Loss of miR-132/212 Has No Long-Term Beneficial Effect on Cardiac Function After Permanent Coronary Occlusion in Mice

Background: Myocardial infarction (MI) is caused by occlusion of the coronary artery and induces ischemia in the myocardium and eventually a massive loss in cardiomyocytes. Studies have shown many factors or treatments that can affect the healing and remodeling of the heart upon infarction, leading to better cardiac performance and clinical outcome. Previously, miR-132/212 has been shown to play an important role in arteriogenesis in a mouse model of hindlimb ischemia and in the regulation of cardiac contractility in hypertrophic cardiomyopathy in mice. In this study, we explored the role of miR-132/212 during ischemia in a murine MI model. Methods and Results: miR-132/212 knockout mice show enhanced cardiac contractile function at baseline compared to wild-type controls, as assessed by echocardiography. One day after induction of MI by permanent occlusion, miR-132/212 knockout mice display similar levels of cardiac damage as wild-type controls, as demonstrated by infarction size quantification and LDH release, although a trend toward more cardiomyocyte cell death was observed in the knockout mice as shown by TUNEL staining. Four weeks after MI, miR-132/212 knockout mice show no differences in terms of cardiac function, expression of cardiac stress markers, and fibrotic remodeling, although vascularization was reduced. In line with these in vivo observation, overexpression of miR-132 or miR-212 in neonatal rat cardiomyocyte suppress hypoxia induced cardiomyocyte cell death. Conclusion: Although we previously observed a role in collateral formation and myocardial contractility, the absence of miR-132/212 did not affect the overall myocardial performance upon a permanent occlusion of the coronary artery. This suggests an interplay of different roles of this miR-132/212 before and during MI, including an inhibitory effect on cell death and angiogenesis, and a positive effect on cardiac contractility and autophagic response. Thus, spatial or tissue specific manipulation of this microRNA family may be essential to fully understand the roles and to develop interventions to reduce infarct size

Setting the stones to restore and monitor European flat oyster reefs in the German North Sea

Ecological restoration includes specific technical phases over the course of an ecosystem recovery process. In the marine environment and for oyster reef restoration, the installation and implementation of pilot reefs close the gap between feasibility studies with small-scale experiments and designated upscaling for marine conservation measures. Against this background, this study presents the design, planning and installation of the first pilot oyster reef in offshore sublittoral regions of the North Sea. The work was conducted as part of marine protected area management in the Natura 2000 site Borkum Reef Ground in the German Bight, in the area of historical offshore oyster grounds. It includes logistical considerations, material selection, methodology for reef base construction and deployment of European flat oysters Ostrea edulis as spat-on-shell, young and adult single seed oysters, and spat-on-reef, as well as the development of an efficient monitoring approach for reef-associated biodiversity. Native Oyster Restoration Alliance monitoring methodologies, such as underwater visual census and seabed images were selected, tested and successfully adapted for the pilot oyster reef and study site. The evaluation and optimization of offshore sublittoral oyster reef monitoring are presented here, and biodiversity metrics are put into perspective with data from recent and historical studies. Results show a few mobile fauna species (e.g., fish and decapods) as first colonizers after reef construction. One year later, biodiversity increased due to a larger number of invertebrate and fish species. However, the pilot oyster reef community still represents an early recolonization stage, with lower biodiversity than historical records. This study presents a proof of concept for the design, planning and construction of an offshore oyster reef and indicates stages in the recovery process. Strategies to optimize and to complement reef-monitoring in challenging environments are discussed, emphasizing additional molecular and functional analyses for future assessments

Cardiac tissue engineering using tissue printing technology and human cardiac progenitor cells

Tissue engineering is emerging as a potential therapeutic approach to overcome limitations of cell therapy, like cell retention and survival, as well as to mechanically support the ventricular wall and thereby prevent dilation. Tissue printing technology (TP) offers the possibility to deliver, in a defined and organized manner, scaffolding materials and living cells. The aim of our study was to evaluate the combination of TP, human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) and biomaterials to obtain a construct with cardiogenic potential for in vitro use or in vivo application. With this approach, we were able to generate an in vitro tissue with homogenous distribution of cells in the scaffold. Cell viability was determined after printing and showed that 92% and 89% of cells were viable at I and 7 days of culturing, respectively. Moreover, we demonstrated that printed hCMPCs retained their commitment for the cardiac lineage. In particular, we showed that 3D culture enhanced gene expression of the early cardiac transcription factors Nkx2.5, Gata-4 and Mef-2c as well as the sarcomeric protein TroponinT. Printed cells were also able to migrate from the alginate matrix and colonize a matrigel layer, thereby forming tubular-like structures. This indicated that printing can be used for defined cell delivery, while retaining functional properties. (C) 2011 Elsevier Ltd. All rights reserved

MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

To understand and assess the roles of miRNAs, visualization of the expression patterns of specific miRNAs is needed at the cellular level in a wide variety of different tissue types. Although miRNA in situ hybridization techniques have been greatly improved in recent years, they remain difficult to routinely perform due to the complexity of the procedure. In addition, as it is crucial to define which tissues or cells are expressing a particular miRNA in order to elucidate the biological function of the miRNA, incorporation of additional stainings for different cellular markers is necessary. Here, we describe a robust and flexible multicolor miRNA in situ hybridization (MMISH) technique for paraffin embedded sections. We show that the miRNA in situ protocol is sensitive and highly specific and can successfully be combined with both immunohistochemical and immunofluorescent stainings

MMISH : Multicolor microRNA in situ hybridization for paraffin embedded samples

To understand and assess the roles of miRNAs, visualization of the expression patterns of specific miRNAs is needed at the cellular level in a wide variety of different tissue types. Although miRNA in situ hybridization techniques have been greatly improved in recent years, they remain difficult to routinely perform due to the complexity of the procedure. In addition, as it is crucial to define which tissues or cells are expressing a particular miRNA in order to elucidate the biological function of the miRNA, incorporation of additional stainings for different cellular markers is necessary. Here, we describe a robust and flexible multicolor miRNA in situ hybridization (MMISH) technique for paraffin embedded sections. We show that the miRNA in situ protocol is sensitive and highly specific and can successfully be combined with both immunohistochemical and immunofluorescent stainings

Melt Electrowriting Allows Tailored Microstructural and Mechanical Design of Scaffolds to Advance Functional Human Myocardial Tissue Formation

Engineering native-like myocardial muscle, recapitulating its fibrillar organization and mechanical behavior is still a challenge. This study reports the rational design and fabrication of ultrastretchable microfiber scaffolds with controlled hexagonal microstructures via melt electrowriting (MEW). The resulting structures exhibit large biaxial deformations, up to 40% strain, and an unprecedented compliance, delivering up to 40 times more elastic energy than rudimentary MEW fiber scaffolds. Importantly, when human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are encapsulated in a collagen-based hydrogel and seeded on these microstructured and mechanically tailored fiber scaffolds, they show an increase in beating rate (1.5-fold), enhanced cell alignment, sarcomere content and organization as well as an increase in cardiac maturation-related marker expression (Cx43 1.8-fold, cardiac Actin 1.5-fold, SERCA2a 2.5-fold, KCNJ2 1.5-fold, and PPARGC1a 3.6-fold), indicative of enhanced iPSC-CM maturation, as compared to rudimentary fiber scaffolds. By combining these novel fiber scaffolds with clinically relevant human iPSC-CMs, a heart patch that allows further maturation of contractile myocytes for cardiac tissue engineering is generated. Moreover, the designed scaffold allows successful shape recovery after epicardial delivery on a beating porcine heart, without negative effects on the engineered construct and iPSC-CM viability

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA-155 (miR-155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre-miR-155, anti-miR-155 or control-miR (ctrl-miR), followed by scratch- and transwell- assays. These functional assays displayed that miR-155 over-expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR-155 transfected cells was collected and zymography analysis showed a significant decrease in MMP-2 and MMP-9 activities. The predicted 3′-UTR of MMP-16, an activator of MMP-2 and -9, was cloned into the pMIR-REPORT vector and luciferase assays were performed. Introduction of miR-155 significantly reduced luciferase activity which could be abolished by cotransfection with anti-miR-155 or target site mutagenesis. By using MMP-16 siRNA to reduce MMP-16 levels or by using an MMP-16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP-16, miR-155 efficiently inhibits cell migration via a reduction in MMP-2 and -9 activities. Our study shows that miR-155 might be used to improve local retention of hCMPCs after intramyocardial delivery

Melt electrowriting allows tailored microstructural and mechanical design of scaffolds to advance functional human myocardial tissue formation

\u3cp\u3eEngineering native-like myocardial muscle, recapitulating its fibrillar organization and mechanical behavior is still a challenge. This study reports the rational design and fabrication of ultrastretchable microfiber scaffolds with controlled hexagonal microstructures via melt electrowriting (MEW). The resulting structures exhibit large biaxial deformations, up to 40% strain, and an unprecedented compliance, delivering up to 40 times more elastic energy than rudimentary MEW fiber scaffolds. Importantly, when human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are encapsulated in a collagen-based hydrogel and seeded on these microstructured and mechanically tailored fiber scaffolds, they show an increase in beating rate (1.5-fold), enhanced cell alignment, sarcomere content and organization as well as an increase in cardiac maturation-related marker expression (Cx43 1.8-fold, cardiac Actin 1.5-fold, SERCA2a 2.5-fold, KCNJ2 1.5-fold, and PPARGC1a 3.6-fold), indicative of enhanced iPSC-CM maturation, as compared to rudimentary fiber scaffolds. By combining these novel fiber scaffolds with clinically relevant human iPSC-CMs, a heart patch that allows further maturation of contractile myocytes for cardiac tissue engineering is generated. Moreover, the designed scaffold allows successful shape recovery after epicardial delivery on a beating porcine heart, without negative effects on the engineered construct and iPSC-CM viability.\u3c/p\u3