Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration.

Heart failure is a serious condition that results from the extensive loss of specialized cardiac muscle cells called cardiomyocytes (CMs), typically caused by myocardial infarction (MI). Messenger RNA (mRNA) therapeutics are emerging as a very promising gene medicine for regenerative cardiac therapy. To date, lipid nanoparticles (LNPs) represent the most clinically advanced mRNA delivery platform. Yet, their delivery efficiency has been limited by their endosomal entrapment after endocytosis. Previously, we demonstrated that a pair of complementary coiled-coil peptides (CPE4/CPK4) triggered efficient fusion between liposomes and cells, bypassing endosomal entrapment and resulting in efficient drug delivery. Here, we modified mRNA-LNPs with the fusogenic coiled-coil peptides and demonstrated efficient mRNA delivery to difficult-to-transfect induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). As proof of in vivo applicability of these fusogenic LNPs, local administration via intramyocardial injection led to significantly enhanced mRNA delivery and concomitant protein expression. This represents the successful application of the fusogenic coiled-coil peptides to improve mRNA-LNPs transfection in the heart and provides the potential for the advanced development of effective regenerative therapies for heart failure.

Extracellular vesicle-mediated protein delivery to the liver.

Extracellular vesicles (EVs) are nanoscale particles that facilitate intercellular communication. They are regarded as a promising natural drug delivery system for transporting and delivering bioactive macromolecules to target cells. Recently, researchers have engineered EVs with FKBP12/FRB heterodimerization domains that interact with rapamycin to load and deliver exogenous proteins for both in vitro and in vivo applications. In this study, we examined the tissue distribution of EVs using near-infrared fluorescent imaging. We evaluated the effectiveness of EV-mediated delivery of Cre recombinase specifically to hepatocytes in the livers of Ai9 Cre-loxP reporter mice. Intravenous injection resulted in more efficient Cre protein delivery to the liver than intraperitoneal injections. Depleting liver-resident macrophages with clodronate-encapsulated liposome pre-treatment did not enhance EV-mediated Cre delivery to hepatocytes. Moreover, we demonstrated that multiple intravenous injections of Cre-EVs facilitated functional Cre delivery to hepatocytes. To the best of our knowledge, this is the first study to simultaneously investigate the tissue distribution of FKBP12/FRB-engineered EVs and their subsequent intracellular protein delivery in Ai9 Cre-loxP reporter mice. These insights can inform preclinical research and contribute to developing next-generation EV-based platforms for delivering therapeutic proteins or genome editing technologies targeting the liver.

Evaluation and manipulation of tissue and cellular distribution of cardiac progenitor cell-derived extracellular vesicles.

Cardiac progenitor cell-derived extracellular vesicles (CPC-EVs) have been successfully applied via different delivery routes for treating post-myocardial infarction injury in several preclinical models. Hence, understanding the in vivo fate of CPC-EVs after systemic or local, i.e. myocardial, delivery is of utmost importance for the further therapeutic application of CPC-EVs in cardiac repair. Here, we studied the tissue- and cell distribution and retention of CPC-EVs after intramyocardial and intravenous injection in mice by employing different EV labeling and imaging techniques. In contrast to progenitor cells, CPC-EVs demonstrated no immediate flush-out from the heart upon intramyocardial injection and displayed limited distribution to other organs over time, as determined by near-infrared imaging in living animals. By employing CUBIC tissue clearing and light-sheet fluorescent microscopy, we observed CPC-EV migration in the interstitial space of the myocardium shortly after EV injection. Moreover, we demonstrated co-localization with cTnI and CD31-positive cells, suggesting their interaction with various cell types present in the heart. On the contrary, after intravenous injection, most EVs accumulated in the liver. To potentiate such a potential systemic cardiac delivery route, targeting the cardiac endothelium could provide openings for directed CPC-EV therapy. We therefore evaluated whether decorating EVs with targeting peptides (TPs) RGD-4C or CRPPR connected to Lamp2b could enhance EV delivery to endothelial cells. Expression of both TPs enhanced CPC-EV uptake under in vitro continuous flow, but did not affect uptake under static cell culture conditions. Together, these data demonstrate that the route of administration influences CPC-EV biodistribution pattern and suggest that specific TPs could be used to target CPC-EVs to the cardiac endothelium. These insights might lead to a better application of CPC-EV therapeutics in the heart.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Intratracheal administration of solutions in mice; development and validation of an optimized method with improved efficacy, reproducibility and accuracy.

Animal models are still vital in the field of respiratory disease research. To improve the accuracy and consistency of the dose of specific compounds administered specifically in the respiratory tract, it is important to optimize and to compare the technique to currently available techniques. In this study, an optimized intubation-mediated intratracheal administration (IMIT) technique is described and compared to oropharyngeal aspiration (OA). Adult female Balb/c mice were treated with Evans Blue using IMIT or OA and sacrificed after a short recovery to observe the distribution of solutions throughout the lungs. Additionally, mice were treated with increasing doses of lipopolysaccharide (LPS) or saline to compare efficacy of both techniques. Inflammatory cell numbers in bronchoalveolar lavage were quantified 24 h post-administration. Evans Blue staining revealed a more homogeneous distribution and less variability among animals treated using IMIT as compared to OA. Higher inflammatory cell numbers were observed in IMIT mice compared to OA mice after exposure to vehicle or the lowest LPS concentration. This study shows that the optimized IMIT is superior to OA with regards to efficacy, reproducibility and accuracy. This IMIT method can be deployed to refine 3R animal welfare aspects of the experimental design and improve the reproducibility of respiratory disease mouse models.

Ventricular TLR4 Levels Abrogate TLR2-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice.

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1ß, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

Neutral Effects of Combined Treatment With GLP-1R Agonist Exenatide and MR Antagonist Potassium Canrenoate on Cardiac Function in Porcine and Murine Chronic Heart Failure Models.

Background: Ischemia-reperfusion and cardiac remodeling is associated with cardiomyocyte death, excessive fibrosis formation, and functional decline, eventually resulting in heart failure (HF). Glucagon-like peptide (GLP)-1 agonists are reported to reduce apoptosis and myocardial infarct size after ischemia-reperfusion. Moreover, mineralocorticoid receptor antagonists (MRAs) have been described to reduce reactive fibrosis and improve cardiac function. Here, we investigated whether combined treatment with GLP-1R agonist exenatide and MRA potassium canrenoate could minimize cardiac injury and limit HF progression in animal models of chronic HF. Methods and Results: Forty female Topigs Norsvin pigs were subjected to 150 min balloon occlusion of the left anterior descending artery (LAD). Prior to reperfusion, pigs were randomly assigned to placebo or combination therapy (either low dose or high dose). Treatment was applied for two consecutive days or for 8 weeks with a continued high dose via a tunneled intravenous catheter. Using 2,3,5-Triphenyltetrazolium chloride (TTC) staining we observed that combination therapy did not affect the scar size after 8 weeks. In line, left ventricular volume and function assessed by three-dimensional (3D) echocardiography (baseline, 7 days and 8 weeks), and cardiac magnetic resonance imaging (CMR, 8 weeks) did not differ between experimental groups. In addition, 36 C57Bl/6JRj mice underwent permanent LAD-occlusion and were treated with either placebo or combination therapy prior to reperfusion, for two consecutive days via intravenous injection, followed by continued treatment via placement of osmotic mini-pumps for 28 days. Global cardiac function, assessed by 3D echocardiography performed at baseline, 7, 14, and 28 days, did not differ between treatment groups. Also, no differences were observed in cardiac hypertrophy, assessed by heart weight/bodyweight and heart weight/tibia length ratio. Conclusion: In the current study, combined treatment with GLP-1R agonist exenatide and MR antagonist potassium canrenoate did not show beneficial effects on cardiac remodeling nor resulted in functional improvement in a small and large animal chronic HF model.

Mildly Increased Renin Expression in the Absence of Kidney Injury in the Murine Transverse Aortic Constriction Model.

Cardiorenal syndrome type 2 is characterized by kidney failure as a consequence of heart failure that affects >50% of heart failure patients. Murine transverse aortic constriction (TAC) is a heart failure model, where pressure overload is induced on the heart without any systemic hypertension or its consequences. Whether renal function is altered in this model is debated, and if so, at which time post-TAC renal dysfunction starts to contribute to worsening of cardiac function. We therefore studied the effects of progressive heart failure development on kidney function in the absence of chronically elevated systemic blood pressure and renal perfusion pressure. C57BL/6J mice (N = 129) were exposed to TAC using a minimally invasive technique and followed from 3 to 70 days post-TAC. Cardiac function was determined with 3D ultrasound and showed a gradual decrease in stroke volume over time. Renal renin expression and plasma renin concentration increased with progressive heart failure, suggesting hypoperfusion of the kidney. In addition, plasma urea concentration, a surrogate marker for renal dysfunction, was increased post-TAC. However, no structural abnormalities in the kidney, nor albuminuria were present at any time-point post-TAC. Progressive heart failure is associated with increased renin expression, but only mildly affected renal function without inducing structural injury. In combination, these data suggest that heart failure alone does not contribute to kidney dysfunction in mice.

A combined CaMKII inhibition and mineralocorticoid receptor antagonism via eplerenone inhibits functional deterioration in chronic pressure overloaded mice.

In the diseased and remodelled heart, increased activity and expression of Ca2+/ calmodulin-dependent protein kinase II (CaMKII), an excess of fibrosis, and a decreased electrical coupling and cellular excitability leads to disturbed calcium homeostasis and tissue integrity. This subsequently leads to increased arrhythmia vulnerability and contractile dysfunction. Here, we investigated the combination of CaMKII inhibition (using genetically modified mice expressing the autocamtide-3-related-peptide (AC3I)) together with eplerenone treatment (AC3I-Epler) to prevent electrophysiological remodelling, fibrosis and subsequent functional deterioration in a mouse model of chronic pressure overload. We compared AC3I-Epler mice with mice only subjected to mineralocorticoid receptor (MR) antagonism (WT-Epler) and mice with only CaMKII inhibition (AC3I-No). Our data show that a combined CaMKII inhibition together with MR antagonism mitigates contractile deterioration as was manifested by a preservation of ejection fraction, fractional shortening, global longitudinal strain, peak strain and contractile synchronicity. Furthermore, patchy fibrosis formation was reduced, potentially via inhibition of pro-fibrotic TGF-ß/SMAD3 signalling, which related to a better global contractile performance and a slightly depressed incidence of arrhythmias. Furthermore, the level of patchy fibrosis appeared significantly correlated to eplerenone dose. The addition of eplerenone to CaMKII inhibition potentiates the effects of CaMKII inhibition on pro-fibrotic pathways. As a result of the applied strategy, limiting patchy fibrosis adheres to a higher synchronicity of contraction and an overall better contractile performance which fits with a tempered arrhythmogenesis.

Complement 5a Receptor deficiency does not influence adverse cardiac remodeling after pressure-overload in mice.

Hypertension is one of the most common risk factors for the development heart failure in the general population. Inflammation plays a central role in this adverse remodeling and eventually to the development of heart failure. Circulating levels of Complement factor 5a (C5a) are increased in hypertensive patients and the C5a receptor is associated with the presence of cardiac fibrosis and inflammation in an experimental hypertension model. To test if C5aR is involved in adverse cardiac remodeling following pressure-overload, we induced transverse aortic constriction (TAC) in wildtype and C5a receptor deficient mice (C5aR-/-). Six weeks after TAC, C5aR-/- animals showed a similar degree of cardiac hypertrophy and decrease in cardiac function as wild type mice (End Systolic Volume; 50.30±5.32 µl vs. 55.81±8.16 µl). In addition, other features of adverse cardiac remodeling like cardiomyocyte cell size (WGA staining), fibrosis (picrosirius red staining) or collagen degradation (matrix metalloproteinase activity assay) did not differ either. In conclusion, full body C5aR deficiency does not affect adverse cardiac remodeling after pressure-overload. However, our finding are in contrast with C5a inhibition studies. Our observations do present the role of C5a-C5aR in adverse cardiac remodeling and heart failure as controversial at the least.

Leukocyte-Associated Immunoglobulin-like Receptor-1 is regulated in human myocardial infarction but its absence does not affect infarct size in mice.

Heart failure after myocardial infarction (MI) depends on infarct size and adverse left ventricular (LV) remodelling, both influenced by the inflammatory response. Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an inhibitory receptor of ITAM-dependent cell activation, present on almost all immune cells. We investigated regulation of LAIR-1 leukocyte expression after MI in patients and hypothesized that its absence in a mouse model of MI would increase infarct size and adverse remodelling. In patients, LAIR-1 expression was increased 3 days compared to 6 weeks after MI on circulating monocytes (24.8 ± 5.3 vs. 21.2 ± 5.1 MFI, p = 0.008) and neutrophils (12.9 ± 4.7 vs. 10.6 ± 3.1 MFI, p = 0.046). In WT and LAIR-1-/- mice, infarct size after ischemia-reperfusion injury was comparable (37.0 ± 14.5 in WT vs. 39.4 ± 12.2% of the area at risk in LAIR-1-/-, p = 0.63). Remodelling after permanent left coronary artery ligation did not differ between WT and LAIR-1-/- mice (end-diastolic volume 133.3 ± 19.3 vs. 132.1 ± 27.9 µL, p = 0.91 and end-systolic volume 112.1 ± 22.2 vs. 106.9 ± 33.5 µL, p = 0.68). Similarly, no differences were observed in inflammatory cell influx or fibrosis. In conclusion, LAIR-1 expression on monocytes and neutrophils is increased in the acute phase after MI in patients, but the absence of LAIR-1 in mice does not influence infarct size, inflammation, fibrosis or adverse cardiac remodelling.

Targeting chronic cardiac remodeling with cardiac progenitor cells in a murine model of ischemia/reperfusion injury.

BACKGROUND: Translational failure for cardiovascular disease is a substantial problem involving both high research costs and an ongoing lack of novel treatment modalities. Despite the progress already made, cell therapy for chronic heart failure in the clinical setting is still hampered by poor translation. We used a murine model of chronic ischemia/reperfusion injury to examine the effect of minimally invasive application of cardiac progenitor cells (CPC) in cardiac remodeling and to improve clinical translation. METHODS: 28 days after the induction of I/R injury, mice were randomized to receive either CPC (0.5 million) or vehicle by echo-guided intra-myocardial injection. To determine retention, CPC were localized in vivo by bioluminescence imaging (BLI) two days after injection. Cardiac function was assessed by 3D echocardiography and speckle tracking analysis to quantify left ventricular geometry and regional myocardial deformation. RESULTS: BLI demonstrated successful injection of CPC (18/23), which were mainly located along the needle track in the anterior/septal wall. Although CPC treatment did not result in overall restoration of cardiac function, a relative preservation of the left ventricular end-diastolic volume was observed at 4 weeks follow-up compared to vehicle control (+5.3 ± 2.1 µl vs. +10.8 ± 1.5 µl). This difference was reflected in an increased strain rate (+16%) in CPC treated mice. CONCLUSIONS: CPC transplantation can be adequately studied in chronic cardiac remodeling using this study set-up and by that provide a translatable murine model facilitating advances in research for new therapeutic approaches to ultimately improve therapy for chronic heart failure.

Increased local delivery of antagomir therapeutics to the rodent myocardium using ultrasound and microbubbles.

Recent developments in microRNA (miRNA) research have identified these as important mediators in the pathophysiological response upon myocardial infarction (MI). Specific miRNAs can inhibit the translation of entire groups of mRNAs, which are involved in specific processes in the pathophysiology after MI, e.g. the fibrotic, apoptotic or angiogenic response. By modulating miRNAs in the heart, these processes can be tuned to improve cardiac function. Antagomirs are effective miRNA-inhibitors, but have a low myocardial specificity and cardiac antagomir treatment therefore requires high doses, which causes side effects. In the present study, ultrasound-triggered microbubble destruction (UTMD) was studied to increase specific delivery of antagomir to the myocardium. Healthy control mice were treated with UTMD and sacrificed at 30min, 24h and 48h, after which antagomir delivery in the heart was analyzed, both qualitatively and quantitatively. Additionally, potential harmful effects of treatment were analyzed by monitoring ECG, analyzing neutrophil invasion and cell death in the heart, and measuring troponin I after treatment. Finally, UTMD was tested for delivery of antagomir in a model of ischemia-reperfusion (I/R) injury. We found that UTMD can significantly increase local antagomir delivery to the non-ischemic heart with modest side-effects like neutrophil invasion without causing apoptosis. Delivered antagomirs enter cardiomyocytes within 30min after treatment and remains there for at least 48h. Interestingly, after I/R injury antagomir already readily enters the infarcted zone and we observed no additional benefit of UTMD for antagomir delivery. This study is the first to explore cardiac antagomir delivery using UTMD. In addition, it is the first to study tissue distribution of short RNA based therapeutics (~22 base pairs) at both the cellular and organ levels after UTMD to the heart in general. In summary, UTMD provides a myocardial delivery strategy for non-vascular permeable cardiac conditions later in the I/R response or chronic conditions like cardiac hypertrophy.

CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload.

BACKGROUND: One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS: A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION: Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.

Central melanocortins regulate the motivation for sucrose reward.

The role of the melanocortin (MC) system in feeding behavior is well established. Food intake is potently suppressed by central infusion of the MC 3/4 receptor agonist α-melanocyte stimulating hormone (α-MSH), whereas the MC 3/4 receptor inverse-agonist Agouti Related Peptide (AGRP) has the opposite effect. MC receptors are widely expressed in both hypothalamic and extra-hypothalamic brain regions, including nuclei involved in food reward and motivation, such as the nucleus accumbens (NAc) and the ventral tegmental area. This suggests that MCs modulate motivational aspects of food intake. To test this hypothesis, rats were injected intracerebroventricularly with α-MSH or AGRP and their motivation for sucrose was tested under a progressive ratio schedule of reinforcement. Food motivated behavior was dose-dependently decreased by α-MSH. Conversely, AGRP increased responding for sucrose, an effect that was blocked by pretreatment with the dopamine receptor antagonist α-flupenthixol. In contrast to progressive ratio responding, free intake of sucrose remained unaltered upon α-MSH or AGRP infusion. In addition, we investigated whether the effects of α-MSH and AGRP on food motivation were mediated by the NAc shell. In situ hybridization of MC3 and MC4 receptor expression confirmed that the MC4 receptor was expressed throughout the NAc, and injection of α-MSH and AGRP into the NAc shell caused a decrease and an increase in motivation for sucrose, respectively. These data show that the motivation for palatable food is modulated by MC4 receptors in the NAc shell, and demonstrate cross-talk between the MC and dopamine system in the modulation of food motivation.

Ghrelin mediates anticipation to a palatable meal in rats.

Food anticipatory activity (FAA) is displayed in rats when access to food is restricted to a specific time frame of their circadian phase, a behavior thought to reflect both hunger and the motivation to eat. Rats also display FAA in a feeding schedule with ad libitum access to normal chow, but limited availability of a palatable meal, which is thought to involve mainly motivational aspects. The orexigenic hormone ghrelin has been implicated in FAA in rodents with restricted access to chow. Because ghrelin plays an important role not only in the control of food intake, but also in reward, we sought to determine the role of ghrelin in anticipation to a palatable meal. Plasma ghrelin levels of non-restricted rats that anticipated chocolate correlated positively with FAA and were increased compared with chow-fed control rats. Furthermore, centrally injected ghrelin increased, whereas an antagonist of the ghrelin receptor decreased, the anticipation to chocolate. Therefore, we hypothesize that central ghrelin signaling is able to mediate the motivational drive to eat.

Melanocortin receptor 4 deficiency affects body weight regulation, grooming behavior, and substrate preference in the rat.

Obesity is caused by an imbalance between energy intake and expenditure and has become a major health-care problem in western society. The central melanocortin system plays a crucial role in the regulation of feeding and energy expenditure, and functional loss of melanocortin receptor 4 (MC4R) is the most common genetic cause of human obesity. In this study, we present the first functional Mc4r knockout model in the rat, resulting from an N-ethyl-N-nitrosourea mutagenesis-induced point mutation. In vitro observations revealed impaired membrane-binding and subsequent nonfunctionality of the receptor, whereas in vivo observations showed that functional loss of MC4R increased body weight, food intake, white adipose mass, and changed substrate preference. In addition, intracerebroventricular (ICV) administration of Agouti-Related Protein(79-129) (AgRP(79-129)), an MC4R inverse agonist, or Melanotan-II (MTII), an MC4R agonist, did affect feeding behavior in wild-type rats but not in homozygous mutant rats, confirming complete loss of MC4R function in vivo. Finally, ICV administration of MTII induced excessive grooming behavior in wild-type rats, whereas this effect was absent in homozygous mutant rats, indicating that MTII-induced grooming behavior is exclusively regulated via MC4R pathways. Taken together, we expect that the MC4R rat model described here will be a valuable tool for studying monogenic obesity in humans. More specifically, the relative big size and increased cognitive capacity of rats as compared to mice will facilitate complex behavioral studies and detailed mechanistic studies regarding central function of MC4R, both of which ultimately may help to further understand the specific mechanisms that induce obesity during loss of MC4R function.

Neuropeptide delivery to the brain: a von Willebrand factor signal peptide to direct neuropeptide secretion.

BACKGROUND: Multiple neuropeptides, sometimes with opposing functions, can be produced from one precursor gene. To study the roles of the different neuropeptides encoded by one large precursor we developed a method to overexpress minigenes and establish local secretion. RESULTS: We fused the signal peptide from the Von Willebrand Factor (VWF) to a furin site followed by a processed form of the Agouti related protein (AgRP), AgRP(83-132) or alpha-melanocyte stimulating hormone. In vitro, these minigenes were secreted and biologically active. Additionally, the proteins of the minigenes were not transported into projections of primary neurons, thereby ensuring local release. In vivo administration of VWF-AgRP(83-132), using an adeno-associated viral vector as a delivery vehicle, into the paraventricular hypothalamus increased body weight and food intake of these rats compared to rats which received a control vector. CONCLUSIONS: This study demonstrated that removal of the N-terminal part of full length AgRP and addition of a VWF signal peptide is a successful strategy to deliver neuropeptide minigenes to the brain and establish local neuropeptide secretion.