Beyond Erectile Dysfunction: cGMP-Specific Phosphodiesterase 5 Inhibitors for Other Clinical Disorders.

Cyclic guanosine monophosphate (cGMP), an important intracellular second messenger, mediates cellular functional responses in all vital organs. Phosphodiesterase 5 (PDE5) is one of the 11 members of the cyclic nucleotide phosphodiesterase (PDE) family that specifically targets cGMP generated by nitric oxide-driven activation of the soluble guanylyl cyclase. PDE5 inhibitors, including sildenafil and tadalafil, are widely used for the treatment of erectile dysfunction, pulmonary arterial hypertension, and certain urological disorders. Preclinical studies have shown promising effects of PDE5 inhibitors in the treatment of myocardial infarction, cardiac hypertrophy, heart failure, cancer and anticancer-drug-associated cardiotoxicity, diabetes, Duchenne muscular dystrophy, Alzheimer's disease, and other aging-related conditions. Many clinical trials with PDE5 inhibitors have focused on the potential cardiovascular, anticancer, and neurological benefits. In this review, we provide an overview of the current state of knowledge on PDE5 inhibitors and their potential therapeutic indications for various clinical disorders beyond erectile dysfunction.

Acute endoplasmic reticulum stress-induced mitochondria respiratory chain damage: The role of activated calpains.

The induction of acute endoplasmic reticulum (ER) stress damages the electron transport chain (ETC) in cardiac mitochondria. Activation of mitochondria-localized calpain 1 (CPN1) and calpain 2 (CPN2) impairs the ETC in pathological conditions, including aging and ischemia-reperfusion in settings where ER stress is increased. We asked if the activation of calpains causes the damage to the ETC during ER stress. Control littermate and CPNS1 (calpain small regulatory subunit 1) deletion mice were used in the current study. CPNS1 is an essential subunit required to maintain CPN1 and CPN2 activities, and deletion of CPNS1 prevents their activation. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 72 h of TUNI treatment. ER stress was increased in both control littermate and CPNS1 deletion mice with TUNI treatment. The TUNI treatment activated both cytosolic and mitochondrial CPN1 and 2 (CPN1/2) in control but not in CPNS1 deletion mice. TUNI treatment led to decreased oxidative phosphorylation and complex I activity in control but not in CPNS1 deletion mice compared to vehicle. The contents of complex I subunits, including NDUFV2 and ND5, were decreased in control but not in CPNS1 deletion mice. TUNI treatment also led to decreased oxidation through cytochrome oxidase (COX) only in control mice. Proteomic study showed that subunit 2 of COX was decreased in control but not in CPNS1 deletion mice. Our results provide a direct link between activation of CPN1/2 and complex I and COX damage during acute ER stress.

Treating diabetes with combination of phosphodiesterase 5 inhibitors and hydroxychloroquine-a possible prevention strategy for COVID-19?

Type 2 diabetes (T2D) is one of the major risk factors for developing cardiovascular disease and the resultant devastating morbidity and mortality. The key features of T2D are hyperglycemia, hyperlipidemia, insulin resistance, and impaired insulin secretion. Patients with diabetes and myocardial infarction have worse prognosis than those without T2D. Moreover, obesity and T2D are recognized risk factors in developing severe form of COVID-19 with higher mortality rate. The current lines of drug therapy are insufficient to control T2D and its serious cardiovascular complications. Phosphodiesterase 5 (PDE5) is a cGMP specific enzyme, which is the target of erectile dysfunction drugs including sildenafil, vardenafil, and tadalafil. Cardioprotective effects of PDE5 inhibitors against ischemia/reperfusion (I/R) injury were reported in normal and diabetic animals. Hydroxychloroquine (HCQ) is a widely used antimalarial and anti-inflammatory drug and its hyperglycemia-controlling effect in diabetic patients is also under investigation. This review provides our perspective of a potential use of combination therapy of PDE5 inhibitor with HCQ to reduce cardiovascular risk factors and myocardial I/R injury in T2D. We previously observed that diabetic mice treated with tadalafil and HCQ had significantly reduced fasting blood glucose and lipid levels, increased plasma insulin and insulin-like growth factor-1 levels, and improved insulin sensitivity, along with smaller myocardial infarct size following I/R. The combination treatment activated Akt/mTOR cellular survival pathway, which was likely responsible for the salutary effects. Therefore, pretreatment with PDE5 inhibitor and HCQ may be a potentially useful therapy not only for controlling T2D but also reducing the rate and severity of COVID-19 infection in the vulnerable population of diabetics.

Single-Dose Treatment with Rapamycin Preserves Post-Ischemic Cardiac Function through Attenuation of Fibrosis and Inflammation in Diabetic Rabbit.

Robust activation of mTOR (mammalian target of rapamycin) signaling in diabetes exacerbates myocardial injury following lethal ischemia due to accelerated cardiomyocyte death with cardiac remodeling and inflammatory responses. We examined the effect of rapamycin (RAPA, mTOR inhibitor) on cardiac remodeling and inflammation following myocardial ischemia/reperfusion (I/R) injury in diabetic rabbits. Diabetic rabbits (DM) were subjected to 45 min of ischemia and 10 days of reperfusion by inflating/deflating a previously implanted hydraulic balloon occluder. RAPA (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before the onset of reperfusion. Post-I/R left ventricular (LV) function was assessed by echocardiography and fibrosis was evaluated by picrosirius red staining. Treatment with RAPA preserved LV ejection fraction and reduced fibrosis. Immunoblot and real-time PCR revealed that RAPA treatment inhibited several fibrosis markers (TGF-ß, Galectin-3, MYH, p-SMAD). Furthermore, immunofluorescence staining revealed the attenuation of post-I/R NLRP3-inflammasome formation with RAPA treatment as shown by reduced aggregation of apoptosis speck-like protein with a caspase recruitment domain and active-form of caspase-1 in cardiomyocytes. In conclusion, our study suggests that acute reperfusion therapy with RAPA may be a viable strategy to preserve cardiac function with the alleviation of adverse post-infarct myocardial remodeling and inflammation in diabetic patients.

PDE5 inhibitor sildenafil attenuates cardiac microRNA 214 upregulation and pro-apoptotic signaling after chronic alcohol ingestion in mice.

Abusive chronic alcohol consumption can cause metabolic and functional derangements in the heart and is a risk factor for development of non-ischemic cardiomyopathy. microRNA 214 (miR-214) is a molecular sensor of stress signals that negatively impacts cell survival. Considering cardioprotective and microRNA modulatory effects of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, we investigated the impact of chronic alcohol consumption on cardiac expression of miR-214 and its anti-apoptotic protein target, Bcl-2 and whether sildenafil attenuates such changes. Adult male FVB mice received unlimited access to either normal liquid diet (control), alcohol diet (35% daily calories intake), or alcohol + sildenafil (1 mg/kg/day, p.o.) for 14 weeks (n = 6-7/group). The alcohol-fed groups with or without sildenafil had increased total diet consumption and lower body weight as compared with controls. Echocardiography-assessed left ventricular function was unaltered by 14-week alcohol intake. Alcohol-fed group had 2.6-fold increase in miR-214 and significant decrease in Bcl-2 expression, along with enhanced phosphorylation of ERK1/2 and cleavage of PARP (marker of apoptotic DNA damage) in the heart. Co-ingestion with sildenafil blunted the alcohol-induced increase in miR-214, ERK1/2 phosphorylation, and maintained Bcl-2 and decreased PARP cleavage levels. In conclusion, chronic alcohol consumption triggers miR-214-mediated pro-apoptotic signaling in the heart, which was prevented by co-treatment with sildenafil. Thus, PDE5 inhibition may serve as a novel protective strategy against cardiac apoptosis due to chronic alcohol abuse.

Cardiovascular Complications Associated with COVID-19 and Potential Therapeutic~Strategies.

The outbreak of coronavirus disease 2019 (COVID-19), an infectious disease with severe acute respiratory syndrome, has now become a worldwide pandemic. Despite the respiratory complication, COVID-19 is also associated with significant multiple organ dysfunction, including severe cardiac impairment. Emerging evidence reveals a direct interplay between COVID-19 and dire cardiovascular complications, including myocardial injury, heart failure, heart attack, myocarditis, arrhythmias as well as blood clots, which are accompanied with elevated risk and adverse outcome among infected patients, even sudden death. The proposed pathophysiological mechanisms of myocardial impairment include invasion of SARS-CoV-2 virus via angiotensin-converting enzyme 2 to cardiovascular cells/tissue, which leads to endothelial inflammation and dysfunction, de-stabilization of vulnerable atherosclerotic plaques, stent thrombosis, cardiac stress due to diminish oxygen supply and cardiac muscle damage, and myocardial infarction. Several promising therapeutics are under investigation to the overall prognosis of COVID-19 patients with high risk of cardiovascular impairment, nevertheless to date, none have shown proven clinical efficacy. In this comprehensive review, we aimed to highlight the current integrated therapeutic approaches for COVID-19 and we summarized the potential therapeutic options, currently under clinical trials, with their mechanisms of action and associated adverse cardiac events in highly infectious COVID-19 patients.

Mitochondrial Complex I Inhibition by Metformin Limits Reperfusion Injury.

Transient, reversible blockade of complex I during early reperfusion after ischemia limits cardiac injury. We studied the cardioprotection of high dose of metformin in cultured cells and mouse hearts via the novel mechanism of acute downregulation of complex I. The effect of high dose of metformin on complex I activity was studied in isolated heart mitochondria and cultured H9c2 cells. Protection with metformin was evaluated in H9c2 cells at reoxygenation and at early reperfusion in isolated perfused mouse hearts and in vivo regional ischemia reperfusion. Acute, high-dose metformin treatment inhibited complex I in ischemia-damaged mitochondria and in H9c2 cells following hypoxia. Accompanying the complex I modulation, high-dose metformin at reoxygenation decreased death in H9c2 cells. Acute treatment with high-dose metformin at the end of ischemia reduced infarct size following ischemia reperfusion in vitro and in vivo, including in the AMP kinase-dead mouse. Metformin treatment during early reperfusion improved mitochondrial calcium retention capacity, indicating decreased permeability transition pore (MPTP) opening. Acute, high-dose metformin therapy decreased cardiac injury through inhibition of complex I accompanied by attenuation of MPTP opening. Moreover, in contrast to chronic metformin treatment, protection by acute, high-dose metformin is independent of AMP-activated protein kinase activation. Thus, a single, high-dose metformin treatment at reperfusion reduces cardiac injury via modulation of complex I.

Hydrogen sulfide mediates the cardioprotective effects of gene therapy with PKG-Iα.

Cyclic GMP-dependent protein kinase (PKG) is a serine-threonine kinase that mediates the cardioprotective effect of ischemic and pharmacologic preconditioning. Since hydrogen sulfide (H2S) has been implicated in mediating the cardioprotective effects of the cGMP modulators tadalafil and cinaciguat, we tested the hypothesis that myocardial gene therapy with PKG exerts cardioprotection against ischemia/reperfusion (I/R) injury through a mechanism involving H2S. Adult rat cardiomyocytes were infected with adenoviral vector encoding PKGIα or inactive mutant PKGIαK390A (K390A) for 24 h. Necrosis and apoptosis (n = 6/group) were determined after 90 min of simulated ischemia and 1 or 18 h of reoxygenation, respectively. To study the effect of PKGIα in vivo, mice received intramyocardial injections of adenoviral PKGIα or K390A. Four days later, the hearts were subjected to 30 min of ischemia followed by reperfusion for 24 h. The inhibitor of H2S-producing enzyme, cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG, 50 mg/kg, ip) was given 30 min before ischemia. PKGIα overexpression induced CSE expression, whereas cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase expression was not changed. PKGIα overexpression increased H2S in the heart and cardiomyocytes in relation to control and PKGIαK390A. Moreover, PAG abolished protection with PKGIα in vitro by increasing necrosis (35.2 ± 1.7%, P < 0.05) and apoptosis (23.5 ± 1.8 %, P < 0.05) as compared to PKGIα-overexpressing cells (necrosis: 17.2 ± 0.9% and apoptosis: 13.2 ± 0.8%). In vivo, PKGIα overexpression reduced infarct size and preserved left ventricular fractional shortening as compared with K390A (P < 0.05) and PAG abolished the cardioprotective effect of PKGIα. The protective effect of myocardial gene therapy with PKGIα against I/R injury is mediated through a mechanism involving H2S signaling.

Nuclear localization of vascular endothelial growth factor-D and regulation of c-Myc-dependent transcripts in human lung fibroblasts.

Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (∼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor-binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors.

Integrated Analysis of lncRNA-miRNA-mRNA Regulatory Network in Rapamycin-Induced Cardioprotection against Ischemia/Reperfusion Injury in Diabetic Rabbits.

The inhibition of mammalian target of rapamycin (mTOR) with rapamycin (RAPA) provides protection against myocardial ischemia/reperfusion (I/R) injury in diabetes. Since interactions between transcripts, including long non-coding RNA (lncRNA), microRNA(miRNA) and mRNA, regulate the pathophysiology of disease, we performed unbiased miRarray profiling in the heart of diabetic rabbits following I/R injury with/without RAPA treatment to identify differentially expressed (DE) miRNAs and their predicted targets of lncRNAs/mRNAs. Results showed that among the total of 806 unique miRNAs targets, 194 miRNAs were DE after I/R in diabetic rabbits. Specifically, eight miRNAs, including miR-199a-5p, miR-154-5p, miR-543-3p, miR-379-3p, miR-379-5p, miR-299-5p, miR-140-3p, and miR-497-5p, were upregulated and 10 miRNAs, including miR-1-3p, miR-1b, miR-29b-3p, miR-29c-3p, miR-30e-3p, miR-133c, miR-196c-3p, miR-322-5p, miR-499-5p, and miR-672-5p, were significantly downregulated after I/R injury. Interestingly, RAPA treatment significantly reversed these changes in miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated the participation of miRNAs in the regulation of several signaling pathways related to I/R injury, including MAPK signaling and apoptosis. Furthermore, in diabetic hearts, the expression of lncRNAs, HOTAIR, and GAS5 were induced after I/R injury, but RAPA suppressed these lncRNAs. In contrast, MALAT1 was significantly reduced following I/R injury, with the increased expression of miR-199a-5p and suppression of its target, the anti-apoptotic protein Bcl-2. RAPA recovered MALAT1 expression with its sponging effect on miR-199-5p and restoration of Bcl-2 expression. The identification of novel targets from the transcriptome analysis in RAPA-treated diabetic hearts could potentially lead to the development of new therapeutic strategies for diabetic patients with myocardial infarction.

Cinaciguat, a novel activator of soluble guanylate cyclase, protects against ischemia/reperfusion injury: role of hydrogen sulfide.

Cinaciguat (BAY 58-2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H(2)S). Adult New Zealand White rabbits were pretreated with 1 or 10 µg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 µg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 µg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 µg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H(2)S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H(2)S generation and a powerful protection against I/R injury in heart.

Preclinical model of type 1 diabetes and myocardial ischemia/reperfusion injury in conscious rabbits-demonstration of cardioprotection with rapamycin.

We developed a preclinical model of myocardial ischemia/reperfusion (I/R) injury in conscious diabetic rabbits to identify an early pharmacological intervention for patients with diabetes and acute myocardial infarction (AMI). Here, we describe a reproducible protocol for induction of diabetes with subsequent manifestation of myocardial I/R injury in conscious rabbits to mimic the real-life scenario observed in clinical settings. Further, we demonstrate the efficacy of rapamycin at the onset of reperfusion to limit the adverse effect of AMI. For complete details on the use and execution of this protocol, please refer to Samidurai et al. (2020).

Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities.

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin (CaM), resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.

Reversal of Endothelial Extracellular Vesicle-Induced Smooth Muscle Phenotype Transition by Hypercholesterolemia Stimulation: Role of NLRP3 Inflammasome Activation.

Recent studies reported that vascular endothelial cells (ECs) secrete NLR family pyrin domain-containing 3 (NLRP3) inflammasome products such as interleukin-1ß (IL-1ß) via extracellular vesicles (EVs) under various pathological conditions. EVs represent one of the critical mechanisms mediating the cell-to-cell communication between ECs and vascular smooth muscle cells (VSMCs). However, whether or not the inflammasome-dependent EVs directly participate in the regulation of VSMC function remains unknown. In the present study, we found that in cultured carotid ECs, atherogenic stimulation by oxysterol 7-ketocholesterol (7-Ket) induced NLRP3 inflammasome formation and activation, reduced lysosome-multivesicular bodies (MVBs) fusion, and increased secretion of EVs that contain inflammasome product IL-1ß. These EC-derived IL-1ß-containing EVs promoted synthetic phenotype transition of co-cultured VSMCs, whereas EVs from unstimulated ECs have the opposite effects. Moreover, acid ceramidase (AC) deficiency or lysosome inhibition further exaggerated the 7-Ket-induced release of IL-1ß-containing EVs in ECs. Using a Western diet (WD)-induced hypercholesterolemia mouse model, we found that endothelial-specific AC gene knockout mice (Asah1fl/fl/ECCre) exhibited augmented WD-induced EV secretion with IL-1ß and more significantly decreased the interaction of MVBs with lysosomes in the carotid arterial wall compared to their wild-type littermates (WT/WT). The endothelial AC deficiency in Asah1fl/fl/ECCre mice also resulted in enhanced VSMC phenotype transition and accelerated neointima formation. Together, these results suggest that NLRP3 inflammasome-dependent IL-1ß production during hypercholesterolemia promotes VSMC phenotype transition to synthetic status via EV machinery, which is controlled by lysosomal AC activity. Our findings provide novel mechanistic insights into understanding the pathogenic role of endothelial NLRP3 inflammasome in vascular injury through EV-mediated EC-to-VSMC regulation.

Endoplasmic reticulum stress-mediated mitochondrial dysfunction in aged hearts.

Aging impairs the mitochondrial electron transport chain (ETC), especially in interfibrillar mitochondria (IFM). Mitochondria are in close contact with the endoplasmic reticulum (ER). Induction of ER stress leads to ETC injury in adult heart mitochondria. We asked if ER stress contributes to the mitochondrial dysfunction during aging. Subsarcolemmal mitochondria (SSM) and IFM were isolated from 3, 18, and 24 mo. C57Bl/6 mouse hearts. ER stress progressively increased with age, especially in 24 mo. mice that manifest mitochondrial dysfunction. OXPHOS was decreased in 24 mo. IFM oxidizing complex I and complex IV substrates. Proteomic analysis showed that the content of multiple complex I subunits was decreased in IFM from 24 mo. hearts, but remained unchanged in in 18 mo. IFM without a decrease in OXPHOS. Feeding 24 mo. old mice with 4-phenylbutyrate (4-PBA) for two weeks attenuated the ER stress and improved mitochondrial function. These results indicate that ER stress contributes to the mitochondrial dysfunction in aged hearts. Attenuation of ER stress is a potential approach to improve mitochondrial function in aged hearts.

Differential Regulation of mTOR Complexes with miR-302a Attenuates Myocardial Reperfusion Injury in Diabetes.

Persistent activation of mTOR (mammalian target of rapamycin) in diabetes increases the vulnerability of the heart to ischemia/reperfusion (I/R) injury. We show here that infusion of rapamycin (mTOR inhibitor) at reperfusion following ischemia reduced myocardial infarct size and apoptosis with restoration of cardiac function in type 1 diabetic rabbits. Likewise, treatment with rapamycin protected hyperglycemic human-pluripotent-stem-cells-derived cardiomyocytes (HG-hiPSC-CMs) following simulated ischemia (SI) and reoxygenation (RO). Phosphorylation of S6 (mTORC1 marker) was increased, whereas AKT phosphorylation (mTORC2 marker) and microRNA-302a were reduced with concomitant increase of its target, PTEN, following I/R injury in diabetic heart and HG-hiPSC-CMs. Rapamycin inhibited mTORC1 and PTEN, but augmented mTORC2 with restoration of miRNA-302a under diabetic conditions. Inhibition of miRNA-302a blocked mTORC2 and abolished rapamycin-induced protection against SI/RO injury in HG-hiPSC-CMs. We conclude that rapamycin attenuates reperfusion injury in diabetic heart through inhibition of PTEN and mTORC1 with restoration of miR-302a-mTORC2 signaling.

STAT3-miR-17/20 signalling axis plays a critical role in attenuating myocardial infarction following rapamycin treatment in diabetic mice.

AIMS: Deregulation of mTOR (mammalian target of rapamycin) signalling occurs in diabetes, which exacerbates injury following myocardial infarction (MI). We therefore investigated the infarct-limiting effect of chronic treatment with rapamycin (RAPA, mTOR inhibitor) in diabetic mice following myocardial ischaemia/reperfusion (I/R) injury and delineated the potential protective mechanism. METHODS AND RESULTS: Adult male diabetic (db/db) or wild-type (WT) (C57) mice were treated with RAPA (0.25 mg/kg/day, intraperitoneal) or vehicle (5% DMSO) for 28 days. The hearts from treated mice were subjected to global I/R in Langendorff mode. Cardiomyocytes, isolated from treated mice, were subjected to simulated ischaemia/reoxygenation (SI/RO) to assess necrosis and apoptosis. Myocardial infarct size was increased in diabetic heart following I/R as compared to WT. Likewise, enhanced necrosis and apoptosis were observed in isolated cardiomyocytes of diabetic mice following SI/RO. Treatment with RAPA reduced infarct size as well as cardiomyocyte necrosis and apoptosis of diabetes and WT mice. RAPA increased STAT3 phosphorylation and miRNA-17/20a expression in diabetic hearts. In addition, RAPA restored AKT phosphorylation (target of mTORC2) but suppressed S6 phosphorylation (target of mTORC1) following I/R injury. RAPA-induced cardioprotection against I/R injury as well as the induction of miR-17/20a and AKT phosphorylation were abolished in cardiac-specific STAT3-deficient diabetic mice, without alteration of S6 phosphorylation. The infarct-limiting effect of RAPA was obliterated in cardiac-specific miRNA-17-92-deficient diabetic mice. The post-I/R restoration of phosphorylation of STAT3 and AKT with RAPA were also abolished in miRNA-17-92-deficient diabetic mice. Additionally, RAPA suppressed the pro-apoptotic prolyl hydroxylase (Egln3/PHD3), a target of miRNA-17/20a in diabetic hearts, which was abrogated in miRNA-17-92-deficient diabetic mice. CONCLUSION: Induction of STAT3-miRNA-17-92 signalling axis plays a critical role in attenuating MI in RAPA-treated diabetic mice. Our study indicates that chronic treatment with RAPA might be a promising pharmacological intervention for attenuating MI and improving prognosis in diabetic patients.

Remote Ischemic Pre-Conditioning Attenuates Adverse Cardiac Remodeling and Mortality Following Doxorubicin Administration in Mice.

OBJECTIVES: Because of its multifaceted cardioprotective effects, remote ischemic pre-conditioning (RIPC) was examined as a strategy to attenuate doxorubicin (DOX) cardiotoxicity. BACKGROUND: The use of DOX is limited by dose-dependent cardiotoxicity and heart failure. Oxidative stress, mitochondrial dysfunction, inflammation, and autophagy modulation have been proposed as mediators of DOX cardiotoxicity. METHODS: After baseline echocardiography, adult male CD1 mice were randomized to either sham or RIPC protocol (3 cycles of 5 min femoral artery occlusion followed by 5 min reperfusion) 1 h before receiving DOX (20 mg/kg, intraperitoneal). The mice were observed primarily for survival over 85 days (86 mice). An additional cohort of 50 mice was randomized to either sham or RIPC 1 h before DOX treatment and was followed for 25 days, at which time cardiac fibrosis, apoptosis, and mitochondrial oxidative phosphorylation were assessed, as well as the expression profiles of apoptosis and autophagy markers. RESULTS: Survival was significantly improved in the RIPC cohort compared with the sham cohort (p = 0.007). DOX-induced cardiac fibrosis and apoptosis were significantly attenuated with RIPC compared with sham (p < 0.05 and p < 0.001, respectively). Although no mitochondrial dysfunction was detected at 25 days, there was a significant increase in autophagy markers with DOX that was attenuated with RIPC. Moreover, DOX caused a 49% decline in cardiac BCL2/BAX expression, which was restored with RIPC (p < 0.05 vs. DOX). DOX also resulted in a 17% reduction in left ventricular mass at 25 days, which was prevented with RIPC (p < 0.01), despite the lack of significant changes in left ventricular ejection fraction. CONCLUSIONS: Our preclinical results suggested that RIPC before DOX administration might be a promising approach for attenuating DOX cardiotoxicity.

Phosphodiesterase 1 upregulation in pulmonary arterial hypertension: target for reverse-remodeling therapy.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a life-threatening disease, characterized by vascular smooth muscle cell hyperproliferation. The calcium/calmodulin-dependent phosphodiesterase 1 (PDE1) may play a major role in vascular smooth muscle cell proliferation. METHODS AND RESULTS: We investigated the expression of PDE1 in explanted lungs from idiopathic PAH patients and animal models of PAH and undertook therapeutic intervention studies in the animal models. Strong upregulation of PDE1C in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was noted on the mRNA level by laser-assisted vessel microdissection and on the protein level by immunohistochemistry. In chronically hypoxic mouse lungs and lungs from monocrotaline-injected rats, PDE1A upregulation was detected in the structurally remodeled arterial muscular layer. Long-term infusion of the PDE1 inhibitor 8-methoxymethyl 3-isobutyl-1-methylxanthine in hypoxic mice and monocrotaline-injected rats with fully established pulmonary hypertension reversed the pulmonary artery pressure elevation, structural remodeling of the lung vasculature (nonmuscularized versus partially muscularized versus fully muscularized small pulmonary arteries), and right heart hypertrophy. CONCLUSIONS: Strong upregulation of the PDE1 family in pulmonary artery smooth muscle cells is noted in human idiopathic PAH lungs and lungs from animal models of PAH. Inhibition of PDE1 reverses structural lung vascular remodeling and right heart hypertrophy in 2 animal models. The PDE1 family may thus offer a new target for therapeutic intervention in pulmonary hypertension.