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BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC(50) values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (I(Na) ) and hERG K(V) 11.1 currents (I(hERG) ) in a concentration-dependent manner; IC(50) values were 23.2 and 10.0 μM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 μM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.

The left atrial appendage (LAA) of the adult heart has been shown to contain cardiac and myeloid progenitor cells. The resident myeloid progenitor population expresses an array of pro-regenerative paracrine factors. Cardiac constructs have been shown to inhibit deleterious remodeling of the heart using physical support. Due to these aspects, LAA holds promise as a regenerative transplant. LAAs from adult mT/mG mice were transplanted to the recipient 129X1-SvJ mice simultaneously as myocardial infarction (MI) was performed. A decellularized LAA patch was implanted in the control group. Two weeks after MI, the LAA patch had integrated to the ventricular wall, and migrated cells were seen in the MI area. The cells had two main phenotypes: small F4/80+ cells and large troponin C+ cells. After follow-up at 8 weeks, the LAA patch remained viable, and the functional status of the heart improved. Cardiac echo demonstrated that, after 6 weeks, the mice in the LAA-patch-treated group showed an increasing and statistically significant improvement in cardiac performance when compared to the MI and MI + decellularized patch controls. Physical patch-support (LAA and decellularized LAA patch) had an equal effect on the inhibition of deleterious remodeling, but only the LAA patch inhibited the hypertrophic response. Our study demonstrates that the LAA transplantation has the potential for use as a treatment for myocardial infarction. This method can putatively combine cell therapy (regenerative effect) and physical support (inhibition of deleterious remodeling).

Background: Recent discoveries have indicated that, in the developing heart, sinus venosus and endocardium provide major sources of endothelium for coronary vessel growth that supports the expanding myocardium. Here we set out to study the origin of the coronary vessels that develop in response to vascular endothelial growth factor B (VEGF-B) in the heart and the effect of VEGF-B on recovery from myocardial infarction. Methods: We used mice and rats expressing a VEGF-B transgene, VEGF-B-gene–deleted mice and rats, apelin-CreERT, and natriuretic peptide receptor 3–CreERT recombinase-mediated genetic cell lineage tracing and viral vector–mediated VEGF-B gene transfer in adult mice. Left anterior descending coronary vessel ligation was performed, and 5-ethynyl-2’-deoxyuridine–mediated proliferating cell cycle labeling; flow cytometry; histological, immunohistochemical, and biochemical methods; single-cell RNA sequencing and subsequent bioinformatic analysis; microcomputed tomography; and fluorescent- and tracer-mediated vascular perfusion imaging analyses were used to study the development and function of the VEGF-B–induced vessels in the heart. Results: We show that cardiomyocyte overexpression of VEGF-B in mice and rats during development promotes the growth of novel vessels that originate directly from the cardiac ventricles and maintain connection with the coronary vessels in subendocardial myocardium. In adult mice, endothelial proliferation induced by VEGF-B gene transfer was located predominantly in the subendocardial coronary vessels. Furthermore, VEGF-B gene transduction before or concomitantly with ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function. Conclusions: The myocardial VEGF-B transgene promotes the formation of endocardium-derived coronary vessels during development, endothelial proliferation in subendocardial myocardium in adult mice, and structural and functional rescue of cardiac tissue after myocardial infarction. VEGF-B could provide a new therapeutic strategy for cardiac neovascularization after coronary occlusion to rescue the most vulnerable myocardial tissue.

Aging, obesity, hypertension, and physical inactivity are major risk factors for endothelial dysfunction and cardiovascular disease (CVD). We applied fluorescence-activated cell sorting (FACS), RNA sequencing, and bioinformatic methods to investigate the common effects of CVD risk factors in mouse cardiac endothelial cells (ECs). Aging, obesity, and pressure overload all upregulated pathways related to TGF-β signaling and mesenchymal gene expression, inflammation, vascular permeability, oxidative stress, collagen synthesis, and cellular senescence, whereas exercise training attenuated most of the same pathways. We identified collagen chaperoneCardiovascular diseases are the number one cause of death in the western world. Endothelial cells that line the blood vessels of the heart play a central role in the development of these diseases. In addition to helping transport blood, these cells support the normal running of the heart, and help it to grow and regenerate. Over time as the body ages and experiences stress, endothelial cells start to deteriorate. This can cause the cells to undergo senescence and stop dividing, and lay down scar-like tissue via a process called fibrosis. As a result, the blood vessels start to stiffen and become less susceptible to repair. Ageing, obesity, high blood pressure, and inactivity all increase the risk of developing cardiovascular diseases, whereas regular exercise has a protective effect. But it was unclear how these different factors affect endothelial cells. To investigate this, Hemanthakumar et al. compared the gene activity of different sets of mice: old vs young, obese vs lean, heart problems vs healthy, and fit vs sedentary. All these risk factors – age, weight, inactivity and heart defects – caused the mice’s endothelial cells to activate mechanisms that lead to stress, senescence and fibrosis. Whereas exercise training had the opposite effect, and turned off the same genes and pathways. All of the at-risk groups also had high levels of a gene called SerpinH1, which helps produce tissue fiber and collagen. Experiments increasing the levels of SerpinH1 in human endothelial cells grown in the laboratory recreated the effects seen in mice, and switched on markers of stress, senescence and fibrosis. According to the World Health Organization, cardiovascular disease now accounts for 10% of the disease burden worldwide. Revealing the affects it has on gene activity could help identify new targets for drug development, such as SerpinH1. Understanding the molecular effects of exercise on blood vessels could also aid in the design of treatments that mimic exercise. This could help people who are unable to follow training programs to reduce their risk of cardiovascular disease.

Background Ischemic heart disease remains the leading cause of mortality and morbidity worldwide despite improved possibilities in medical care. Alongside interventional therapies, such as coronary artery bypass grafting, adjuvant tissue-engineered and cell-based treatments can provide regenerative improvement. Unfortunately, most of these advanced approaches require multiple lengthy and costly preparation stages without delivering significant clinical benefits. Methods We evaluated the effect of epicardially delivered minute pieces of atrial appendage tissue material, defined as atrial appendage micrografts (AAMs), in a mouse myocardial infarction model. An extracellular matrix patch was used to cover and fix the AAMs onto the surface of the infarcted heart. Results The matrix-covered AAMs salvaged the heart from the infarction-induced loss of functional myocardium and attenuated scarring. Site-selective proteomics of injured ischemic and uninjured distal myocardium from AAMs-treated and -untreated tissue sections revealed increased expression of several cardiac regeneration-associated proteins (i.e., periostin, transglutaminases, and glutathione peroxidases) and activation of pathways responsible for angiogenesis and cardiogenesis in relation to AAMs therapy. Conclusions Epicardial delivery of AAMs encased in an extracellular matrix patch scaffold salvages functional cardiac tissue from ischemic injury and restricts fibrosis after myocardial infarction. Our results support the use of AAMs as tissue-based therapy adjuvants for salvaging the ischemic myocardium.

Summary: Chemical modifications of RNA provide an additional, epitranscriptomic, level of control over cellular functions. N-6-methylated adenosines (m6As) are found in several types of RNA, and their amounts are regulated by methyltransferases and demethylases. One of the most important enzymes catalyzing generation of m6A on mRNA is the trimer N-6-methyltransferase METTL3-14-WTAP complex. Its activity has been linked to such critical biological processes as cell differentiation, proliferation, and death.We used in silico-based discovery to identify small-molecule ligands that bind to METTL3-14-WTAP and determined experimentally their binding affinity and kinetics, as well as their effect on enzymatic function. We show that these ligands serve as activators of the METTL3-14-WTAP complex. : The methyltransferase complex METTL3-14-WTAP catalyzes generation of m6A on mRNA. Selberg et al. report the in silico discovery and experimental characterization of small-molecule compounds with exceptionally high binding efficiencies to METTL3-14-WTAP. Remarkably, these compounds act as enzyme activators and lead to increased m6A levels in RNA. Keywords: RNA methylation, N6-adenosine-methyltransferases, N-6-methyladenosine, ligand binding, computer-aided design, epitranscriptomics

Aging, obesity, hypertension and physical inactivity are major risk factors for endothelial dysfunction and cardiovascular disease (CVD). We applied fluorescence-activated cell sorting (FACS), RNA sequencing and bioinformatic methods to investigate the common effects of CVD risk factors on cardiac endothelial cells (ECs). Aging, obesity and pressure overload all upregulated pathways related to TGF-β signaling and mesenchymal gene expression, inflammation, vascular permeability, oxidative stress, collagen synthesis and cellular senescence, whereas exercise training downregulated most of the same pathways. We identified collagen chaperone SerpinH1/HSP47 to be significantly increased by aging and obesity and repressed by exercise training. Mechanistic studies demonstrated that SERPINH1/HSP47 in human ECs changed cell morphology and increased mesenchymal gene expression, while its silencing inhibited collagen deposition. Our data demonstrate that CVD risk factors significantly remodel the transcriptomic landscape of cardiac ECs to acquire senescence and mesenchymal features. SERPINH1/HSP47 was identified as a potential therapeutic target in ECs.

Significance Compromised vascular integrity is associated with capillary leakage in sepsis, but effective therapies stabilizing the vasculature are lacking. Here, we show that targeting β1-integrin in vivo with inhibitory antibodies or deletion of a single allele of endothelial β1-integrin inhibits lipopolysaccharide (LPS)-induced vascular leakage in murine endotoxemia. The inflammatory agents IL-1β, thrombin, and LPS induced changes in endothelial cell–extracellular matrix (ECM) adhesion via β1-integrin, angiopoietin-2, and the adapter protein tensin-1, leading to increased endothelial cell contractility and permeability. These results indicate that β1-integrin actively promotes vascular leakage and that targeting β1-integrin signaling could be a novel means of achieving vascular stabilization in pathological vascular leak., Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against β1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. β1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell–cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial β1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1β decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via β1- and α5-integrins and ANGPT2. Additionally, β1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5β1-integrin into tensin-1–positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, β1-integrin promotes endothelial barrier disruption during inflammation, and targeting β1-integrin signaling could serve as a novel means of blocking pathological vascular leak.

Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l(p.S78G) knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.

Ischemic heart disease remains the leading cause of mortality and morbidity worldwide despite improved possibilities in medical care. Alongside interventional therapies, such as coronary artery bypass grafting, adjuvant tissue-engineered and cell-based treatments can provide regenerative improvement. Unfortunately, most of these advanced approaches require multiple lengthy and costly preparation stages without delivering significant clinical benefits.We evaluated the effect of epicardially delivered minute pieces of atrial appendage tissue material, defined as atrial appendage micrografts (AAMs), in mouse myocardial infarction model. An extracellular matrix patch was used to cover and fix the AAMs onto the surface of the infarcted heart. The matrix-covered AAMs salvaged the heart from infarction-induced loss of functional myocardium and attenuated scarring. Site-selective proteomics of injured ischemic and uninjured distal myocardium from AAM-treated and untreated tissue sections revealed an increased expression of several cardiac regeneration-associated proteins (i.e. periostin, transglutaminases and glutathione peroxidases) as well as activation of pathways responsible for angio- and cardiogenesis in relation to AAMs therapy.Epicardial delivery of AAMs encased in an extracellular matrix patch scaffold salvages functional cardiac tissue from ischemic injury and restricts fibrosis after myocardial infarction. Our results support the use of AAMs as tissue-based therapy adjuvants for salvaging the ischemic myocardium.

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating rates (1, 2 Hz). Cumulating doses of E-4031 produced prolonged APDs, followed by EADs and drug-induced quiescence. These observations were corroborated by patch clamp and contractility measurements. Similar responses, although more modest were seen with the IKs potassium channel blocker JNJ-303. In conclusion, optogenetic measurements of AP waveforms combined with optical pacing compare well with the patch clamp gold standard. Combined with video motion contractile measurements, optogenetic imaging provides an appealing alternative for electrophysiological screening of human cardiomyocyte responses in pharmacological efficacy and safety testings. publishedVersion

Background and aim: We hypothesised that the use of 50% compared to 100% oxygen maintains cerebral oxygenation and ameliorates the disturbance of cardiac mitochondrial respiration during cardiopulmonary resuscitation (CPR). Methods: Ventricular fibrillation (VF) was induced electrically in anaesthetised healthy adult pigs and left untreated for seven minutes followed by randomisation to manual ventilation with 50% or 100% oxygen and mechanical chest compressions (LUCAS (R)). Defibrillation was performed at thirteen minutes and repeated if necessary every two minutes with 1 mg intravenous adrenaline. Cerebral oxygenation was measured with near-infrared spectroscopy (rSO(2), INVOS (TM) 5100C Cerebral Oximeter) and with a probe (NEUROVENT-PTO, RAUMEDIC) in the frontal brain cortex (PbO2). Heart biopsies were obtained 20 min after the return of spontaneous circulation (ROSC) with an analysis of mitochondrial respiration (OROBOROS Instruments Corp., Innsbruck, Austria), and compared to four control animals without VF and CPR. Brain rSO(2) and PbO2 were log transformed and analysed with a mixed linear model and mitochondrial respiration with an analysis of variance. Results: Of the twenty pigs, one had a breach of protocol and was excluded, leaving nine pigs in the 50% group and ten in the 100% group. Return of spontaneous circulation (ROSC) was achieved in six pigs in the 50% group and eight in the 100% group. The rSO(2) (p = 0.007) was lower with FiO(2) 50%, but the PbO2 was not (p = 0.93). After ROSC there were significant interactions between time and FiO(2) regarding both rSO(2) (p = 0.001) and PbO2 (p = 0.004). Compared to the controls, mitochondrial respiration was decreased, with adenosine diphosphate (ADP) levels of 57 (17) pmol s(-1) mg(-1) compared to 92 (23) pmol s(-1) mg(-1) (p = 0.008), but there was no difference between different oxygen fractions (p = 0.79). Conclusions: The use of 50% oxygen during CPR results in lower cerebral oximetry values compared to 100% oxygen but there is no difference in brain tissue oxygen. Cardiac arrest disturbs cardiac mitochondrial respiration, but it is not alleviated with the use of 50% compared to 100% oxygen (Ethical and hospital approvals ESAVI/1077/04.10.07/2016 and HUS/215/2016, 7 30.3.2016, Funding Helsinki University and others). (C) 2017 Elsevier B.V. All rights reserved.

Hypertension and persistent activation of the renin–angiotensin system (RAS) are predisposing factors for the development of acute kidney injury (AKI). Although bone-marrow-derived stromal cells (BMSCs) have shown therapeutic promise in treatment of AKI, the impact of pathological RAS on BMSC functionality has remained unresolved. RAS and its local components in the bone marrow are involved in several key steps of cell maturation processes. This may also render the BMSC population vulnerable to alterations even in the early phases of RAS pathology. We isolated transgenic BMSCs (TG-BMSCs) from young end-organ-disease-free rats with increased RAS activation [human angiotensinogen/renin double transgenic rats (dTGRs)] that eventually develop hypertension and die of end-organ damage and kidney failure at 8 weeks of age. Control cells (SD-BMSCs) were isolated from wild-type Sprague–Dawley rats. Cell phenotype, mitochondrial reactive oxygen species (ROS) production and respiration were assessed, and gene expression profiling was carried out using microarrays. Cells’ therapeutic efficacy was evaluated in a rat model of acute ischaemia/reperfusion-induced AKI. Serum urea and creatinine were measured at 24 h and 48 h. Acute tubular damage was scored and immunohistochemistry was used for evaluation for markers of inflammation [monocyte chemoattractant protein (MCP-1), ED-1], and kidney injury [kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL)]. TG-BMSCs showed distinct mitochondrial morphology, decreased cell respiration and increased production of ROS. Gene expression profiling revealed a pronounced pro-inflammatory phenotype. In contrast with the therapeutic effect of SD-BMSCs, administration of TG-BMSCs in the AKI model resulted in exacerbation of kidney injury and high mortality. Our results demonstrate that early persistent RAS activation can dramatically compromise therapeutic potential of BMSCs by causing a shift into a pro-inflammatory phenotype with mitochondrial dysfunction.

Local anesthetic toxicity is thought to be mediated partly by inhibition of cardiac mitochondrial function. Intravenous (i.v.) lipid emulsion may overcome this energy depletion, but doses larger than currently recommended may be needed for rescue effect. In this randomized study with anesthetized pigs, we compared the effect of a large dose, 4 mL/kg, of i.v. 20% Intralipid® ( n = 7) with Ringer’s acetate ( n = 6) on cardiovascular recovery after a cardiotoxic dose of bupivacaine. We also examined mitochondrial respiratory function in myocardial cell homogenates analyzed promptly after needle biopsies from the animals. Bupivacaine plasma concentrations were quantified from plasma samples. Arterial blood pressure recovered faster and systemic vascular resistance rose more rapidly after Intralipid than Ringer’s acetate administration ( p < 0.0001), but Intralipid did not increase cardiac index or left ventricular ejection fraction. The lipid-based mitochondrial respiration was stimulated by approximately 30% after Intralipid ( p < 0.05) but unaffected by Ringer’s acetate. The mean (standard deviation) area under the concentration–time curve (AUC) of total bupivacaine was greater after Intralipid (105.2 (13.6) mg·min/L) than after Ringer’s acetate (88.1 (7.1) mg·min/L) ( p = 0.019). After Intralipid, the AUC of the lipid-un-entrapped bupivacaine portion (97.0 (14.5) mg·min/L) was 8% lower than that of total bupivacaine ( p < 0.0001). To conclude, 4 mL/kg of Intralipid expedited cardiovascular recovery from bupivacaine cardiotoxicity mainly by increasing systemic vascular resistance. The increased myocardial mitochondrial respiration and bupivacaine entrapment after Intralipid did not improve cardiac function.

Doxorubicin is a widely used and effective chemotherapy drug. However, cardiac and skeletal muscle toxicity of doxorubicin limits its use. Inhibiting myostatin/activin signalling can prevent muscle atrophy, but its effects in chemotherapy-induced muscle wasting are unknown. In the present study we investigated the effects of doxorubicin administration alone or combined with activin receptor ligand pathway blockade by soluble activin receptor IIB (sACVR2B-Fc). Doxorubicin administration decreased body mass, muscle size and bone mineral density/content in mice. However, these effects were prevented by sACVR2B-Fc administration. Unlike in many other wasting situations, doxorubicin induced muscle atrophy without markedly increasing typical atrogenes or protein degradation pathways. Instead, doxorubicin decreased muscle protein synthesis which was completely restored by sACVR2B-Fc. Doxorubicin administration also resulted in impaired running performance without effects on skeletal muscle mitochondrial capacity/function or capillary density. Running performance and mitochondrial function were unaltered by sACVR2B-Fc administration. Tumour experiment using Lewis lung carcinoma cells demonstrated that sACVR2B-Fc decreased the cachectic effects of chemotherapy without affecting tumour growth. These results demonstrate that blocking ACVR2B signalling may be a promising strategy to counteract chemotherapy-induced muscle wasting without damage to skeletal muscle oxidative capacity or cancer treatment.

The β-keto amphetamine (cathinone, β-KA) designer drugs such as mephedrone (4-methylmethcathinone, 4-MMC) show a large degree of structural similarity to amphetamines like methamphetamine (METH). However, little is currently known about whether these substances also share the potential neurotoxic properties of their non-keto amphetamine counterparts, or what mechanisms could be involved. Here, we evaluate the cytotoxicity of β-KAs in SH-SY5Y cells using lactate dehydrogenase (LDH) assays, assess the redox potential of a range of β-KAs and non-keto amphetamines using the sensitive redox indicator 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and explore the effect of 4-MMC on the formation of protein adducts using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) and on the mitochondrial respiratory chain using high-resolution respirometry. We show that treatment with β-KAs increases LDH release. Further, we demonstrate that even under physiological pH, β-KAs are effective and selective—as compared with their non-keto analogues—reductants in the presence of electron acceptors. Increased pH (range 7.6–8.0) greatly enhanced the reactivity up to sixfold. We found no evidence of protein adduct formation, suggesting the reactivity is due to direct electron transfer by the β-KAs. Finally, we show that 4-MMC and METH produce dissimilar effects on the respiratory chain. Our results indicate that β-KAs such as 4-MMC possess cytotoxic properties in vitro. Furthermore, in the presence of an electron-accepting redox partner, the ketone moiety of β-KAs is vital for pH-dependent redox reactivity. Further work is needed to establish the importance of β-KA redox properties and its potential toxicological importance in vivo.

The increased use of cathinone-type designer drugs, known as legal highs, has led to concerns about their potential neurotoxicity due to their similarity to methamphetamine (METH). Therefore, closer investigations of their toxic effects are needed. We investigated the effects of the cathinones 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxymethcathinone (MDMC) and the amphetamine METH on cytotoxicity and mitochondrial respiration in SH-SY5Y neuroblastoma cells. We also investigated the contribution of reactive species, dopamine, Bcl-2 and tumor necrosis factor α (TNFα) on toxicity. Finally, we investigated the effect of cathinone breakdown products using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry and studied their involvement in toxicity. We observed dose-dependent increases in cytotoxicity and decreases in mitochondrial respiration following treatment with all cathinones and amphetamines. Glutathione depletion increases amphetamine, but not cathinone toxicity. Bcl-2 and TNFα pathways are involved in toxicity but dopamine levels are not. We also show that cathinones, but not amphetamines, spontaneously produce reactive species and cytotoxic methylbenzamide breakdown products when in aqueous solution. These results provide an important first insight into the mechanisms of cathinone cytotoxicity and pave the way for further studies on cathinone toxicity in vivo.

Congestive heart failure is one of the leading causes of disability in long-term survivors of cancer. The anthracycline antibiotic doxorubicin (DOX) is used to treat a variety of cancers, but its utility is limited by its cumulative cardiotoxicity. As advances in cancer treatment have decreased cancer mortality, DOX-induced cardiomyopathy has become an increasing problem. However, the current means to alleviate the cardiotoxicity of DOX are limited. We considered that vascular endothelial growth factor-B (VEGF-B), which promotes coronary arteriogenesis, physiological cardiac hypertrophy, and ischemia resistance, could be an interesting candidate for prevention of DOX-induced cardiotoxicity and congestive heart failure. To study this, we administered an adeno-associated viral vector expressing VEGF-B or control vector to normal and tumor-bearing mice 1 wk before DOX treatment, using doses mimicking the concentrations used in the clinics. VEGF-B treatment completely inhibited the DOX-induced cardiac atrophy and whole-body wasting. VEGF-B also prevented capillary rarefaction in the heart and improved endothelial function in DOX-treated mice. VEGF-B also protected cultured endothelial cells from apoptosis and restored their tube formation. VEGF-B increased left ventricular volume without compromising cardiac function, reduced the expression of genes associated with pathological remodeling, and improved cardiac mitochondrial respiration. Importantly, VEGF-B did not affect serum or tissue concentrations of DOX or augment tumor growth. By inhibiting DOX-induced endothelial damage, VEGF-B could provide a novel therapeutic possibility for the prevention of chemotherapy-associated cardiotoxicity in cancer patients.

e00045 Kidney ischemia-reperfusion (I/R) injury is a common cause of acute kidney injury. We tested whether dexmedetomidine (Dex), an alpha2 adrenoceptor (α2-AR) agonist, protects against kidney I/R injury. Sprague–Dawley rats were divided into four groups: (1) Sham-operated group; (2) I/R group (40 min ischemia followed by 24 h reperfusion); (3) I/R group + Dex (1 μg/kg i.v. 60 min before the surgery), (4) I/R group + Dex (10 μg/kg). The effects of Dex postconditiong (Dex 1 or 10 μg/kg i.v. after reperfusion) as well as the effects of peripheral α2-AR agonism with fadolmidine were also examined. Hemodynamic effects were monitored, renal function measured, and acute tubular damage along with monocyte/macrophage infiltration scored. Kidney protein kinase B, toll like receptor 4, light chain 3B, p38 mitogen-activated protein kinase (p38 MAPK), sirtuin 1, adenosine monophosphate kinase (AMPK), and endothelial nitric oxide synthase (eNOS) expressions were measured, and kidney transciptome profiles analyzed. Dex preconditioning, but not postconditioning, attenuated I/R injury-induced renal dysfunction, acute tubular necrosis and inflammatory response. Neither pre- nor postconditioning with fadolmidine protected kidneys. Dex decreased blood pressure more than fadolmidine, ameliorated I/R-induced impairment of autophagy and increased renal p38 and eNOS expressions. Dex downregulated 245 and upregulated 61 genes representing 17 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, in particular, integrin pathway and CD44. Ingenuity analysis revealed inhibition of Rac and nuclear factor (erythroid-derived 2)-like 2 pathways, whereas aryl hydrocarbon receptor (AHR) pathway was activated. Dex preconditioning ameliorates kidney I/R injury and inflammatory response, at least in part, through p38-CD44-pathway and possibly also through ischemic preconditioning.

USF1 (upstream stimulatory factor 1) is a transcription factor associated with familial combined hyperlipidemia and coronary artery disease in humans. However, whether USF1 is beneficial or detrimental to cardiometabolic health has not been addressed. By inactivating USF1 in mice, we demonstrate protection against diet-induced dyslipidemia, obesity, insulin resistance, hepatic steatosis, and atherosclerosis. The favorable plasma lipid profile, including increased high-density lipoprotein cholesterol and decreased triglycerides, was coupled with increased energy expenditure due to activation of brown adipose tissue (BAT). Usf1 inactivation directs triglycerides from the circulation to BAT for combustion via a lipoprotein lipase-dependent mechanism, thus enhancing plasma triglyceride clearance. Mice lacking Usf1 displayed increased BAT-facilitated, diet-induced thermogenesis with up-regulation of mitochondrial respiratory chain complexes, as well as increased BAT activity even at thermoneutrality and after BAT sympathectomy. A direct effect of USF1 on BAT activation was demonstrated by an amplified adrenergic response in brown adipocytes after Usf1 silencing, and by augmented norepinephrine-induced thermogenesis in mice lacking Usf1. In humans, individuals carrying SNP (single-nucleotide polymorphism) alleles that reduced USF1 mRNA expression also displayed a beneficial cardiometabolic profile, featuring improved insulin sensitivity, a favorable lipid profile, and reduced atherosclerosis. Our findings identify a new molecular link between lipid metabolism and energy expenditure, and point to the potential of USF1 as a therapeutic target for cardiometabolic disease.

BACKGROUND AND PURPOSE Renal ischaemia/reperfusion (RI/R) injury is a major cause of acute kidney injury (AKI) and an important determinant of long-term kidney dysfunction. AMP-kinase and histone deacetylase sirtuin 1 (SIRT1) regulate cellular metabolism and are activated during hypoxia. We investigated whether AMP-kinase activator AICAR (5-amino-4-imidazolecarboxamide riboside-1-β-D-ribofuranoside) ameliorates RI/R injury and whether SIRT1 is involved in the pathogenesis. EXPERIMENTAL APPROACH Eight-week-old Sprague Dawley rats were divided into five groups: (i) sham-operated group; (ii) I/R group (40 min bilateral ischaemia followed by 24 h of reperfusion; (iii) I/R group + AICAR 50 mg·kg(-1) i.v. given 60 min before operation; (iv). I/R group + AICAR 160 mg·kg(-1) i.v; (v) I/R group + AICAR 500 mg·kg(-1) i.v. Serum creatinine and urea levels were measured. Acute tubular necrosis (ATN), monocyte/macrophage infiltration and nitrotyrosine expression were scored. Kidney AMP-activated protein kinase (AMPK) and SIRT1 expressions were measured. KEY RESULTS Highest dose of AICAR decreased serum creatinine and urea levels, attenuated I/R injury-induced nitrosative stress and monocyte/macrophage infiltration, and ameliorated the development of ATN. Kidney I/R injury was associated with decreased AMPK phosphorylation and a fivefold increase in kidney SIRT1 expression. AICAR increased pAMPK/AMPK ratio and prevented the I/R-induced increase in renal SIRT1 expression. CONCLUSIONS AND IMPLICATIONS AICAR protects against the development of ATN after kidney I/R injury. Activators of kidney AMP kinase may thus represent a novel therapeutic approach to patients susceptible to AKI and to those undergoing kidney transplantation. The present study also suggests a role for SIRT1 in the pathogenesis of RI/R injury.

Microfiltered native whey protein (MFNW) exerts superior in vitro functional properties compared to traditional cheese whey protein. We here investigated the metabolic effects of a novel MFNW, produced by economic polymeric membranes and rich in α-lactalbumin (α-lactalbumin 24% and β-lactoglobulin 65% of the protein), in a mice model of diet-induced obesity during weight gain and weight loss. Furthermore, the effects of MFNW were compared with those of α-lactalbumin. Replacement of casein in the diet (18% of energy) by MFNW prevented weight gain and body fat accumulation without influencing food intake, body temperature or glucose homeostasis. However, the anti-obesity effect of MFNW was inferior compared to α-lactalbumin (approximately 1/3). During weight loss (70% of ad libitum energy intake) the beneficial effects of both MFNW and α-lactalbumin were attenuated. Our findings indicate that MFNW protects against diet-induced obesity, and suggest that the beneficial effects of MFNW are due, to a great extent, to its α-lactalbumin content.

Lactoferrin (LF) is an iron-binding glycoprotein exerting several physiological functions such as antimicrobial activity, modulatory effects on lipid metabolism and anti-inflammatory properties. We examined whether LF prevents diet-induced obesity and development of fatty liver formation in C57Bl/6J mice. Lactoferrin (15% of dietary casein replaced by LF) enhanced weight loss and decreased body fat content during energy restriction compared to casein without influencing energy intake. During the ad libitum feeding after weight loss, LF suppressed weight regain and protected against the development of fatty liver formation compared to casein without influencing energy intake or fecal fat excretion. During the weight regain phase LF also improved glucose tolerance, decreased skeletal muscle leptin mRNA expression, and ameliorated adipose tissue inflammation. In conclusion , LF supplementation enhances the outcome of weight loss and subsequent weight regain, ameliorates fatty liver formation, and exerts beneficial effects on glucose tolerance and adipocyte tissue inflammation without interfering energy intake.

The calcium sensitizer levosimendan has shown beneficial effects on cardiac remodelling in spontaneously diabetic Goto-Kakizaki (GK) rats 12 weeks after experimental myocardial infarction (MI). However, the short-term effects and the cellular mechanisms remain partially unresolved. The aim was to study the effects of oral levosimendan treatment on the myocardial gene expression profile in diabetic GK rats 4 weeks after MI/sham operation. MI was induced to diabetic GK rats. Twenty-four hours after surgery, rats were randomized into four groups: MI, MI +levosimendan (1 mg/kg/day), sham-operated and sham-operated +levosimendan. Cardiac function and histology were examined 1, 4 and 12 weeks after MI. The effects of levosimendan on cardiac gene expression profile were investigated by microarray analysis. Levosimendan ameliorated post-infarct heart failure and cardiac remodelling. Levosimendan altered the expression of 264 of MI and sham rats, respectively; these changes were associated with alterations in two Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Levosimendan up-regulated 3 genes in the renin-angiotensin system pathway [angiotensin receptor 1 (Agtr1), chymase 1 (Cma1) and thimet oligopeptidase 1 (Thop1)] and down-regulated 3 genes in the glycerolipid metabolism pathway [diacylglycerol kinase gamma (Dgkg), carboxyl ester lipase (Cel) and Diacylglycerol kinase iota]. Levosimendan induced opposite effects on the gene expression of pleckstrin homology (PH) domain containing family f (Plekhf1), carboxymethylenebutenolidase homologue (Cmbl) (up-regulation) and hydroxyprostaglandin dehydrogenase 15 (Hpgd) (down-regulation) as compared with MI. MI versus sham affected 420 genes and was associated with alterations in 12 KEGG pathways. The beneficial effects of levosimendan on cardiac hypertrophy in sham-operated GK rats was associated with altered expression in 522 genes and associated with three KEGG pathways including purine metabolism, cell cycle pathway and pathways in cancer. Levosimendan protects against post-infarct heart failure and cardiac remodelling. Analysis of the cardiac transcriptome revealed several genes that are regulated by levosimendan. These genes may represent novel drug targets for heart failure and diabetic cardiomyopathy.

We investigated the potential of a novel whey protein isolate (WPI) rich in lactoperoxidase, lactoferrin, growth factors and immunoglobulins, in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. Compared with casein, WPI dose-dependently enhanced weight loss and decreased body fat content during energy restriction. WPI decreased hepatic S6 ribosomal protein phosphorylation and increased SIRT3 expression. WPI did not influence energy intake, apparent fat digestibility, or adipose tissue inflammation. Compared with casein, WPI dose-dependently prevented weight regain and protected against the development of fatty liver formation during ad libitum feeding after weight loss. WPI also modestly improved glucose tolerance. It was concluded that compared with casein, a diet based on WPI as protein source significantly improved outcome of weight loss and subsequent weight regain in high-fat-fed C57Bl/6J mice. The effects are mediated, at least partly, via inhibition of mTOR nutrient sensing pathway and activation of SIRT3 in the liver.

The effects of milk-derived whey protein α-lactalbumin (α-lac) and amino acid mixture with equal amino acid profile (AA) on weight loss and weight regain in a model of diet-induced obesity were compared. Obesity was induced in C57BL/6J mice with a high-fat diet. Energy restriction was performed for 50 days on casein (calcium 0.8%) and two different high-calcium (1.8%) whey protein-based (α-lac + Ca and AA + Ca) diets, after which the animals were fed with the same diets ad libitum for additional 50 days. The mice on α-lac and AA lost significantly more weight and body fat than mice on casein diet (P

Dehydroepiandrosterone (DHEA) fatty acyl esters once incorporated in high density lipoprotein (HDL) induce a stronger vasodilatory response in rat mesenteric arteries ex vivo compared to native HDL. We studied the role of HDL receptor, scavenger receptor class B, type 1 (SR-B1), as well as estrogen and androgen receptors in the vasodilatory response of HDL-associated DHEA fatty acyl esters. Using cultured human vascular endothelial cells (HUVEC), we investigated the possible internalization and cellular response of HDL-associated DHEA esters. We prepared DHEA ester-enriched HDL by incubating human plasma in the presence of DHEA. After isolation and purification, HDL was added in cumulative doses to arterial rings precontracted with noradrenaline. Inhibition of the function of SR-B1 almost completely abolished maximal vasorelaxation by DHEA-enriched HDL while estrogen or androgen receptor blockage had no significant effect. When HUVECs were incubated in the presence of [ 3 H]DHEA ester-enriched HDL, the amount of intracellular [ 3 H]-radioactivity increased steadily during 24 h. Blocking of SR-B1 reduced this uptake by a mean of 30%. The proportion of unesterified [ 3 H]DHEA, as analyzed by thin-layer chromatography, increased intracellularly and in the cell culture media after several hours of incubation of the cells in the presence of [ 3 H]DHEA ester-enriched HDL. This indicated slow hydrolysis of DHEA fatty acyl esters and subsequent excretion of unesterified DHEA by the cells. In conclusion, DHEA-enriched HDL induced vasorelaxation via the SR-B1-facilitated pathway. However, this vasodilation is not likely to be attributed to rapid hydrolysis of HDL-associated DHEA esters by the vascular endothelium.

Background/Aims: The potential of whey protein and calcium to modify skeletal muscle gene expression during energy restriction (ER) was investigated in a model of diet-induced obesity. Methods: Obese C57BL/6J mice received casein (calcium 0.4%) and two different high-calcium (1.8%) whey protein-based [whey protein isolate (WPI) + Ca and α-lactalbumin + Ca] diets for ER. Results: Compared to casein, WPI and α-lactalbumin-based diets altered 208 and 287 genes, respectively, of which 186 genes were common to WPI and α-lactalbumin diets. These genes represented 31 KEGG pathways. The Wnt signaling was the most enriched pathway among the 101 genes regulated by α-lactalbumin only, whereas the 22 genes regulated by WPI only were not associated with KEGG pathways. Unlike casein, WPI and α-lactalbumin diets decreased Aldh1a7, Fasn, leptin, Nr4a3 and Scd1 mRNA expression, indicating dietary protein source-dependent alterations in muscle lipid and fatty acid metabolism. Muscle weight or lean body mass maintenance did not differ between groups although modest changes in hypertrophy/atrophy signaling were found. Conclusion: The skeletal muscle gene expression profile is modified by the dietary protein source and calcium during ER which may explain, at least in part, the greater anti-obesity effect of whey proteins and calcium compared to casein.

Background— Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is incompletely understood. Methods and Results— Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation. As previously reported for the VEGF-B 167 isoform, transgenic mice and rats expressing both isoforms of VEGF-B in the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1 tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant capillary angiogenesis, increased permeability, or inflammatory cell recruitment. Conclusions— VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the VEGF-B–induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B–induced increased arterial supply to this area could have therapeutic potential in ischemic heart disease.

Calcium-sensitizing agents improve cardiac function in acute heart failure; however, their long-term effects on cardiovascular mortality are unknown. We tested the hypothesis that levosimendan, an inodilator that acts through calcium sensitization, opening of ATP-dependent potassium channels and phosphodiesterase III inhibition, improves cardiac function and survival in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs), a model of angiotensin II (Ang II)-induced hypertensive heart failure. Levosimendan (1 mg kg(-1)) was administered orally to 4-week-old dTGRs and normotensive Sprague-Dawley rats for 4 weeks. Untreated dTGRs developed severe hypertension, cardiac hypertrophy, heart failure with impaired diastolic relaxation, and exhibited a high mortality rate at the age of 8 weeks. Levosimendan did not decrease blood pressure and did not prevent cardiac hypertrophy. However, levosimendan improved systolic function, decreased cardiac atrial natriuretic peptide mRNA expression, ameliorated Ang II-induced cardiac damage and decreased mortality. Levosimendan did not correct Ang II-induced diastolic dysfunction and did not influence heart rate. In a separate survival study, levosimendan increased dTGR survival by 58% and median survival time by 27% (P=0.004). Our findings suggest that levosimendan ameliorates Ang II-induced hypertensive heart failure and reduces mortality. The results also support the notion that the effects of levosimendan in dTGRs are mediated by blood pressure-independent mechanisms and include improved systolic function and amelioration of Ang II-induced coronary and cardiomyocyte damage.

Cardiac hypertrophy is classically considered as an adaptive and compensatory response enabling cardiomyocytes to increase their work output and thus cardiac function. Biomechanical stress and neurohumoral activation are the most important triggers of pathological hypertrophy and the transition of cardiac hypertrophy to heart failure. Several novel regulators and putative drug targets of cardiac hypertrophy have been found by using gene-modified and acquired models of cardiac hypertrophy. Recent studies have also revealed distinct patterns of cardiac substrate utilization in cardiac hypertrophy and heart failure. The use of novel systems biology techniques such as metabolomics may therefore in future provide insights into the metabolic processes and cardiovascular biology related to cardiac hypertrophy and also extend the ability to discover circulating biomarkers for cardiovascular diseases. The present review discusses current knowledge on molecular mechanisms of cardiac hypertrophy, with special emphasis on novel regulators and putative drug targets of cardiac hypertrophy such as the tissue renin-angiotensin-aldosterone system, calcineurin/nuclear factor of activated T cells pathway, phosphatidylinositol 3-kinase/growth promoting protein kinase B, mammalian target of rapamycin, histone deacetylases, AMPkinases, microRNAs and angiogenetic factors.

Increased capillary leakage is a potentially life-threatening condition associated with sepsis and cytokine release syndrome (CRS) that develops as a side effect of novel chimeric antigen receptor (CAR) T-cell cancer immunotherapies. CRS and sepsis are characterized by immune activation, resulting in elevated circulating levels of inflammatory cytokines including IL-6 and IL-1β, and thrombin that promote vascular leakage and the development of hypovolemic shock and multiorgan failure. However, molecular mechanisms behind capillary leakage remain unknown, and importantly, targeted approaches for the management of capillary leakage are limited. Here, we found that β1-integrin acts as a critical universal mediator of endothelial permeability induced by various inflammatory agents, including LPS, IL-6, IL-1β and thrombin. In a mouse model of LPS-induced systemic inflammation, resulting in increased inflammatory cytokine production and vascular leakage, function blocking antibodies against β1-integrin prevented vascular leakage and improved integrity of vascular endothelium, leading to protection from LPS-induced cardiac failure. β1-integrin blocking antibodies were effective in reducing vascular leakage even when administered after the onset of systemic inflammation and increase in the serum levels of IL-6, IL-1β and TNF-α. By using an in vitro model, we found that inflammatory agents generated mechanical stress, in a β1-integrin dependent manner, leading to endothelial cell contractility and permeability. Conclusively, our data indicates that β1-integrin promotes vascular leakage in inflammation, and that targeting this mechanism may have therapeutic utility for vascular stabilization in CRS and capillary leak syndromes associated with immune activation and systemic inflammation. Citation Format: Elina A. Kiss, Laura Hakanpää, Guillaume Jacquemet, Ilkka Miinalainen, Martina Lerche, Camilo Guzman, Eero Mervaala, Lauri Eklund, Johanna Ivaska, Pipsa Saharinen. b1-Integrin promotes inflammatory cytokine-induced vascular leakage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5067.

Obesity is frequently associated with endothelial dysfunction. We hypothesized that high-fat feeding dysregulates the balance between endothelial derived nitric oxide and superoxide formation. Furthermore, we examined whether caloric restriction could reverse the detrimental vascular effects related to obesity. Male C57Bl/6 mice were fed with normal-fat diet (fat 17%) or high-fat diet (fat 60%) for 150 days. After establishment of obesity at day 100, a subgroup of obese mice were put on caloric restriction (CR) (70% of ad libitum energy intake) for an additional 50 days. At day 100, aortic rings from obese mice receiving high-fat diet showed impaired endothelium-dependent vasodilation in response to acetylcholine (ACh). Caloric restriction reversed high-fat diet-induced endothelial dysfunction. At day 150, impaired vasodilatory responses to ACh in obese mice without caloric restriction were markedly improved by preincubation with the tetrahydrobiopterin (BH(4)) precursor sepiapterin and L-arginine, a substrate for endothelial nitric oxide synthase (eNOS). Additionally, inhibition of vascular arginase by L-norvaline partially, and superoxide scavenging by Tiron completely, restored endothelial cell function. Obese mice showed increased vascular superoxide production, which was diminished by endothelial denudation, pretreated of the vascular rings with apocynin (an inhibitor of reduced nicotinamide adenine dinucleotide phosphate [NADPH] oxidase), oxypurinol (an inhibitor of xanthine oxidase), N(G)-nitro-L-arginine methyl ester (LNAME; an inhibitor of eNOS), or by adding the BH(4) precursor sepiapterin. Caloric restriction markedly attenuated vascular superoxide production. In obese mice on CR, endothelial denudation increased superoxide formation whereas vascular superoxide production was unaffected by L-NAME. Western blot analysis revealed decreased phosphorylated eNOS (Ser1177)-to-total eNOS expression ratio in obese mice as compared to lean controls, whereas the phospho-eNOS/NOS ratio in obese mice on CR did not differ from the lean controls. In conclusion, the present study suggests that caloric restriction reverses obesity induced endothelial dysfunction and vascular oxidative stress, and underscores the importance of uncoupled eNOS in the pathogenesis.

Background and purpose: Levosimendan is a novel, short half-life calcium sensitizer used as pharmacological inotropic support in acute decompensated heart failure. After oral administration, levosimendan is metabolized to OR-1855, which, in rats, is further metabolized into OR-1896. OR-1896 is a long-lasting metabolite of levosimendan sharing the pharmacological properties of the parent compound. Experimental approach: Effects of oral OR-1896 treatment on post-infarct heart failure and cardiac remodelling were assessed in diabetic Goto–Kakizaki (GK) rats, an animal model of type II diabetes. Myocardial infarction (MI) was produced to GK rats by coronary ligation. Twenty-four hours after MI or sham operation, the rats were randomized into four groups: (i) MI; (ii) MI + OR-1896 treatment; (iii) sham; and (iv) sham + OR-1896. Cardiac function and markers of cardiac remodelling were assessed 1, 4 and 12 weeks after MI. Key results: OR-1896 increased ejection fraction and fractional shortening in GK rats with MI. OR-1896 ameliorated post-infarct cardiac hypertrophy, and prevented the MI-induced increase in cardiac mRNA for atrial natriuretic peptide, monocyte chemoattractant protein-1 and connective tissue growth factor, markers of pressure/volume overload, inflammation and fibrosis respectively. OR-1896 also suppressed mRNA for senescence-associated p16INK4A and p19ARF. The beneficial effects of OR-1896 were more prominent at week 12 than at week 4. OR-1896 did not influence systolic blood pressure, blood glucose level, myocardial infarct size or cardiovascular mortality. Conclusions and implications: Oral treatment with calcium sensitizer OR-1896 protects against post-infarct heart failure and cardiac remodelling in experimental model of type II diabetes.

Angiotensin II (Ang II) induces mitochondrial dysfunction. We tested whether Ang II alters the “metabolomic” profile. We harvested hearts from 8-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGRs) and controls (Sprague-Dawley), all with or without Ang II type 1 receptor (valsartan) blockade. We used gas chromatography coupled with time-of-flight mass spectrometry to detect 247 intermediary metabolites. We used a partial least-squares discriminate analysis and identified 112 metabolites that differed significantly after corrections (false discovery rate q P

There is compelling evidence to indicate an important role for increased local renin-angiotensin system activity in the pathogenesis of cardiac hypertrophy and heart failure. Resveratrol is a natural polyphenol that activates SIRT1, a novel cardioprotective and longevity factor having NAD(+)-dependent histone deacetylase activity. We tested the hypothesis whether resveratrol could prevent from angiotensin II (Ang II)-induced cardiovascular damage. Four-week-old double transgenic rats harboring human renin and human angiotensinogen genes (dTGR) were treated for 4 weeks either with SIRT1 activator resveratrol or SIRT1 inhibitor nicotinamide. Untreated dTGR and their normotensive Sprague-Dawley control rats (SD) received vehicle. Untreated dTGR developed severe hypertension as well as cardiac hypertrophy, and showed pronounced cardiovascular mortality compared with normotensive SD rats. Resveratrol slightly but significantly decreased blood pressure, ameliorated cardiac hypertrophy and prevented completely Ang II-induced mortality, whereas nicotinamide increased blood pressure without significantly influencing cardiac hypertrophy or survival. Resveratrol decreased cardiac ANP mRNA expression and induced cardiac mRNA expressions of mitochondrial biogenesis markers peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha), mitochondrial transcription factor (Tfam), nuclear respiratory factor 1 (NRF-1) and cytochrome c oxidase subunit 4 (cox4). Resveratrol dose-dependently increased SIRT1 activity in vitro. Our findings suggest that the beneficial effects of SIRT1 activator resveratrol on Ang II-induced cardiac remodeling are mediated by blood pressure-dependent pathways and are linked to increased mitochondrial biogenesis.

Background Diabetes increases the risk for fatal myocardial infarction and development of heart failure. Levosimendan, an inodilator acting both via calcium sensitization and opening of ATP-dependent potassium channels, is used intravenously for acute decompensated heart failure. The long-term effects of oral levosimendan on postinfarct heart failure are largely unknown. Objective To examine whether oral treatment with levosimendan could improve cardiac functions and prevent cardiac remodeling after myocardial infarction in a rodent model of type 2 diabetes, the Goto-Kakizaki rat. Methods Myocardial infarction (MI) was induced to diabetic Goto-Kakizaki and nondiabetic Wistar rats by coronary ligation. Twenty-four hours after surgery, Goto-Kakizaki and Wistar rats were randomized into four groups: MI group without treatment, MI group with levosimendan for 12 weeks (1 mg/kg per day), sham-operated group, sham-operated group with levosimendan. Blood pressure, cardiac functions as wells as markers of cardiac remodeling were determined. Results In Goto-Kakizaki rats, MI induced systolic heart failure, pronounced cardiac hypertrophy in the remote area, and sustained cardiomyocyte apoptosis. Postinfarct cardiac remodeling was associated with increased atrial natriuretic peptide, interleukin-6 and connective tissue growth factor mRNA expressions, as well as three-fold increased cardiomyocyte senescence, measured as cardiac p16 INK4a mRNA expression. Levosimendan improved cardiac function and prevented postinfarct cardiomyocyte hypertrophy, cardiomyocyte apoptosis, and cellular senescence. Levosimendan also ameliorated MI-induced atrial natriuretic peptide, IL-6, and connective tissue growth factor overexpression as well as MI-induced disturbances in calcium-handling proteins (SERCA2, Na + -Ca 2+ exchanger) without changes in diabetic status or systemic blood pressure. In nondiabetic Wistar rats, MI induced systolic heart failure; however, the postinfarct cardiac remodeling was associated with less pronounced cardiac hypertrophy, cardiomyocyte apoptosis, inflammatory reaction, and induction of cellular senescence. Levosimendan only partially prevented postinfarct heart failure and cardiac remodeling in Wistar rats. Conclusion Our findings suggest a therapeutic role for oral levosimendan in prevention of postinfarct heart failure and cardiac remodeling in type 2 diabetes and underscore the importance of sustained cardiomyocyte apoptosis and induction of cellular senescence in the pathogenesis.