Development of the Clinical Application of miRNA with Proregenerative Effect on the Heart

Rationale: Cardiovascular disorders are the first cause of mortality and morbidity worldwide. This is due, at least in part, to the poor regenerative capacity of the heart and the lack of drugs able to foster cardiac regeneration. Our recent work has identified a few human microRNAs (miRNAs), in particular hsa-miR-199a-3p, able to stimulate proliferation of cardiomyocytes and, once expressed in the mouse heart using viral vectors, to induce cardiac regeneration after myocardial infarction. Objective: As a first step towards clinical translation, in this study we assess the efficacy of the pro-regenerative miR-199a after myocardial infarction, delivered using an AAV vector in a pig model of ischemia-reperfusion and as a synthetic RNA molecule after a single intracardiac injection in mice. Methods and Results: We evaluated the efficacy of miR-199a in a pig model of ischemiareperfusion after direct myocardial injection of an AAV6 vectors carrying the miR-199a precursor. Cardiac function was evaluated at days 2 and 28 by gadolinium-enhanced cardiac magnetic resonance imaging (cMRI). The results showed significant reduction of infarct size and increased cardiac function in the animals treated with AAV6 miR-199a at one month after treatment. Histological analysis uncovered a significant increase in cardiomyocyte proliferation in the infarct border zone, paralleled by the expression of markers of cardiomyocyte de-differentiation. Despite these remarkably positive signs of cardiac regeneration, pigs treated with AAV6-miR-199a died of sudden death at weeks 7-8 after treatment. In three of these pigs, clusters of small, proliferating cells with a phenotype of undifferentiated myogenic progenitors were apparent. In parallel, we comparatively analyzed the efficacy different lipid formulations in delivering hsa-miR-199a-3p as a naked RNA mimic in both primary neonatal rat cardiomyocytes and in vivo. We established a transfection protocol allowing persistence of miR-199a-3p mimics, carrying different chemical modifications, for at least 12 days after a single intracardiac injection, with minimal dispersion to other organs and long-term preservation of miRNA functional activity, as assessed by monitoring the expression of two direct mRNA targets. We administered this synthetic formulation immediately after myocardial infarction in mice and found that a single intracardiac injection was sufficient to reduce scar size and improve global cardiac function up to two months. Histology confirmed reduced scar extension and immunofluorescence showed increased cardiomyocyte proliferation in the miR-199a-3p mimic injected animals. Conclusions: AAV6-miR-199a injection after ischemia-reperfusion in pigs significantly improves cardiac function and reduce scar size. However, AAV6-driven miR-199a persistent expression in the pig myocardium is fraught with safety issues. A single administration of mir-199a-3p mimic is sufficient to stimulate adult mouse cardiac repair and restoration of cardiac function. Together, these results are concordant in indicating that a miRNA-based therapy aimed at inducing cardiomyocyte proliferation might be pursued to stimulate cardiac regeneration, however that the duration of the miRNA effect needs to be tightly controlled

Single-Dose Intracardiac Injection of Pro-Regenerative MicroRNAs Improves Cardiac Function After Myocardial Infarction

Rationale: Recent evidence indicates that a few human microRNAs (miRNAs), in particular hsa-miR-199a-3p and hsa-miR-590-3p, stimulate proliferation of cardiomyocytes and, once expressed in the mouse heart using viral vectors, induce cardiac regeneration after myocardial infarction. Viral vectors, however, are not devoid of safety issues and, more notably, drive expression of the encoded miRNAs for indefinite periods of time, which might not be desirable in light of human therapeutic application. Objective: As an alternative to the use of viral vectors, we wanted to assess the efficacy of synthetic miRNA mimics in inducing myocardial repair after single intracardiac injection using synthetic lipid formulations. Methods and Results: We comparatively analyzed the efficacy of different lipid formulations in delivering hsa-miR-199a-3p and hsa-miR-590-3p both in primary neonatal mouse cardiomyocytes and in vivo. We established a transfection protocol allowing persistence of these two mimics for at least 12 days after a single intracardiac injection, with minimal dispersion to other organs and long-term preservation of miRNA functional activity, as assessed by monitoring the expression of two direct mRNA targets. Administration of this synthetic formulation immediately after myocardial infarction in mice resulted in marked reduction of infarct size and persistent recovery of cardiac function. Conclusions: A single administration of synthetic miRNA-lipid formulations is sufficient to stimulate cardiac repair and restoration of cardiac function

KLF15 Is a Molecular Link between Endoplasmic Reticulum Stress and Insulin Resistance

Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15-/- mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15-/- liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action

KLF15 and PPAR α

The metabolic myocardium is an omnivore and utilizes various carbon substrates to meet its energetic demand. While the adult heart preferentially consumes fatty acids (FAs) over carbohydrates, myocardial fuel plasticity is essential for organismal survival. This metabolic plasticity governing fuel utilization is under robust transcriptional control and studies over the past decade have illuminated members of the nuclear receptor family of factors (e.g., PPARα) as important regulators of myocardial lipid metabolism. However, given the complexity of myocardial metabolism in health and disease, it is likely that other molecular pathways are likely operative and elucidation of such pathways may provide the foundation for novel therapeutic approaches. We previously demonstrated that Kruppel-like factor 15 (KLF15) is an independent regulator of cardiac lipid metabolism thus raising the possibility that KLF15 and PPARα operate in a coordinated fashion to regulate myocardial gene expression requisite for lipid oxidation. In the current study, we show that KLF15 binds to, cooperates with, and is required for the induction of canonical PPARα-mediated gene expression and lipid oxidation in cardiomyocytes. As such, this study establishes a molecular module involving KLF15 and PPARα and provides fundamental insights into the molecular regulation of cardiac lipid metabolism

Kruppel-like Factor 15 Is a Critical Regulator of Cardiac Lipid Metabolism

Background: Metabolic homeostasis is central to normal cardiac function. The molecular mechanisms underlying metabolic plasticity in the heart remain poorly understood. Results: Kruppel-like factor 15 (KLF15) is a direct and independent regulator of myocardial lipid flux. Conclusion: KLF15 is a core component of the transcriptional circuitry that governs cardiac metabolism. Significance: This work is the first to implicate the KLF transcription factor family in cardiac metabolism. The mammalian heart, the body\u27s largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism

Action of growth promoters on the intestinal mucosa of broiler chickens

La búsqueda de productos naturales para reemplazar eluso de antibióticos de síntesis química en la producciónaviar se ha incrementado últimamente. Los flavonoidesde los extractos vegetales contienen catequinas conpropiedades antibacterianas y antioxidantes. En el presentetrabajo estudiamos un extracto vegetal polifenólico (EVP)derivado del quebracho colorado (Schinopsis lorentzii)como aditivo dietario. Se analizó su efecto (500 g/toneladade alimento) en pollos parrilleros hasta el día 35 de vida,respecto a un promotor antibiótico, bacitracina metilenodisalicilato (BMD) (500 g/tonelada de alimento) y ungrupo control que no recibió aditivos. No se observarondiferencias significativas en los parámetros productivos nien los estudios histológicos e histomorfométricos en variostramos intestinales, salvo al día 35 en que se encontró unincremento significativo en la relación vellosidad?cripta delíleon, en las aves que recibieron el EVP con respecto alresto de los grupos estudiados. Por otro lado, las aves querecibieron BMD presentaron una disminución significativaen el nivel de IgA secretoria. De esta manera, se postulaque el EVP es un excelente candidato para reemplazar a losantibióticos de origen sintético ya que mejoraría la absorciónde nutrientes sin interferir en los niveles de IgA secretoria.The search for natural products to replace the use of chemically synthesized antibiotics in avian production has increased lately. Flavonoids in plant extracts contain catechins with antibacterial and antioxidant properties. In the present work we study a polyphenolic vegetable extract (PVE) derived from the quebracho red wood (Schinopsis lorentzii) as a dietary additive. Its effect (500 g/ton of feed) in broiler chickens until day 35 of life was analyzed with respect to an antibiotic promoter, bacitracin methylene disalicylate (BMD) (500 g/ton of feed) and a control group that did not receive additives. No significant differences were observed in the productive parameters nor in the histological and histomorphometric studies in various intestinal sections, except for a significant increase in the ileum villus-crypt ratio found in the chickens that received the PVE, on day 35, compared to the rest of the groups under study. On the other hand, the poultry that received BMD presented a significant decrease in the level of secretory IgA. Thus, it is postulated that PVE is an excellent candidate to replace synthetic antibiotics since it would improve the absorption of nutrients without interfering with secretory IgA levels.Fil: Pinto, Silvina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Vignoni, Ernesto. Universidad Nacional de Luján. Departamento de Tecnología; ArgentinaFil: Esquivel, Cecilia María. Universidad Nacional de Luján. Departamento de Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Prosdocimo, Florencia Maria. Universidad Nacional de Luján. Departamento de Tecnología; ArgentinaFil: Mitarotonda, Romina. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Cerny, Natacha. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Barrios, Hebe Alicia. Universidad Nacional de Luján. Departamento de Ciencias Básicas; ArgentinaFil: De Franceschi, Mauricio. Universidad Nacional de Luján. Departamento de Tecnología; ArgentinaFil: de Marzi, Mauricio Cesar. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; Argentin

Interventions targeting glucocorticoid-Krüppel-like factor 15-branched-chain amino acid signaling improve disease phenotypes in spinal muscular atrophy mice

The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn−/−;SMN2 and Smn2B/− mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling

Matricellular protein CCN3 mitigates abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease

Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α

Glucocorticoid levels rise dramatically in late gestation to mature foetal organs in readiness for postnatal life. Immature heart function may compromise survival. Cardiomyocyte glucocorticoid receptor (GR) is required for the structural and functional maturation of the foetal heart in vivo, yet the molecular mechanisms are largely unknown. Here we asked if GR activation in foetal cardiomyocytes in vitro elicits similar maturational changes. We show that physiologically relevant glucocorticoid levels improve contractility of primary-mouse-foetal cardiomyocytes, promote Z-disc assembly and the appearance of mature myofibrils, and increase mitochondrial activity. Genes induced in vitro mimic those induced in vivo and include PGC-1α, a critical regulator of cardiac mitochondrial capacity. SiRNA-mediated abrogation of the glucocorticoid induction of PGC-1α in vitro abolished the effect of glucocorticoid on myofibril structure and mitochondrial oxygen consumption. Using RNA sequencing we identified a number of transcriptional regulators, including PGC-1α, induced as primary targets of GR in foetal cardiomyocytes. These data demonstrate that PGC-1α is a key mediator of glucocorticoid-induced maturation of foetal cardiomyocyte structure and identify other candidate transcriptional regulators that may play critical roles in the transition of the foetal to neonatal heart