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

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

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

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

Human cardiomyocyte progenitor cell-derived cardiomyocytes display a maturated electrical phenotype

Cardiomyocyte progenitor cells (CMPCs) can be isolated from the human heart and differentiated into cardiomyocytes in vitro. A comprehensive assessment of their electrical phenotype upon differentiation is essential to predict potential future applications of this cell source. CMPCs isolated from human fetal heart were differentiated in vitro and examined using immunohistochemistry, Western blotting, RT-PCR, voltage clamp and current clamp techniques. Differentiated cultures presented up to 95% alpha-actinin positive cardiomyocytes. Adherens junction and desmosomal proteins beta-catenin, N-cadherin, desmin and plakophilin2 were upregulated. Expression levels of cardiac connexins were not affected by differentiation, however Cx43 phosphorylation was increased upon differentiation, accompanied by translocation of connexins to the cell border. RT-PCR analysis demonstrated upregulation of all major cardiac ion channel constituents during differentiation. Patch clamp experiments showed that cardiomyocytes had a stable resting membrane potential of -73.4+/-1.8 mV. Infusion of 1 mM BaCl(2) resulted in depolarization to -59.9+/-2.8 mV, indicating I(K1) channel activity. Subsequent voltage clamp experiments confirmed presence of near mature I(Na), I(Ca,L) and I(K1) current densities. Infusion of the I(Kr) blocker Almokalant caused prolongation of the action potential by 40%. Differentiated monolayers were not spontaneously contracting in the absence of serum, but responded to field stimulation, displaying adult ventricular-like action potentials. Human fetal CMPC-derived cardiomyocytes have a homogenous and rather mature electrical phenotype that benefits to in vitro physiology and pharmacology. In the context of cardiac repair, their properties may translate into a reduced pro-arrhythmic risk and enhanced electrical integration upon transplantatio

Exosomes from Cardiomyocyte Progenitor Cells and Mesenchymal Stem Cells Stimulate Angiogenesis Via EMMPRIN

To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI