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Heart failure is the culmination of diverse cardiovascular diseases, including hypertension, ischaemic disease and atherosclerosis, valvular insufficiency, myocarditis, and contractile protein mutations, and is uniformly characterized by a progressive loss [...], Heart failure is characterized by a debilitating decline in cardiac function (1), and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy (2,3). MicroRNAs (miRNAs) are dysregulated in heart failure (4,5) but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes invitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.

Abstract: 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. [ABSTRACT FROM AUTHOR]

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the Western population, claiming 17000 deaths per year in the United States and affecting 25% of people older than 65 years of age. Contrary to traditional belief, CAVD is not a passive, degenerative disease but rather a dynamic disease, where initial cellular changes in the valve leaflets progress into fibrotic lesions that induce valve thickening and calcification. Advanced thickening and calcification impair valve function and lead to aortic stenosis (AS). Without intervention, progressive ventricular hypertrophy ensues, which ultimately results in heart failure and death. Currently, aortic valve replacement (AVR), surgical or transcatheter, is the only effective therapy to treat CAVD. However, these costly interventions are often delayed until the late stages of the disease. Nonetheless, 275000 are performed per year worldwide, and this is expected to triple by 2050. Given the current landscape, next-generation therapies for CAVD are needed to improve patient outcome and quality of life. Here, we first provide a background on the aortic valve (AV) and the pathobiology of CAVD as well as highlight current directions and future outlook on the development of functional 3D models of CAVD in vitro. We then consider an often-overlooked aspect contributing to CAVD: miRNA (mis)regulation. Therapeutics could potentially normalize miRNA levels in the early stages of the disease and may slow its progression or even reverse calcification. We close with a discussion of strategies that would enable the use of miRNA as a therapeutic for CAVD. This focuses on an overview of controlled delivery technologies for nucleic acid therapeutics to the valve or other target tissues. [ABSTRACT FROM AUTHOR]

Cardiac thyroid-hormone signaling is a critical determinant of cellular metabolism and function in health and disease. A local hypothyroid condition within the failing heart in rodents has been associated with the re-expression of the fetally expressed thyroid-hormone- inactivating enzyme deiodinase type III (Dio3). While this enzyme emerges as a common denominator in the development of heart failure, the mechanism underlying its regulation remains largely unclear. In the present study, we investigated the involvement of microRNAs (miRNAs) in the regulation of Dio3 mRNA expression in the remodeling left ventricle (LV) of the mouse heart following myocardial infarction (MI). In silico analysis indicated that of the miRNAs that are differentially expressed in the post-MI heart, miR- 214 has the highest potential to target Dio3 mRNA. In accordance, a luciferase reporter assay, including the full-length 3'UTR of mouse Dio3 mRNA, showed a 30% suppression of luciferase activity by miR-214. In the post-MI mouse heart, miR-214 and Dio3 protein were shown to be co-expressed in cardiomyocytes, while time-course analysis revealed that Dio3 mRNA expression precedes miR-214 expression in the post-MI LV. This suggests that a Dio3-induced decrease of T3 levels is involved in the induction of miR-214, which was supported by the finding that cardiac miR-214 expression is down regulated by T3 in mice. In vitro analysis of human DIO3 mRNA furthermore showed that miR-214 is able to suppress both mRNA and protein expression. Dio3 mRNA is a target of miR-214 and the Dio3-dependent stimulation of miR-214 expression in post-MI cardiomyocytes supports the involvement of a negative feedback mechanism regulating Dio3 expression. [ABSTRACT FROM AUTHOR]

Better understanding of the molecular mechanisms of remodeling has become a major objective of heart failure (HF) research to stop or reverse its progression. Left ventricular assist devices (LVADs) are being used in patients with HF, leading to partial reverse remodeling. In the present study, proteomics identified significant changes in α-1-antichymotrypsin (ACT) levels during LVAD support. Moreover, the potential role of ACT in reverse remodeling was studied in detail.Expression of ACT mRNA (quantitative-polymerase chain reaction) decreased significantly in post-LVAD myocardial tissue compared with pre-LVAD tissue (n=15; P<0.01). Immunohistochemistry revealed that ACT expression and localization changed during LVAD support. Circulating ACT levels were elevated in HF patients (n=18) as compared with healthy controls (n=6; P=0.001) and normalized by 6 months of LVAD support. Because increasing evidence implicates that microRNAs (miRs) are involved in myocardial disease processes, we also investigated whether ACT is post-transcriptionally regulated by miRs. Bioinformatics analysis pointed miR-137 as a potential regulator of ACT. The miR-137 expression is inversely correlated with ACT mRNA in myocardial tissue. Luciferase activity assays confirmed ACT as a direct target for miR-137, and in situ hybridization indicated that ACT and miR-137 were mainly localized in cardiomyocytes and stromal cells.High ACT plasma levels in HF normalized during LVAD support, which coincides with decreased ACT mRNA in heart tissue, whereas miR-137 levels increased. MiR-137 directly targeted ACT, thereby indicating that ACT and miR-137 play a role in the pathophysiology of HF and reverse remodeling during mechanical support. [ABSTRACT FROM AUTHOR]

Generation of upscaled quantities of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), for therapeutic or testing applications, is both expensive and time-consuming. Herein, a scalable bioprocess for hiPSC-CM expansion in stirred-tank bioreactors (STB) is developed. By combining the continuous activation of the Wnt pathway, through perfusion of CHIR99021, within a mild hypoxia environment, the expansion of hiPSC-CM as aggregates is maximized, reaching 4 billion of pure hiPSC-CM in 2L STB. In particular, the importance of i) controlling the dissolved oxygen at 10% O 2 to reduce reactive oxygen species production and upregulate genes involved in cell proliferation, resulting in higher expansion rates (tenfold) compared to normoxic conditions, and ii) maintaining constant power input per volume as a scale-up criteria is demonstrated. After expansion, hiPSC-CM further mature in culture, revealing more mature transcriptional signatures, higher sarcomere alignment and improved calcium handling. This new bioprocess opens the door to time- and cost-effective generation of hiPSC-CM., (© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml -1 collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml -1 collagen and 1 mg·ml -1 fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct., (Creative Commons Attribution license.)

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

The development of new cardioprotective approaches using in vivo models of ischemic heart disease remains challenging as differences in cardiac physiology, phenotype, and disease progression between humans and animals influence model validity and prognostic value. Furthermore, economical and ethical considerations have to be taken into account, especially when using large animal models with relevance for conducting preclinical studies. The development of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has opened new opportunities for in vitro studies on cardioprotective compounds. However, the immature cellular phenotype of iPSC-CMs remains a roadblock for disease modeling. Here, we show that metabolic maturation renders the susceptibility of iPSC-CMs to hypoxia further toward a clinically representative phenotype. iPSC-CMs cultured in a conventional medium did not show significant cell death after exposure to hypoxia. In contrast, metabolically matured (MM) iPSC-CMs showed inhibited mitochondrial respiration after exposure to hypoxia and increased cell death upon increased durations of hypoxia. Furthermore, we confirmed the applicability of MM iPSC-CMs for in vitro studies of hypoxic damage by validating the known cardioprotective effect of necroptosis inhibitor necrostatin-1. Our results provide important steps to improving and developing valid and predictive human in vitro models of ischemic heart disease., (© The Author(s) 2022. Published by Oxford University Press.)

Worldwide, over 26 million patients suffer from heart failure (HF). One strategy aspiring to prevent or even to reverse HF is based on the transplantation of cardiac tissue-engineered (cTE) constructs. These patient-specific constructs aim to closely resemble the native myocardium and, upon implantation on the diseased tissue, support and restore cardiac function, thereby preventing the development of HF. However, cTE constructs off-the-shelf availability in the clinical arena critically depends on the development of efficient preservation methodologies. Short- and long-term preservation of cTE constructs would enable transportation and direct availability. Herein, currently available methods, from normothermic- to hypothermic- to cryopreservation, for the preservation of cardiomyocytes, whole-heart, and regenerative materials are reviewed. A theoretical foundation and recommendations for future research on developing cTE construct specific preservation methods are provided. Current research suggests that vitrification can be a promising procedure to ensure long-term cryopreservation of cTE constructs, despite the need of high doses of cytotoxic cryoprotective agents. Instead, short-term cTE construct preservation can be achieved at normothermic or hypothermic temperatures by administration of protective additives. With further tuning of these promising methods, it is anticipated that cTE construct therapy can be brought one step closer to the patient., (© 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Aims: Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure., Methods and Results: We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10-11 and 7.7 × 10-4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10-8 and 1.4 × 10-3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene., Conclusion: This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Compromised cardiac function is a hallmark for heart failure, mostly appearing as decreased contractile capacity due to dysregulated calcium handling. Unfortunately, the underlying mechanism causing impaired calcium handling is still not fully understood. Previously the miR-132/212 family was identified as a regulator of cardiac function in the failing mouse heart, and pharmaceutically inhibition of miR-132 is beneficial for heart failure. In this study, we further investigated the molecular mechanisms of miR-132/212 in modulating cardiomyocyte contractility in the context of the pathological progression of heart failure. We found that upregulated miR-132/212 expressions in all examined hypertrophic heart failure mice models. The overexpression of miR-132/212 prolongs calcium decay in isolated neonatal rat cardiomyocytes, whereas cardiomyocytes isolated from miR-132/212 KO mice display enhanced contractility in comparison to wild type controls. In response to chronic pressure-overload, miR-132/212 KO mice exhibited a blunted deterioration of cardiac function. Using a combination of biochemical approaches and in vitro assays, we confirmed that miR-132/212 regulates SERCA2a by targeting the 3'-end untranslated region of SERCA2a. Additionally, we also confirmed PTEN as a direct target of miR-132/212 and potentially participates in the cardiac response to miR132/212. In end-stage heart failure patients, miR-132/212 is upregulated and correlates with reduced SERCA2a expression. The up-regulation of miR-132/212 in heart failure impairs cardiac contractile function by targeting SERCA2a, suggesting that pharmaceutical inhibition of miR-132/212 might be a promising therapeutic approach to promote cardiac function in heart failure patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lei, Wahlquist, el Azzouzi, Deddens, Kuster, van Mil, Rojas-Munoz, Huibers, Mercola, de Weger, Van der Velden, Xiao, Doevendans and Sluijter.)

Heart failure (HF) is a leading cause of death worldwide. The most common conditions that lead to HF are coronary artery disease, myocardial infarction, valve disorders, high blood pressure, and cardiomyopathy. Due to the limited regenerative capacity of the heart, the only curative therapy currently available is heart transplantation. Therefore, there is a great need for the development of novel regenerative strategies to repair the injured myocardium, replace damaged valves, and treat occluded coronary arteries. Recent advances in manufacturing technologies have resulted in the precise fabrication of 3D fiber scaffolds with high architectural control that can support and guide new tissue growth, opening exciting new avenues for repair of the human heart. This review discusses the recent advancements in the novel research field of fiber patterning manufacturing technologies for cardiac tissue engineering (cTE) and to what extent these technologies could meet the requirements of the highly organized and structured cardiac tissues. Additionally, future directions of these novel fiber patterning technologies, designs, and applicability to advance cTE are presented., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the past few years, the use of specific cell types derived from induced pluripotent stem cells (iPSCs) has developed into a powerful approach to investigate the cellular pathophysiology of numerous diseases. Despite advances in therapy, heart disease continues to be one of the leading causes of death in the developed world. A major difficulty in unravelling the underlying cellular processes of heart disease is the extremely limited availability of viable human cardiac cells reflecting the pathological phenotype of the disease at various stages. Thus, the development of methods for directed differentiation of iPSCs to cardiomyocytes (iPSC-CMs) has provided an intriguing option for the generation of patient-specific cardiac cells. In this review, a comprehensive overview of the currently published iPSC-CM models for hereditary heart disease is compiled and analysed. Besides the major findings of individual studies, detailed methodological information on iPSC generation, iPSC-CM differentiation, characterization, and maturation is included. Both, current advances in the field and challenges yet to overcome emphasize the potential of using patient-derived cell models to mimic genetic cardiac diseases.

It is estimated that a typical myocardial infarction results in the loss of approximately one billion functional cardiomyocytes, which are replaced by a non-contractile fibrous scar, eventually leading to heart failure. The currently available surgical, drug, and device-based therapies cannot reverse the loss of functional myocardium, which is the fundamental cause of the problem. As a result of this lack of an available medical solution, heart failure has evolved into a global epidemic. Therefore, the development of regenerative therapeutic strategies to halt the progression of ischemic heart disease to advanced heart failure has become one of the most urgent medical needs of this century. This review first addresses the extremely limited endogenous regenerative capacity of the mammalian heart, and the benefits and limitations of stem cell-based therapies for cardiac repair. Then it discusses the known roles of microRNAs after cardiac injury and the possibility of employing microRNAs to enhance cardiac regeneration.

Mammalian spermatogonial stem cells are a special type of adult stem cells because they can contribute to the next generation. Knockout studies have indicated a role for TRP53 and PTEN in insulating male germ cells from pluripotency, but the mechanism by which this is achieved is largely unknown. To get more insight in these processes, an RNAi experiment was performed on the mouse spermatogonial stem cell line GSDG1. Lipofectaminemediated transfection of siRNAs directed against Trp53 and Pten resulted in decreased expression levels as determined by quantitative RT-PCR and immunoblotting. The effects of knockdown were examined by determining the expression levels of genes that are involved in reprogramming and pluripotency of cells, specifically Nanog, Eras, c-Myc, Klf4, Oct4, and Sox2. Additionally, the effects of TRP53 or PTEN knockdown on Plzf and Ddx4 expression were measured, which are highly expressed in spermatogonial stem cells and differentiating male germ cells, respectively. The main finding of this study is that knockdown of Trp53 and Pten independently resulted in significantly higher expression levels of the pluripotency-associated gene Nanog, and we hypothesize that TRP53 and PTEN mediated repression is important for the insulation of male germ cells from pluripotency.

Objective: To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell transplantation. Cardiomyocyte progenitor cells (CMPCs) are a promising cell population, easily expanded and efficiently differentiated into beating cardiomyocytes. Recently, several studies have demonstrated that microRNAs (miRNAs) are important for stem cell maintenance and differentiation via translational repression. We hypothesize that miRNAs are also involved in proliferation/differentiation of the human CMPCs in vitro., Methods and Results: Human fetal CMPCs were isolated, cultured, and efficiently differentiated into beating cardiomyocytes. miRNA expression profiling demonstrated that muscle-specific miR-1 and miR-499 were highly upregulated in differentiated cells. Transient transfection of miR-1 and -499 in CMPC reduced proliferation rate by 25% and 15%, respectively, and enhanced differentiation into cardiomyocytes in human CMPCs and embryonic stem cells, likely via the repression of histone deacetylase 4 or Sox6. Histone deacetylase 4 and Sox6 protein levels were reduced, and small interference RNA (siRNA)-mediated knockdown of Sox6 strongly induced myogenic differentiation., Conclusions: miRNAs regulate the proliferation of human CMPC and their differentiation into cardiomyocytes. By modulating miR-1 and -499 expression levels, human CMPC function can be altered and differentiation directed, thereby enhancing cardiomyogenic differentiation.

Small RNAs have shown to be ubiquitous, useful, post-transcriptional gene silencers in a diverse array of living organisms. As a result of homologous sequence interactions, these small RNAs repress gene expression. Through a process called RNA interference (RNAi), double strand RNA molecules are processed by an enzyme called Dicer, which cleaves RNA duplexes into 21-23 base pair oligomers. Depending on their end-point functions, these oligomers are named differently, the two most common being small interfering RNAs (siRNAs) and microRNAs (miRNAs). These small RNAs are the effector molecules for inducing RNAi, leading to post-transcriptional gene silencing by guiding the RNAi-induced silencing complex (RISC) to the target mRNA. By exploiting these small RNAs, it is possible to regulate the expression of genes related to human disease. The knockdown of such target genes can be achieved by transfecting cells with synthetically engineered small RNAs or small RNA expressing vectors. Within recent years, studies have also shown the important role of miRNAs in different diseases. By using several chemically engineered anti-miRNA oligonucleotides, disease related miRNAs can be specifically and effectively silenced. Since RNAi has developed into an everyday method for in vitro knockdown of any target gene of interest, the next step is to further explore its potential in vivo and the unique opportunities it holds for the development of novel therapeutic strategies. This review explores the various applications of small RNA technology in in vivo studies, and its potential for silencing genes associated with various human diseases. We describe the latest development in small RNA technology for both gene knockdown, and the inhibition of translational silencing in animal studies. A variety of small RNA formulations and modifications will be reviewed for their improvement on stability and half-life, their safety and off-target effects, and their efficiency and specificity of gene silencing.