhibition of GLUT1 upregulation during acute PO. Furthermore, we found that YAP promotes glycolysis by upregulating GLUT1 in cultured rat ventricular myocytes. Glycolysis is intimately involved in cell growth. Thus, we examined whether AAV-mediated GLUT1 overexpression rescues adaptive cardiac hypertrophy during acute PO in YAPchKO mice. Wild-type (WT) or YAPchKO mice were injected with AAV-control or AAV-GLUT. After 14 days, these mice were subjected to sham or transverse artic constriction (TAC) for 7 days. TAC induced adaptive cardiac hypertrophy in WT mice injected with both AAVs. In contrast, left ventricular (LV) dysfunction, LV dilation, and inhibition of cardiac hypertrophy were observed in YAPchKO mice injected with AAV-control after TAC. AAV-GLUT1 injection successfully rescued GLUT1 upregulation, LV function, and cardiac hypertrophy in YAPchKO mice after TAC. These results suggest that YAP-induced upregulation of GLUT1 plays a critical role in promoting adaptive cardiac hypertrophy during acute PO.

Matrix metalloproteinases (MMPs) and tumor necrosis factor (TNF)-α contribute to the pathogenesis of several ocular diseases. In this study, we aimed to determine the role of interaction between TNF-α and MMP-9 in capillary degeneration. In rats, retinal injury was induced by intravitreal injection of N-methyl-D-aspartic acid (NMDA) at postnatal day 7. We examined 1) the effects of blocking MMP-9 and TNF-α signaling pathway on capillary degeneration, 2) changes in protein levels and distribution of MMP-9 and TNF-α, and 3) the interaction between MMP-9 and TNF-α in regulating the expression level of each protein in retinas of NMDA-injected eyes. Intravitreal injection of GM6001, an MMP inhibitor, or TNF-α neutralizing antibody (anti-TNF-α Ab) attenuated capillary degeneration in retinas of NMDA-injected eyes. Protein levels of TNF-α increased 2 h after NMDA injection, whereas those of MMP-9 increased 4 h after the injection. Anti-TNF-α Ab suppressed activation of MMP-9 in retinas of NMDA-injected eyes, whereas GM6001 diminished the TNF-α protein expression. Incubation of recombinant TNF-α with supernatants of homogenized retina increased protein levels and activity of MMP-9. These results suggest that TNF-α and MMP-9 collaboratively contributes to the progressive capillary degeneration in injured retinas.

Yes-associated protein 1 (YAP), a major transcriptional cofactor in the Hippo signaling pathway, is known to regulate cell growth and homeostasis. We have shown previously that YAP is activated and mediates adaptive cardiac hypertrophy in response to acute pressure overload (PO). However, how YAP induces adaptive hypertrophy is unknown. Glycolysis is intimately involved in cell growth, including cardiac hypertrophy. Here we examined whether YAP regulates glycolysis during acute PO to promote adaptive cardiac hypertrophy. Evaluation of extracellular acidification rate using the Seahorse XF analyzer in freshly isolated adult ventricular myocytes (AVMs) from wild-type mice revealed that glycolytic flux was increased by acute PO. The PO-induced glycolysis was attenuated in isolated AVMs from cardiac-specific heterozygous YAP knockout mice. We found that YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of glucose metabolites, including L-serine, L-aspartate, and malate, during acute PO. YAP overexpression increased GLUT1 protein levels, glycolytic flux, and cardiomyocytes size in isolated AVMs. These results suggest that YAP induces adaptive cardiac hypertrophy through activation of aerobic glycolysis in response to acute PO.