Current Pharmaceutical Design - Volume 19, Issue 39, 2013

The phosphodiesterese-5 (PDE5) inhibitors, including sildenafil (Viagrat), vardenafil (Levitrat), tadalafil (Cialist) and avanafil (Stendrat) have been developed for the treatment of erectile dysfunction. Moreover, sildenafil and tadalafil have been approved for the management of pulmonary arterial hypertension. A number of preclinical studies have shown that PDE5 inhibitors have powerful protective effect against several clinical scenarios including myocardial ischemia/reperfusion injury, doxorubicin and post-MI, heart failure, cardiac hypertrophy, heart transplantation, Duchenne muscular dystrophy and preconditioning of stem cells. Based on these studies, it appears that sildenafil and other PDE5 inhibitors hold promise for further development as novel drug therapies in cardioprotection.

Cardiomyocytes and neurons are highly susceptible to ischemia-reperfusion injury; accordingly, considerable effort has been devoted to elucidating the cellular mechanisms responsible for ischemia-reperfusion-induced cell death and developing novel strategies to minimize ischemia-reperfusion injury. Maintenance of mitochondrial integrity is, without question, a critical determinant of cell fate. However, there is emerging evidence of a novel and intriguing extension to this paradigm: mitochondrial dynamics (that is, changes in mitochondrial morphology achieved by fission and fusion) may play an important but as-yet poorly understood role as a determinant of cell viability. Focusing on heart and brain, our aims in this review are to provide a synopsis of the molecular mechanisms of fission and fusion, summarize our current understanding of the complex relationships between mitochondrial dynamics and the pathogenesis of ischemia-reperfusion injury, and speculate on the possibility that targeted manipulation of mitochondrial dynamics may be exploited for the design of novel therapeutic strategies for cardio- and neuroprotection.

Fatty acids have an important role in providing energy for sustained contractile activity and viability of the heart. However, considerable evidence now supports a role for fatty acids in the modulation of cardiovascular pathology as well. This may be beneficial or detrimental due to the structural differences in the various fatty acids. Polyunsaturated fatty acids appear to provide important protection to the heart during ischemic reperfusion challenge. Conversely, trans fatty acids are thought to mediate detrimental cardiac effects. Potentially pathological features of ischemic cardiomyocytes may be manifested as qualitative findings in studies of myocardial infarction and atherosclerosis. These general conclusions, however, are complicated by opposing effects that different fatty acids have even within the same group (i.e. n-6 versus n-3 fatty acids within the polyunsaturated fatty acids group, and industrial versus ruminant trans fats). Understanding more about how these fatty acid species alter ischemic myocardial injury is an increasingly attractive area of research. The identification of further targets of fatty acid interactions has great potential to provide valuable information for the modulation of cardiovascular disease.

Sudden cardiac death (SCD) from ventricular fibrillation during myocardial infarction is a leading cause of total and cardiovascular mortality. It has a multifactorial, complex nature and aggregates in families, implicating the involvement of heritable factors in the determination of risk. During the last few years, genome-wide association studies have uncovered common genetic variants modulating risk of SCD. We here review the current insight on genetic determinants of SCD in the community and describe the genome-wide association approaches undertaken thus far in uncovering genetic determinants of SCD risk.

Background/Aims. The simultaneous supplementation of creatine and D-ribose has been shown to reduce apoptosis in vitro in non-irreversibly injured cultured ischemic cardiomyocytes through down-regulation of the signaling mechanisms governing adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (Akt). Here, we test the hypothesis that an analogous mechanism exists in vivo when the challenge is chronic exposure to hypoxia. Methods. Five week-old mice were exposed to an atmosphere containing 10% O2 for 10 days. Mice were gavaged daily with vehicle, creatine, D-ribose or creatine + D-ribose. After sacrifice, myocardial and pulmonary tissue were harvested for structural and biochemical analyses. Results. Hypoxia induced right ventricle hypertrophy and left ventricle apoptosis. Both phenotypes were slightly reduced by either creatine or D-ribose, whereas the simultaneous administration of creatine + D-ribose almost completely reversed the effects of hypoxia. Furthermore, creatine + D-ribose diminished the hypoxia-induced increases in the activity of AMPK, Akt and JNK, but not of ERK. Finally, the hypoxia-induced pulmonary overexpression of endothelin-1 mRNA was markedly reduced by creatine + D-ribose. Conclusion. The simultaneous administration of creatine + D-ribose confers additional cardiovascular protection with respect to that observed with either creatine or D-ribose. The mechanism stems from the AMPK and Akt signaling pathways. These findings may form the basis of a paradigm to re-energize non-irreversibly damaged cardiomyocytes, counteracting injury by triggering specific signaling pathways.

The purpose of the present study was to assess the impact of brief daily reoxygenation during adaptation to chronic continuous hypoxia (CCH) on protective cardiac phenotype. Adult male Wistar rats were kept at CCH (10% oxygen) for 5, 15 or 30 days; a subgroup of animals was exposed to room air daily for a single 60-min period. While 5 days of CCH did not affect myocardial infarction induced by 20-min coronary artery occlusion and 3-h reperfusion, 15 days reduced infarct size from 62% of the area at risk in normoxic controls to 5%, and this protective effect was more pronounced after 30 days (41%). Susceptibility to ischemic ventricular arrhythmias exhibited reciprocal development. CCH increased myocardial abundance of mitochondrial superoxide dismutase (MnSOD) without affecting malondialdehyde concentration. Daily reoxygenation abolished both the infarct size-limiting effect of CCH and MnSOD upregulation, and increased malondialdehyde (by 53%). Ventricular cardiomyocytes isolated from CCH rats exhibited better survival and lower lactate dehydrogenase release caused by simulated ischemia/reperfusion than cells from normoxic and daily reoxygenated groups. The cytoprotective effects of CCH were attenuated by the large-conductance Ca2+-activated K+ (BKCa) channel blocker paxilline, while the opener NS1619 reduced cell injury in the normoxic group but not in the CCH group. Daily reoxygenation restored the NS1619- induced protection, whereas paxilline had no effect, resembling the pattern observed in the normoxic group. The results suggest that CCH is cardioprotective and brief daily reoxygenation blunts its salutary effects, possibly by a mechanism involving oxidative stress and attenuation of the activation of mitochondrial BKCa channels.

The signal transducer and activator of transcription 3 (STAT3) transduces stress signals from the plasma membrane to the nucleus but has recently also been identified in mitochondria. Inhibition of cardiomyocyte mitochondrial STAT3 with the STAT3-specific inhibitor Stattic decreases ADP-stimulated respiration and enhances calcium-induced mitochondrial permeability transition pore (MPTP) opening. The aim of the present study was to analyze whether or not these effects of STAT3 inhibition by Stattic are mediated by the formation of reactive oxygen species (ROS). The H2O2 formation from isolated rat left ventricular mitochondria was measured continuously in the presence of the complex 1 substrates glutamate and malate using the H2O2 indicator Amplex UltraRed. Stattic dose-dependently increased mitochondrial ROS formation (slope of Amplex UltraRed fluorescence/time; DMSO: 0.39±0.01; 1 μ M Stattic: 0.40±0.03; 10 μ M Stattic: 0.71±0.04; 25 μ M Stattic: 1.43±0.05; 50 μ M Stattic: 3.53±0.23; 100 μ M Stattic: 9.23±0.69, n=5 mitochondrial preparations, p<0.05 for 10-100 μ M Stattic). The increase in the ROS signal by 100 μ M Stattic was abolished in the presence of the ROS scavenger N-acetylcysteine (Nac, 0.46±0.02, n=7, p<0.05). Mitochondria treated with 100 μ M Stattic produced less ATP than control mitochondria (86±3 arbitrary units (a.u.) vs. 128±7 a.u., n=9, p<0.05). Again, in the presence of Nac ATP production was similar between Stattic-treated and control mitochondria (142±4 a.u. vs. 147±12 a.u., n=5, p=ns). MPTP opening was induced by lower amounts of calcium in Stattic-treated than in control mitochondria (in nmol CaCl2/mg protein, Stattic: 507±57, n=7; control: 857±70, n=7, p<0.05). There was no difference in calcium-induced MPTP opening between Stattic-treated (833±57, n=6) and control mitochondria (921±75, n=7, p=ns) in the presence of Nac. Taken together, our data show that inhibition of mitochondrial STAT3 by Stattic impacts on mitochondrial ATP production and MPTP opening through enhanced ROS formation.

Hypothesis. The present study evaluates the hypothesis that sour cherry seed extract (SCSE) protects against cardiovascular disease and inflammation in hypercholesterolemic rabbits, and that this protection correlates with SCSE-induced activity of heme oxygenase- 1 (HO-1), a cytoprotective enzyme contributing to oxidative stress responses. Methods: 18 New Zealand white rabbits were divided into three groups receiving: I. cholesterol-free rabbit chow; II. chow containing 2% cholesterol; or III. 2% cholesterol plus SCSE for 16 weeks. Heart functions were monitored by echocardiography 0, 4, and 16 weeks after the initiation of cholesterol-supplemented feeding. At the 16-week time-point, isolated hearts were subjected to ischemia-reperfusion (I/R), followed by measurement of heart rate (HR), aortic flow (AF), coronary flow (CF), aortic pressure (AoP), and left ventricular developed pressure (LVDP). Myocardial infarct size was determined using triphenyl tetrazolium chloride (TTC). Quantification of fatty streaks was assessed using Sudan-III staining. Western blot analysis was used to determine the content of cytochrome c oxidase III (COX III), vascular endothelial growth factor (VEGF), and HO-1 in the myocardium. Results: Relative to cholesterol-treated animals not receiving SCSE, SCSE-treated animals exhibited significantly improved cardiac function and improved peak early diastolic velocity to peak atrial velocity ratio (E'/A'), along with decreased atherosclerotic plaque formation and infarct size. Increased HO-1 and COX III protein expression and COX activity were also noted in hearts from SCSE-treated rabbits. Conclusions: This study demonstrates SCSE cardioprotective effects on hypercholesterolemic hearts. Correlation of these outcomes with HO-1 expression suggests that the effect may be mediated by activity of this enzyme. However, definitive proof of HO-1 dependence requires further investigation.

Metabolic disorders such as insulin resistance (IR) and dyslipidemia (DL) might contribute to the induction of diabetic cardiomyopathy (DCM). However, few relevant animal models are currently available for studying the time-course of DCM and evaluating experimental therapeutics. The present study proposes a rodent model of dietary-induced IR combined or not with DL in order to investigate the impact of chronic IR and DL on in vivo myocardial function. Male rats were fed a western-type diet (65% fat; 15% fructose; WD). DL was induced by combining the western diet with i.p. injections of a nonionic surface-active agent (P-407; 0.2 mg/kg, 3 times/wk; P-407). A chow diet was used as control. At 11 and 14 weeks, cardiac function was assessed by echocardiography. Fasting blood glucose increased in WD group while plasma lipids markedly accumulated in P-407 treated rats. Echocardiographic data showed no significant difference in cardiac geometry under basal conditions. Diastolic dysfunction was evidenced at 14 weeks by a significant decrease in E/A ratio in the P-407 group. Moreover, fractional shortening was significantly depressed under dobutamine stress in WD group at 14 weeks whereas systolic dysfunction appeared as early as 11 weeks and worsened at 14 weeks in P-407 animals. Finally, myocardial TNF-alpha tissue content increased in P-407 group. In conclusion, DL exacerbated cardiac lipotoxicity and functional complications associated with IR. This experimental model of combined IR and DL closely mimics the main clinical manifestations of DCM and might therefore constitute a useful tool for the evaluation of pharmacological treatments.

Autophagy is an intracellular bulk degradation process for elimination of damaged macromolecules and organelles. In the past decades, the scientific community has gained increasingly detailed understanding of the role of autophagy in myocardial homeostasis, although still many controversies remain. In the ischemic myocardium, autophagy appears to be beneficial for survival, whereas upon reperfusion the process may induce cell death. However, the overall effect of autophagy seems to depend on the duration and intensity of stress, as along with the extent of autophagy within myocardial tissue. Reperfusion of an ischemic heart maybe harmful, but it is an essential process for myocardial survival. One of the major adverse consequences of reperfusion is the occurrence of ventricular fibrillation (VF). In the present study, we investigated the possible connection between autophagy and VF. Isolated mouse hearts were subjected to ischemia/reperfusion (I/R) and divided into two groups based on the development of VF at the beginning of reperfusion. Western blot analysis was conducted for autophagy-associated proteins LC3B, ATG-5, ATG-7, ATG-12, Bcl-2 and Beclin-1 proteins. Significantly higher level of Beclin-1 and LC3B-II/LC3B-I ratio (both definitive autophagy biomarkers) was observed in the fibrillated myocardium, versus tissue from the nonfibrillated hearts. Interestingly, although Bcl-2 is a major regulator of Beclin-1, level of this protein was not significantly altered in tissue from fibrillated, versus non-fibrillated hearts. Moreover, Atg7 expression showed a trend, albeit nonsignificant, towards elevation in fibrillated versus non-fibrillated hearts. Results of the present investigation demonstrate a possible link between VF and autophagy. Studies by authors of this report to evaluate potential etiologic relationships between the two processes are ongoing.

Obesity arises from an impairment of energy homeostasis, which essentially involves the balance of food intake and energy dissipation. Some secreted molecules in the hypothalamus have become the focus of recent attention for their important roles in the regulation of food intake. One such molecule, nesfatin-1, is a novel molecule originally expressed in the hypothalamic nuclei of the brain, which exerts its satiety function in conjunction with other molecules, including oxytocin and pro-opiomelanocortin (POMC). Nesfatin-1 is processed from its precursor, DNA binding/EF-hand/acidic protein (NEFA)/nucleobindin 2 (NUCB2), and its mRNA is unexpectedly stabilized by troglitazone, a ligand for peroxisome proliferator-activated receptor γ (PPARγ). Subsequent analyses and observations have demonstrated that nesfatin-1 is also located in brain nuclei outside the hypothalamus and in peripheral tissues, and that nesfatin-1 neurons in the brain receive several signals. These findings imply that nesfatin-1 is an endogenous molecule important for the regulation of not only food intake but also other physiological functions. We discuss what is currently known about nesfatin-1, including new developments in our understanding of its distribution, regulation, and biological function.

Nesfatin-1 is an eighty two amino acid, naturally occurring multifunctional protein encoded in the precursor nucleobindin-2 (NUCB2). A comparison of sequences indicates that NUCB2 is present in a number of animals, from hydra to humans. The 30 amino acid mid-segment of nesfatin-1 is considered to be the bioactive core of the protein, and this region displays the highest identity among nesfatin-1 sequences reported thus far. Similar to the sequence relationships observed, the tissue-specific expression and biological actions of nesfatin-1 also appear to be highly conserved across species. For example, brain is a major tissue abundantly expressing nesfatin- 1 in several species. It has been shown that various key regions of the rat, mouse and goldfish brain, which are involved in the regulation of feeding and metabolism express nesfatin-1. Exogenous administration of nesfatin-1 results in a decrease in the food intake of rats, mice and goldfish. In addition, nesfatin-1 has been shown to regulate a number of other physiological processes including hormone secretion from the pancreatic islets and pituitary gland, stress and behavior. While nesfatin-1 research still remains an emerging area in physiology, the literature available thus far clearly shows that nesfatin-1 is an important regulator of homeostasis in animals.

Nesfatin-1 is the N-terminal fragment of nucleobindin-2 (NUCB2). The antibody against nesfatin-1 recognizes both full length of NUCB2 and nesfatin-1, thus the immunolabeling represents NUCB2/nesfatin-1. It has been found that NUCB2/nesfatin-1 is widely distributed in the rodent central nervous system. The immunoreactivity is more intensive in the brain autonomic centers that regulate feeding, neuroendocrine and cardiovascular functions, such as the hypothalamic paraventricular nucleus, supraoptic nucleus, lateral hypothalamic area, Edinger-Westphal nucleus, locus coeruleus, dorsal vagal complex and medullary raphe nuclei. In neurons, NUCB2/nesfatin-1 is located in the soma and primary dendrites, not in nerve fibers. NUCB2/nesfatin-1 is co-localized with several neurotransmitters involved in regulation of food intake, autonomic and neuroendocrine functions, including oxytocin, vasopressin, neuropeptide Y, cocaine- and amphetamine-regulated transcript, proopiomelanocortin, α-melanocyte-stimulating hormone, melanin-concentrating hormone, leptin, mammalian target of rapamycin, urocortin-1, corticotropin-releasing factor and serotonin. In the periphery, NUCB2/nesfatin-1 is located mainly in the pituitary, gastric mucosa where it coexists with ghrelin, and pancreatic endocrine cells containing insulin. Nesfatin-1 is detectable in the cerebrospinal fluid of rats. NUCB2/nesfatin-1 is measurable in the plasma, and altered under different conditions in rodents and humans, such as immune challenge, high fat diet and exercise, anorexia nervosa, anxiety and depression. Anatomical data suggest that nesfatin-1 is a unique neuroendocrine peptide that may be involved in regulation of homeostasis.

Nesfatin-1, derived from an 82-amino-acid peptide precursor protein nucleobindin-2 (NUCB2), is a highly conserved peptide across mammalian species. Initial functional and neuroanatomical studies on NUCB2/nesfatin-1 in the central nervous system have supported a role for NUCB2/nesfatin-1 as a novel satiety molecule. In recent years, however, it has become apparent that this neuropeptide is involved in various other processes, one of which is the stress response. Stress-associated activation of NUCB2/nesfatin-1 neurons, together with nesfatin-1’s central actions in the brain, is indicative of its significance in the stress adaptation response. Interestingly, increasing body of evidence implicates also NUCB2/nesfatin-1 in various forms of stress-associated psychopathologies, such as anxiety and depression. In this review, we will outline evidence that has significantly broadened our understanding of the biological significance of NUCB2/nesfatin-1 far beyond to be only a hypothalamic peptide with potent anorexigenic actions. NUCB2/nesfatin-1 neurons in the brain seem to emerge as novel, integral regulators of the stress adaptation response.

The present review summarizes the current understanding of the neuronal activation patterns induced by nesfatin-1 in both the hypothalamus and the brainstem, as well as the physiological outcomes caused by the activation of these neuronal populations. Studies using cFos measurements, Ca2+ imaging techniques, electrophysiological recordings, and microinjections have led to the identification of the paraventricular nucleus, arcuate nucleus, lateral and ventromedial hypothalamic areas, as well as medullary centers such as the nucleus of the solitary tract and dorsal motor nucleus of the vagus as targets of central nesfatin-1 actions on food intake, cardiovascular function, stress responses, and glucose homeostasis.

Nesfatin-1 was recently identified in the rat brain as a potential post-translational processing product derived from nucleobindin2 (NUCB2). The first biological action identified for nesfatin-1 was the reduction of nocturnal food intake in rats. The anorexigenic effect of nesfatin-1 was corroborated by several independent laboratories and is now established as a physiological action of this peptide based on the regulation of brain NUCB2/nesfatin-1 under different metabolic conditions and the stimulation of food intake and body weight when endogenous brain NUCB2/nesfatin-1 is blocked. Nesfatin-1 shows extensive co-localization with various other, predominantly food intake inhibitory, hypothalamic peptides including corticotropin-releasing factor (CRF), oxytocin, cholecystokinin, proopiomelanocortin, -αmelanocyte stimulating hormone (α-MSH), thyrotropin-releasing hormone (TRH), the orexigenic neuropeptide Y and brain biogenic amines, histamine, serotonin, and catecholamines. The food intake suppressing effect of centrally injected nesfatin-1 has been established so far to involve several downstream mechanisms including H1, CRF2, TRH, oxytocin as well as melanocortin-3/4 receptor signaling pathways. This intricate embedding of NUCB2/nesfatin-1 in central food intake regulatory pathways recruited during the dark phase corresponding to the eating period in rodents, unlike the orexigenic response to a fast, points towards a role for nesfatin-1 in modulating the nocturnal food intake. Although our knowledge on the regulation and effects of NUCB2/nesfatin-1 as a new anorexic peptide markedly increased during the past five years, several important gaps of knowledge remain to be filled in the near future such as the regulation of NUCB2 processing and nesfatin-1 release as well as the identification, localization and regulation of the nesfatin-1 receptor.

The novel satiety factor nesfatin-1 and its precursor NUCB2 are the neuropeptides widely expressed in the central nervous system. Nesfatin-1/NUCB2 is also localized in peripheral tissues and regulates the glucose and energy metabolism on multiple processes. Nesfatin-1 potentiates both insulin release from pancreatic β-cells and insulin action in liver, contributing to energy storage. Furthermore, nesfatin-1/NUCB2 regulates adipocyte differentiation. The polymorphism of the NUCB2 gene is associated with obesity. Thus, nesfatin- 1/NUCB2 plays a role in integrating feeding, glucose homeostasis, and energy storage/expenditure. Dysfunction of expression, secretion and/or action of nesfatin-1/NUCB2 might be involved in the type 2 diabetes, obesity and metabolic syndrome. Nesfatin-1/NUCB2 and its regulatory processes may provide novel targets for treating associated diseases of the metabolic syndrome. Here, we review the by now published studies on nesfatin-1/NUCB2 localization and action in islets and discuss the physiological and pathophysiological roles of the nesfatin-1/NUCB2 in glucose and energy metabolism.

Nesfatin-1, derived from the precursor NEFA/nucleobindin2 (NUCB2), was initially identified as a feeding-suppressing neuropeptide, acting at central (mainly, hypothalamic) levels in a leptin-independent manner. However, recent experimental evidence strongly suggests that, rather than being a simple anorectic hypothalamic signal, nesfatin-1 operates at different tissues as an integral regulator of energy homeostasis and closely related neuroendocrine functions. On the latter, growing, albeit as yet fragmentary, evidence has pointed out recently that NUCB2/ nesfatin-1 is involved in the regulation of different aspects of reproductive maturation and function, by acting probably at different levels of the hypothalamic-pituitary-gonadal (HPG) axis. As documented by rodent studies, the reproductive facet of nesfatin-1 likely includes (i) a permissive role in (female) pubertal maturation, (ii) stimulatory effects on the gonadotropic axis, whose magnitude, in terms of LH responses, varies depending on the maturational stage and probably the sex and species, and (iii) direct expression and actions in the gonads. These features, together with the proven expression of NUCB2/nesfatin-1 in tissues with essential roles in the metabolic control of reproduction, such as the hypothalamus, adipose and pancreas, support a putative role of nesfatin-1 as neurohormonal signal linking body metabolic status, puberty and fertility. Curiously enough, although its reproductive dimension seems to be conserved in non-mammalian vertebrates, recent studies in goldfish have surfaced predominant inhibitory actions of nesfatin-1 at different levels of the HPG axis in fish. These findings illustrate our as yet limited understanding of this aspect of nesfatin-1 physiology, whose relevance in the joint control of metabolism and reproduction in health and disease warrants further investigation.