Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity.

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; IC50 values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (INa ) and hERG KV 11.1 currents (IhERG ) in a concentration-dependent manner; IC50 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.

Characterizing the Key Metabolic Pathways of the Neonatal Mouse Heart Using a Quantitative Combinatorial Omics Approach.

The heart of a newborn mouse has an exceptional capacity to regenerate from myocardial injury that is lost within the first week of its life. In order to elucidate the molecular mechanisms taking place in the mouse heart during this critical period we applied an untargeted combinatory multiomics approach using large-scale mass spectrometry-based quantitative proteomics, metabolomics and mRNA sequencing on hearts from 1-day-old and 7-day-old mice. As a result, we quantified 1.937 proteins (366 differentially expressed), 612 metabolites (263 differentially regulated) and revealed 2.586 differentially expressed gene loci (2.175 annotated genes). The analyses pinpointed the fructose-induced glycolysis-pathway to be markedly active in 1-day-old neonatal mice. Integrated analysis of the data convincingly demonstrated cardiac metabolic reprogramming from glycolysis to oxidative phosphorylation in 7-days old mice, with increases of key enzymes and metabolites in fatty acid transport (acylcarnitines) and ß-oxidation. An upsurge in the formation of reactive oxygen species and an increase in oxidative stress markers, e.g., lipid peroxidation, altered sphingolipid and plasmalogen metabolism were also evident in 7-days mice. In vitro maintenance of physiological fetal hypoxic conditions retained the proliferative capacity of cardiomyocytes isolated from newborn mice hearts. In summary, we provide here a holistic, multiomics view toward early postnatal changes associated with loss of a tissue regenerative capacity in the neonatal mouse heart. These results may provide insight into mechanisms of human cardiac diseases associated with tissue regenerative incapacity at the molecular level, and offer a prospect to discovery of novel therapeutic targets.

Exacerbation of acute kidney injury by bone marrow stromal cells from rats with persistent renin-angiotensin system activation.

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.

Dexmedetomidine preconditioning ameliorates kidney ischemia-reperfusion injury.

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.

Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

BACKGROUND: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. METHODS: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. RESULTS: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. CONCLUSIONS: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

Distinct effects of calorie restriction on adipose tissue cytokine and angiogenesis profiles in obese and lean mice.

BACKGROUND: Obesity associates with low-grade inflammation and adipose tissue remodeling. Using sensitive high-throughput protein arrays we here investigated adipose tissue cytokine and angiogenesis-related protein profiles from obese and lean mice, and in particular, the influence of calorie restriction (CR). METHODS: Tissue samples from visceral fat were harvested from obese mice fed with a high-fat diet (60% of energy), lean controls receiving low-fat control diet as well as from obese and lean mice kept under CR (energy intake 70% of ad libitum intake) for 50 days. Protein profiles were analyzed using mouse cytokine and angiogenesis protein array kits. RESULTS: In obese and lean mice, CR was associated with 11.3% and 15.6% reductions in body weight, as well as with 4.0% and 4.6% reductions in body fat percentage, respectively. Obesity induced adipose tissue cytokine expressions, the most highly upregulated cytokines being IL-1ra, IL-2, IL-16, MCP-1, MIG, RANTES, C5a, sICAM-1 and TIMP-1. CR increased sICAM-1 and TIMP-1 expression both in obese and lean mice. Overall, CR showed distinct effects on cytokine expressions; in obese mice CR largely decreased but in lean mice increased adipose tissue cytokine expressions. Obesity was also associated with increased expressions of angiogenesis-related proteins, in particular, angiogenin, endoglin, endostatin, endothelin-1, IGFBP-3, leptin, MMP-3, PAI-1, TIMP-4, CXCL16, platelet factor 4, DPPIV and coagulation factor III. CR increased endoglin, endostatin and platelet factor 4 expressions, and decreased IGFBP-3, NOV, MMP-9, CXCL16 and osteopontin expressions both in obese and lean mice. Interestingly, in obese mice, CR decreased leptin and TIMP-4 expressions, whereas in lean mice their expressions were increased. CR decreased MMP-3 and PAI-1 only in obese mice, whereas CR decreased FGF acidic, FGF basic and coagulation factor III, and increased angiogenin and DPPIV expression only in lean mice. CONCLUSIONS: CR exerts distinct effects on adipocyte cytokine and angiogenesis profiles in obese and lean mice. Our study also underscores the importance of angiogenesis-related proteins and cytokines in adipose tissue remodeling and development of obesity.

Caloric restriction ameliorates angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy.

Angiotensin II-induced cardiac damage is associated with oxidative stress-dependent mitochondrial dysfunction. Caloric restriction (CR), a dietary regimen that increases mitochondrial activity and cellular stress resistance, could provide protection. We tested that hypothesis in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs). CR (60% of energy intake for 4 weeks) decreased mortality in dTGRs. CR ameliorated angiotensin II-induced cardiomyocyte hypertrophy, vascular inflammation, cardiac damage and fibrosis, cardiomyocyte apoptosis, and cardiac atrial natriuretic peptide mRNA overexpression. The effects were blood pressure independent and were linked to increased endoplasmic reticulum stress, autophagy, serum adiponectin level, and 5' AMP-activated protein kinase phosphorylation. CR decreased cardiac p38 phosphorylation, nitrotyrosine expression, and serum insulin-like growth factor 1 levels. Mitochondria from dTGR hearts showed clustered mitochondrial patterns, decreased numbers, and volume fractions but increased trans-sectional areas. All of these effects were reduced in CR dTGRs. Mitochondrial proteomic profiling identified 43 dTGR proteins and 42 Sprague-Dawley proteins, of which 29 proteins were in common in response to CR. We identified 7 proteins in CR dTGRs that were not found in control dTGRs. In contrast, 6 mitochondrial proteins were identified from dTGRs that were not detected in any other group. Gene ontology annotations with the Panther protein classification system revealed downregulation of cytoskeletal proteins and enzyme modulators and upregulation of oxidoreductase activity in dTGRs. CR provides powerful, blood pressure-independent, protection against angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy. The findings support the notion of modulating cardiac bioenergetics to ameliorate angiotensin II-induced cardiovascular complications.

Distinct effects of calorie restriction and resveratrol on diet-induced obesity and Fatty liver formation.

The potential of resveratrol to mimic beneficial effects of calorie restriction (CR) was investigated. We compared the effects of both CR (70% of ad libitum energy intake) or resveratrol (2 g/kg or 4 g/kg food) on high-fat diet-induced obesity and fatty liver formation in C57Bl/6J mice, and we examined their effects on calorimetry, metabolic performance, and the expressions of inflammatory genes and SIRT proteins. We found that resveratrol with 4 g/kg dose partially prevented hepatic steatosis and hepatocyte ballooning and induced skeletal muscle SIRT1 and SIRT4 expression while other examined parameter were unaffected by resveratrol. In contrast, CR provided superior protection against diet-induced obesity and fatty liver formation as compared to resveratrol, and the effects were associated with increased physical activity and ameliorated adipose tissue inflammation. CR increased expressions of SIRT3 in metabolically important tissues, suggesting that the beneficial effects of CR are mediated, at least in part, via SIRT3-dependent pathways.

Skeletal muscle gene expression profile is modified by dietary protein source and calcium during energy restriction.

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.

Levosimendan improves cardiac function and survival in rats with angiotensin II-induced hypertensive heart failure.

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.

Novel regulators and drug targets of cardiac hypertrophy.

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.

Resveratrol induces mitochondrial biogenesis and ameliorates Ang II-induced cardiac remodeling in transgenic rats harboring human renin and angiotensinogen genes.

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.

Effects of the calcium sensitizer OR-1896, a metabolite of levosimendan, on post-infarct heart failure and cardiac remodelling in diabetic Goto-Kakizaki rats.

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 p16(INK4A) and p19(ARF). 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.

Metabolomics in angiotensin II-induced cardiac hypertrophy.

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 <0.05). We found great differences in the use of fatty acids as an energy source, namely, decreased levels of octanoic, oleic, and linoleic acids in dTGR (all P<0.01). The increase in cardiac hypoxanthine levels in dTGRs suggested an increase in purine degradation, whereas other changes supported an increased ketogenic amino acid tyrosine level, causing energy production failure. The metabolomic profile of valsartan-treated dTGRs more closely resembled Sprague-Dawley rats than untreated dTGRs. Mitochondrial respiratory chain activity of cytochrome C oxidase was decreased in dTGRs, whereas complex I and complex II were unaltered. Mitochondria from dTGR hearts showed morphological alterations suggesting increased mitochondrial fusion. Cardiac expression of the redox-sensitive and the cardioprotective metabolic sensor sirtuin 1 was increased in dTGRs. Interestingly, valsartan changed the level of 33 metabolites and induced mitochondrial biogenesis in Sprague-Dawley rats. Thus, distinct patterns of cardiac substrate use in Ang II-induced cardiac hypertrophy are associated with mitochondrial dysfunction. The finding underscores the importance of Ang II in the regulation of mitochondrial biogenesis and cardiac metabolomics, even in healthy hearts.

Oral levosimendan prevents postinfarct heart failure and cardiac remodeling in diabetic Goto-Kakizaki rats.

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 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 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.

Metabolomic changes in fatty liver can be modified by dietary protein and calcium during energy restriction.

AIM: To characterise the effect of energy restriction (ER) on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further enhanced by modification of dietary protein source and calcium. METHODS: Liver metabolomic profile of lean and obese C57Bl/6J mice (n = 10/group) were compared with two groups of weight-reduced mice. ER was performed on control diet and whey protein-based high-calcium diet (whey + Ca). The metabolomic analyses were performed using the UPLC/MS based lipidomic platform and the HPLC/MS/MS based primary metabolite platform. RESULTS: ER on both diets significantly reduced hepatic lipid accumulation and lipid droplet size, while only whey + Ca diet significantly decreased blood glucose (P < 0.001) and serum insulin (P < 0.01). In hepatic lipid species the biggest reduction was in the level of triacylglycerols and ceramides while the level of cholesterol esters was significantly increased during ER. Interestingly, diacylglycerol to phospholipid ratio, an indicator of relative amount of diabetogenic diglyceride species, was increased in the control ER group, but decreased in the whey + Ca ER group (P < 0.001, vs obese). ER on whey + Ca diet also totally reversed the obesity induced increase in the relative level of lipotoxic ceramides (P < 0.001, vs obese; P > 0.05, vs lean). These changes were accompanied with up-regulated TCA cycle and pentose phosphate pathway metabolites. CONCLUSION: ER-induced changes on hepatic metabolomic profile can be significantly affected by dietary protein source. The therapeutic potential of whey protein and calcium should be further studied.

Activation of protective and damaging components of the cardiac renin-angiotensin system after myocardial infarction in experimental diabetes.

INTRODUCTION: Diabetes is associated with prolonged apoptotic cell death of cardiac myocytes and adverse remodelling after myocardial infarction (MI). Because the renin-angiotensin system (RAS) has a major role in the remodelling, we studied whether diabetes is associated with altered regulation of RAS after MI in rats. METHODS: Male Wistar rats were randomised to receive either streptozotocin (diabetic group) or citrate buffer (control group) intravenously. MI was produced four weeks later by ligating the left descending coronary artery. The rats were sacrificed 1, 4 and 12 weeks after the operation. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE 2), angiotensin type 1 and 2 receptors (AT(1)-receptor, AT(2)-receptor), and connective tissue growth factor (CTGF) mRNA expression were determined. RESULTS: The expression of both protective and damaging components of RAS increased after MI. However, myocardial ACE 2 and AT(2)-receptor messenger ribonucleic acid (mRNA) expression levels were significantly lower in diabetic compared to non-diabetic rats 1 week after MI. In contrast, AT(1)-receptor, ACE and CTGF mRNA levels were up-regulated in diabetic as compared with non-diabetic rats 12 weeks after MI. CONCLUSION: The activation of the protective components of RAS (ACE 2 and AT(2)-receptor) was blunted early after MI in diabetic rats, whereas the levels of ACE, AT(1)-receptor and CTGF mRNA leading to adverse effects on myocardium, were elevated in diabetic as compared with non-diabetic rats. This unbalanced activation of the RAS may influence the pathophysiology of myocardial injury in diabetes after MI.

Lipoic acid supplementation prevents cyclosporine-induced hypertension and nephrotoxicity in spontaneously hypertensive rats.

BACKGROUND: Cyclosporine (CsA) has significantly improved long-term survival after organ transplantations. Hypertension and nephrotoxicity are common side effects during CsA treatment and are aggravated by high salt intake. OBJECTIVE: To examine whether lipoic acid (LA), a natural antioxidant that scavenges reactive oxygen species and regenerates/recycles endogenous antioxidants, could prevent CsA-induced hypertension and nephrotoxicity. METHODS: Six-week-old spontaneously hypertensive rats (SHR) on a high-sodium diet (NaCl 6%) received CsA [5 mg/kg subcutaneously (s.c.)] alone or in combination with LA (0.5% w/w) for 6 weeks. Blood pressure, arterial functions, and tissue morphology were determined. Immunohistochemistry, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography were used for kidney and heart samples. RESULTS: CsA induced severe hypertension, cardiac hypertrophy, endothelial dysfunction, and pronounced albuminuria. Histologically, the kidneys showed severe thickening of the media of the afferent arteries with fibrinoid necrosis, perivascular monocyte/macrophage infiltration and nitrotyrosine overexpression. CsA induced the expression of fibrogenic connective tissue growth factor both in the heart and kidneys. The detrimental effects of CsA were associated with upregulation of myocardial atrial natriuretic peptide (ANP) mRNA expression, paradoxical activation of the renin-angiotensin system (RAS), induction of renal reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and overexpression of oxidative stress-induced transcription factor NRF2. LA lowered blood pressure, ameliorated cardiac hypertrophy and endothelial dysfunction, and totally normalized albuminuria. In LA-treated rats, renal and cardiac morphologies were indistinguishable from those of SHR controls. CsA-induced myocardial ANP and connective tissue growth factor (CTGF) mRNA overexpression, RAS activation, NADPH oxidase induction, and NRF2 overexpression were prevented by LA. LA induced the mRNA expression of gamma-glutamylcysteine ligase, the rate-limiting enzyme in glutathione synthesis, and markedly increased hepatic cysteine and glutathione concentrations. CONCLUSIONS: Our findings suggest a salutary role for lipoic acid supplementation in the prevention of CsA-induced hypertension and nephrotoxicity, and underscore the importance of increased oxidative stress in the pathogenesis of CsA toxicity.

Vasopeptidase inhibition has beneficial cardiac effects in spontaneously diabetic Goto-Kakizaki rats.

In this study we examined diabetes- and hypertension-induced changes in cardiac structure and function in an animal model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We hypothesized that treatment with omapatrilat, a vasopeptidase inhibitor, which causes simultaneous inhibition of angiotensin converting enzyme and neutral endopeptidase, provides additional cardioprotective effects, during normal- as well as high sodium intake, compared to treatment with enalapril, a selective inhibitor of angiotensin converting enzyme. Fifty-two GK rats were randomized into 6 groups to receive either normal-sodium (NaCl 0.8%) or high-sodium (NaCl 6%) diet and enalapril, omapatrilat or vehicle for 12 weeks. The GK rats developed hypertension, cardiac hypertrophy and overexpression of cardiac natriuretic peptides and profibrotic connective tissue growth factor compared to nondiabetic Wistar rats. The high dietary sodium further increased the systolic blood pressure, and changed the mitral inflow pattern measured by echocardiography towards diastolic dysfunction. Enalapril and omapatrilat equally decreased the systolic blood pressure compared to the control group during normal- as well as high-sodium diet. Both drugs had beneficial cardioprotective effects, which were blunted by the high dietary sodium. Compared to enalapril, omapatrilat reduced the echocardiographically measured left ventricular mass during normal-sodium diet and improved the diastolic function during high-sodium diet in GK rats. Furthermore, omapatrilat reduced relative cardiac weight more effectively than enalapril during high sodium intake. Our results suggest that both the renin-angiotensin and the neutral endopeptidase system are involved in the pathogenesis of diabetic cardiomyopathy since vasopeptidase inhibition was shown to provide additional benefits in comparison with selective angiotensin converting enzyme inhibition alone.

Vascular and renal effects of vasopeptidase inhibition and angiotensin-converting enzyme blockade in spontaneously diabetic Goto-Kakizaki rats.

BACKGROUND: The renin-angiotensin system plays an important role in the pathogenesis of diabetes-induced vascular and renal complications. Vasopeptidase inhibitors simultaneously inhibit angiotensin-converting enzyme (ACE) and neutral endopeptidase. OBJECTIVE: To compare the effectiveness of vasopeptidase inhibition and ACE inhibition in preventing hypertension, endothelial dysfunction and diabetic nephropathy in spontaneously diabetic Goto-Kakizaki (GK) rats. METHODS: Eight-week-old GK rats received omapatrilat (40 mg/kg) or enalapril (30 mg/kg) for 12 weeks, either during a normal-sodium or high-sodium diet (7% w/w). Blood pressure, arterial functions and renal morphology were determined. RESULTS: Blood pressure and albuminuria were increased in GK rats compared to non-diabetic Wistar controls. Endothelium-dependent vascular relaxation in response to acetylcholine (ACh) and endothelium-independent vascular relaxation in response to sodium nitroprusside (SNP) were impaired in GK rats. Experiments with N-nitro-L-arginine methyl ester (L-NAME), diclofenac, and L-NAME + diclofenac suggested that cyclooxygenase and endothelium-derived hyperpolarizing factor components of endothelium-dependent vascular relaxation were also impaired. A high-sodium diet aggravated hypertension and diabetes-induced vascular and renal complications. Omapatrilat and enalapril normalized blood pressure and albuminuria during the normal-sodium diet, and effectively ameliorated diabetes-induced renal complications also during the high-sodium diet. However, omapatrilat improved endothelium-dependent relaxation to ACh to a greater extent (85 +/- 5%) than enalapril (68 +/- 6%, P < 0.05). Diclofenac pre-incubation eliminated this difference between omapatrilat and enalapril in ACh-induced vascular relaxation, suggesting that it was mediated, at least in part, via the cyclooxygenase pathway. CONCLUSIONS: Despite comparable blood pressure-lowering and renoprotective properties, omapatrilat may be more effective in preventing vascular dysfunction during diabetes compared to enalapril in GK rats.