AP-1 signaling modulates cardiac fibroblast stress responses.

Matrix remodeling outcomes largely dictate patient survival post myocardial infarction. Moreover, human-restricted noncoding regulatory elements have been shown to worsen fibrosis, but their mechanism of action remains elusive. Here, we demonstrate, using induced pluripotent stem cell-derived cardiac fibroblasts (iCFs), that inflammatory ligands abundant in the remodeling heart after infarction activate AP-1 transcription factor signaling pathways resulting in fibrotic responses. This observed signaling induces deposition of fibronectin matrix and is further capable of supporting immune cell adhesion; pathway inhibition blocks iCF matrix production and cell adhesion. Polymorphisms in the noncoding regulatory elements within the 9p21 locus (also referred to as ANRIL) redirect stress programs, and in iCFs, they transcriptionally silence the AP-1 inducible transcription factor GATA5. The presence of these polymorphisms modulate iCF matrix production and assembly and reduce cell-cell signaling. These data suggest that this signaling axis is a critical modulator of cardiac disease models and might be influenced by noncoding regulatory elements.

Retinoic acid released from self-assembling peptide activates cardiomyocyte proliferation and enhances repair of infarcted myocardium.

The limited cardiomyocyte proliferation is insufficient for repair of the myocardium. Therefore, activating cardiomyocyte proliferation might be a reasonable option for myocardial regeneration. Here, we investigated effect of retinoic acid (RA) on inducing adult cardiomyocyte proliferation and assessed efficacy of self-assembling peptide (SAP)-released RA in activating regeneration of the infarcted myocardium. Effect of RA on inducing cardiomyocyte proliferation was examined with the isolated cardiomyocytes. Expression of the cell cycle-associated genes and paracrine factors in the infarcted myocardium was examined at one week after treatment with SAP-carried RA. Cardiomyocyte proliferation, myocardial regeneration and improvement of cardiac function were assessed at four weeks after treatment. In the adult rat myocardium, expression of RA synthetase gene Raldh2 and RA concentration were decreased significantly. After treatment with RA, the proliferated cardiomyocytes were increased. The formulated SAP could sustainedly release RA. After treatment with SAP-carried RA, expression of the pro-proliferative genes in cell cycle and paracrine factors in the infarcted myocardium were up-regulated. Myocardial regeneration was enhanced, and cardiac function was improved significantly. These results demonstrate that RA can induce adult cardiomyocytes to proliferate effectively. The sustained release of RA with SAP is a promise strategy to enhance repair of the infarcted myocardium.

Single-cell genomics illustrates heterogeneous phenotypes of myocardial fibroblasts under ischemic insults.

Myocardial regenerative strategies are promising where the choice of ideal cell population is crucial for successful translational applications. Herein, we explored the regenerative/repair responses of infarct zone cardiac fibroblast(s) (CF) by unveiling their phenotype heterogeneity at single-cell resolution. CF were isolated from the infarct zone of Yucatan miniswine that suffered myocardial infarction, cultured under simulated ischemic and reperfusion, and grouped into control, ischemia, and ischemia/reperfusion. The single-cell RNA sequencing analysis revealed 19 unique cell clusters suggesting distinct subpopulations. The status of gene expression (log2 fold change (log2 FC) > 2 and log2 FC < -2) was used to define the characteristics of each cluster unveiling with diverse features, including the pro-survival/cardioprotective (Clusters 1, 3, 5, 9, and 18), vasculoprotective (Clusters 2 and 5), anti-inflammatory (Clusters 4 and 17), proliferative (Clusters 4 and 5), nonproliferative (Clusters 6, 8, 11, 16, 17, and 18), proinflammatory (Cluster 6), profibrotic/pathologic (Clusters 8 and 19), antihypertrophic (Clusters 8 and 10), extracellular matrix restorative (Clusters 9 and 12), angiogenic (Cluster 16), and normal (Clusters 7 and 15) phenotypes. Further understanding of these unique phenotypes of CF will provide significant translational opportunities for myocardial regeneration and cardiac management.

Proteomic analysis of neonatal mouse hearts shows PKA functions as a cardiomyocyte replication regulator.

The ability of the adult mammalian heart to regenerate can save the cardiac muscle from a loss of function caused by injury. Cardiomyocyte regeneration is a key aspect of research for the treatment of cardiovascular diseases. The mouse heart shows temporary regeneration in the first week after birth; thus, the newborn mouse heart is an ideal model to study heart muscle regeneration. In this study, proteomic analysis was used to investigate the differences in protein expression in the hearts of neonatal mice at days 1 (P1 group), 4 (P4 group), and 7 (P7 group). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed changes in several groups of proteins, including the protein kinase A (PKA) signaling pathway. Moreover, it was found that PKA inhibitors and agonists regulated cardiomyocyte replication in neonatal mouse hearts. These findings suggest that PKA may be a target for the regulation of the cardiomyocyte cell cycle.

Modified mRNA as a Treatment for Myocardial Infarction.

Myocardial infarction (MI) is a severe disease with high mortality worldwide. However, regenerative approaches remain limited and with poor efficacy. The major difficulty during MI is the substantial loss of cardiomyocytes (CMs) with limited capacity to regenerate. As a result, for decades, researchers have been engaged in developing useful therapies for myocardial regeneration. Gene therapy is an emerging approach for promoting myocardial regeneration. Modified mRNA (modRNA) is a highly potential delivery vector for gene transfer with its properties of efficiency, non-immunogenicity, transiency, and relative safety. Here, we discuss the optimization of modRNA-based therapy, including gene modification and delivery vectors of modRNA. Moreover, the effective of modRNA in animal MI treatment is also discussed. We conclude that modRNA-based therapy with appropriate therapeutical genes can potentially treat MI by directly promoting proliferation and differentiation, inhibiting apoptosis of CMs, as well as enhancing paracrine effects in terms of promoting angiogenesis and inhibiting fibrosis in heart milieu. Finally, we summarize the current challenges of modRNA-based cardiac treatment and look forward to the future direction of such treatment for MI. Further advanced clinical trials incorporating more MI patients should be conducted in order for modRNA therapy to become practical and feasible in real-world treatment.

Streptozotocin-Induced Type 1 and 2 Diabetes Mellitus Mouse Models Show Different Functional, Cellular and Molecular Patterns of Diabetic Cardiomyopathy.

The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.

Combining stem cells in myocardial infarction: The road to superior repair?

Myocardial infarction irreversibly destroys millions of cardiomyocytes in the ventricle, making it the leading cause of heart failure worldwide. Over the past two decades, many progenitor and stem cell types were proposed as the ideal candidate to regenerate the heart after injury. The potential of stem cell therapy has been investigated thoroughly in animal and human studies, aiming at cardiac repair by true tissue replacement, by immune modulation, or by the secretion of paracrine factors that stimulate endogenous repair processes. Despite some successful results in animal models, the outcome from clinical trials remains overall disappointing, largely due to the limited stem cell survival and retention after transplantation. Extensive interest was developed regarding the combinational use of stem cells and various priming strategies to improve the efficacy of regenerative cell therapy. In this review, we provide a critical discussion of the different stem cell types investigated in preclinical and clinical studies in the field of cardiac repair. Moreover, we give an update on the potential of stem cell combinations as well as preconditioning and explore the future promises of these novel regenerative strategies.

Left Ventricular Hemodynamics and Relationship With Myocardial Recovery and Optimization in Patients Supported on CF-LVAD Therapy.

BACKGROUND: Despite interest in left ventricular (LV) recovery, there is an absence of data on the relationship between intrinsic LV hemodynamics and both reverse remodeling on a continuous flow LV assist device (CF-LVAD) therapy. We hypothesized that the markers of intrinsic LV function would be associated with remodeling, optimization, and outcomes. METHODS AND RESULTS: Patients with continuous flow LVADs between 2015 and 2019 who underwent combined left and right heart catheterization ramp protocol at a single institution were enrolled. Patients were stratified by response to continuous flow LV assist device therapy: full responders, partial responders, or nonresponders per the Utah-Inova criteria. Hemodynamic data, including LV hemodynamics of peak LV dP/dt and tau (τ) were obtained at each phase. The 1-year heart failure hospitalization-free survival was the primary end point. Among 61 patients included in the current study 38 (62%) were classified as nonresponders, 14 as partial responders (23%), and 9 as full responders (15%). The baseline LV dP/dt and τ varied by response status (P ≤ .02) and generally correlated with reverse remodeling on linear regression. Biventricular filling pressures varied with τ and there was an interaction effect of speed on the relationship between τ and pulmonary capillary wedge pressure (P = .04). Last, τ was a prognostic marker and associated with 1-year HF hospital-free survival (odds ratio 1.04, 95% confidence interval 1.00-1.07, P = .02 per millisecond increase). CONCLUSIONS: Significant correlations between τ and LV dP/dt and reverse remodeling were noted, with τ serving as a prognostic marker. A higher LVAD speed was associated with a greater reliance on LVAD for unloading. Future work should focus on defining the optimal level of LVAD support in relation to LV recovery.

A 3D mathematical model of coupled stem cell-nutrient dynamics in myocardial regeneration therapy.

Stem cell therapy is a promising treatment for the regeneration of myocardial tissue injured by an ischemic event. Mathematical modeling of myocardial regeneration via stem cell therapy is a challenging task, since the mechanisms underlying the processes involved in the treatment are not yet fully understood. Many aspects must be accounted for, such as the spread of stem cells and nutrients, chemoattraction, cell proliferation, stages of cell maturation, differentiation, angiogenesis, stochastic effects, just to name a few. In this paper we propose a 3D mathematical model with a free boundary that aims to provide a qualitative description of some main aspects of the stem cell regenerative therapy in a simplified scenario. The paper mainly focuses on the description of the shrinking of the necrotic core during treatment. The stem cell and nutrients dynamics are described through coupled reaction-diffusion problems. Proliferation, chemoattraction, tissue regeneration and nutrient consumption are included in the model.

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

Regenerative cross talk between cardiac cells and macrophages.

Aside from the first week postnatal, murine heart regeneration is restricted and responses to damage follow classic fibrotic remodeling. Recent transcriptomic analyses have suggested that significant cross talk with the sterile immune response could maintain a more embryonic-like signaling network that promotes acute, transient responses. However, with age, this response-likely mediated by neonatal yolk sac macrophages-then transitions to classical macrophage-mediated, cardiac fibroblast (CF)-based remodeling of the extracellular matrix (ECM) after myocardial infarction (MI). The molecular mechanisms that govern the change with age and drive fibrosis via inflammation are poorly understood. Using multiple ribonucleic acid sequencing (RNA-Seq) datasets, we attempt to resolve the relative contributions of CFs and macrophages in the bulk-healing response of regenerative (postnatal day 1) and nonregenerative hearts (postnatal day 8+). We performed an analysis of bulk RNA-Seq datasets from myocardium and cardiac fibroblasts as well as a single-cell RNA-Seq dataset from cardiac macrophages. MI-specific pathway differences revealed that nonregenerative hearts generated more ECM and had larger matricellular responses correlating with inflammation, produced greater chemotactic gradients to recruit macrophages, and expressed receptors for danger-associated molecular patterns at higher levels than neonates. These changes could result in elevated stress-response pathways compared with neonates, converging at NF-κB and activator protein-1 (AP-1) signaling. Profibrotic gene programs, which greatly diverge on day 3 post MI, lay the foundation for chronic fibrosis, and thus postnatal hearts older than 7 days typically exhibit significantly less regeneration. Our analyses suggest that the macrophage ontogenetic shift in the heart postnatally could result in detrimental stress signaling that suppresses regeneration.NEW & NOTEWORTHY Immediately postnatal mammalian hearts are able to regenerate after infarction, but the cells, pathways, and molecules that regulate this behavior are unclear. By comparing RNA-Seq datasets from regenerative mouse hearts and older, nonregenerative hearts, we are able to identify biological processes that are hallmarks of regeneration. We find that sterile inflammatory processes are upregulated in nonregenerative hearts, initiating profibrotic gene programs 3 days after myocardial infarction that can cause myocardial disease.

Reviewing the role of cardiac exosomes in myocardial repair at a glance.

Exosome-based therapy is an emerging novel approach for myocardial infarction (MI) treatment. Exosomes are identified as extracellular vesicles that are produced within multivesicular bodies in the cells' cytosols and then are secreted from the cells. Exosomes are 30-100 nm in diameter that are released from viable cells and are different from other secreted vesicles such as apoptotic bodies and microvesicles in their origin and contents such as RNAs, proteins, and nucleic acid. The recent advances in exosome research have demonstrated the role of these bionanovesicles in the physiological, pathological, and molecular aspects of the heart. The results of in vitro and preclinical models have shown that exosomes from different cardiac cells can improve cardiac function following MI. For example, mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) containing exosomes can affect the proliferation, survival, and differentiation of cardiac fibroblasts and cardiomyocytes. Moreover, MSCs- and CPCs-derived exosomes can enhance the migration of endothelial cells. Exosome-based therapy approaches augment the cardiac function by multiple means, such as reducing fibrosis, stimulation of vascular angiogenesis, and proliferation of cardiomyocytes that result in replacing damaged heart tissue with newly generated functional myocytes. This review article aims to briefly discuss the recent advancements in the role of secreted exosomes in myocardial repair by focusing on cardiac cells-derived exosomes.

Long Non-coding RNA ECRAR Triggers Post-natal Myocardial Regeneration by Activating ERK1/2 Signaling.

Reactivating post-natal myocardial regeneration potential may be a feasible strategy to regenerate the injured adult heart. Long non-coding RNAs (lncRNAs) have been implicated in regulating cellular differentiation, but whether they can elicit a regenerative response in the post-natal heart remains unknown. In this study, by characterizing the lncRNA transcriptome in human hearts during the fetal-to-adult transition, we found that 3,092 lncRNAs were differentially expressed, and we further identified a novel upregulated fetal lncRNA that we called endogenous cardiac regeneration-associated regulator (ECRAR), which promoted DNA synthesis, mitosis, and cytokinesis in post-natal day 7 and adult rat cardiomyocytes (CMs). Overexpression of ECRAR markedly stimulated myocardial regeneration and induced recovery of cardiac function after myocardial infarction (MI). Knockdown of ECRAR inhibited post-natal day 1 CM proliferation and prevented post-MI recovery. ECRAR was transcriptionally upregulated by E2F transcription factor 1 (E2F1). In addition, ECRAR directly bound to and promoted the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), resulting in downstream targets of cyclin D1 and cyclin E1 activation, which, in turn, activated E2F1. The E2F1-ECRAR-ERK1/2 signaling formed a positive feedback loop to drive cell cycle progression, and, therefore, it promoted CM proliferation. These findings indicated that our newly discovered ECRAR may be a valuable therapeutic target for heart failure.

Stem Cell Therapy in Heart Disease: Limitations and Future Possibilities.

Heart diseases are one of the major causes of morbidity and mortality worldwide. Despite major advances in drug and interventional therapies, surgical procedures, and organ transplantation, further research into new therapeutic options is still necessary. Stem cell therapy has emerged as one option for the treatment of a variety of heart diseases. Although a large number of clinical trials have shown stem cell therapy to be a promising therapeutic approach, the results obtained from these clinical studies are inconsistent, and stem cell-based improvements of heart performance and cardiac remodeling were found to be quite limited. Since the precise mechanisms underlying the therapeutic actions of stem cells are still under debate, researchers have developed a variety of strategies to improve and boost the potency of stem cells in repair. In this Reviews, we summarize both the current therapeutic strategies using stem cells and future directions for enhancing stem cell potency.

Effective myocardial perfusion and concomitant haemodynamic status determine the clinical diversity of anomalous left coronary artery from the pulmonary artery.

BACKGROUND: Anomalous left coronary artery from the pulmonary artery is a rare congenital heart disease (CHD) with diverse clinical presentations despite the same anatomy. Factors determining this heterogeneous presentation are not well understood. METHOD AND RESULTS: We retrospectively investigated 14 patients (12 females) who underwent surgical repair of anomalous left coronary artery from the pulmonary artery. These patients were divided into three groups based upon the severity of initial presentation: (1) severe, life-threatening condition (n = 5), (2) mild-to-moderate distress (n = 6), and (3) asymptomatic (n = 3). All patients presented with left ventricular dilation and retrograde flow in left coronary artery by echocardiogram. Eight patients in (1) and (2) presented with severe left ventricular dysfunction. All but one showed abnormal ECG consistent with myocardial ischemia or infarction. Asymptomatic patients had preserved left ventricular systolic function despite ischemic findings on ECG. In 13 patients after surgical repair, all but one normalised left ventricular geometry and systolic function, suggesting nearly full myocardial recovery upon improvement of myocardial perfusion; 8 patients had residual echogenic papillary muscle with variable degree of mitral regurgitation. CONCLUSIONS: Evidence of myocardial ischemic injury was present in all patients with anomalous left coronary artery from the pulmonary artery regardless of their initial presentation. Retrograde flow in left coronary artery, implying collateral vessel development from right coronary artery to left coronary artery, was noted in all patients, yet only few patients had preserved systolic function at the time of diagnosis. The balance between effective myocardial perfusion and a deleterious fistulous flow provided by these collaterals and the simultaneous haemodynamic status are what determine the clinical diversity of anomalous left coronary artery from the pulmonary artery.

Cardiac Stem Cell-Loaded Delivery Systems: A New Challenge for Myocardial Tissue Regeneration.

Cardiovascular disease (CVD) remains the leading cause of death in Western countries. Post-myocardial infarction heart failure can be considered a degenerative disease where myocyte loss outweighs any regenerative potential. In this scenario, regenerative biology and tissue engineering can provide effective solutions to repair the infarcted failing heart. The main strategies involve the use of stem and progenitor cells to regenerate/repair lost and dysfunctional tissue, administrated as a suspension or encapsulated in specific delivery systems. Several studies demonstrated that effectiveness of direct injection of cardiac stem cells (CSCs) is limited in humans by the hostile cardiac microenvironment and poor cell engraftment; therefore, the use of injectable hydrogel or pre-formed patches have been strongly advocated to obtain a better integration between delivered stem cells and host myocardial tissue. Several approaches were used to refine these types of constructs, trying to obtain an optimized functional scaffold. Despite the promising features of these stem cells' delivery systems, few have reached the clinical practice. In this review, we summarize the advantages, and the novelty but also the current limitations of engineered patches and injectable hydrogels for tissue regenerative purposes, offering a perspective of how we believe tissue engineering should evolve to obtain the optimal delivery system applicable to the everyday clinical scenario.

Statins Stimulate New Myocyte Formation After Myocardial Infarction by Activating Growth and Differentiation of the Endogenous Cardiac Stem Cells.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert pleiotropic effects on cardiac cell biology which are not yet fully understood. Here we tested whether statin treatment affects resident endogenous cardiac stem/progenitor cell (CSC) activation in vitro and in vivo after myocardial infarction (MI). Statins (Rosuvastatin, Simvastatin and Pravastatin) significantly increased CSC expansion in vitro as measured by both BrdU incorporation and cell growth curve. Additionally, statins increased CSC clonal expansion and cardiosphere formation. The effects of statins on CSC growth and differentiation depended on Akt phosphorylation. Twenty-eight days after myocardial infarction by permanent coronary ligation in rats, the number of endogenous CSCs in the infarct border zone was significantly increased by Rosuvastatin-treatment as compared to untreated controls. Additionally, commitment of the activated CSCs into the myogenic lineage (c-kitpos/Gata4pos CSCs) was increased by Rosuvastatin administration. Accordingly, Rosuvastatin fostered new cardiomyocyte formation after MI. Finally, Rosuvastatin treatment reversed the cardiomyogenic defects of CSCs in c-kit haploinsufficient mice, increasing new cardiomyocyte formation by endogenous CSCs in these mice after myocardial infarction. In summary, statins, by sustaining Akt activation, foster CSC growth and differentiation in vitro and in vivo. The activation and differentiation of the endogenous CSC pool and consequent new myocyte formation by statins improve myocardial remodeling after coronary occlusion in rodents. Similar effects might contribute to the beneficial effects of statins on human cardiovascular diseases.

Mesenchymal stem cell and bone marrow mononuclear cell therapy for cardiomyopathy: From bench to bedside.

To date, stem cell-based therapies for cardiac diseases have not achieved any significant clinical accomplishment. Globally, numerous patients are currently treated with autologous stem cells. The safety and practicality of this technique have been well-examined, its disadvantages have been recognized, and many trials have been proposed. Inadequate description of the implemented cell types, a variety of cell-handling proficiencies, and concerning factors related to autologous stem cells have been known as the central elements restricting the approval of cell-based therapies. The idea that bone marrow (BM)-derived cells could be applied to regenerate and cure damage in various organs is the basis for bone marrow mononuclear cell (BMMNC) therapy for heart disease. Mesenchymal stem cells (MSCs) are a part of the BMMNCs; on one hand, they have the capability to differentiate into various tissues, and, on the other, their immunomodulatory effects have been considered and clinically confirmed in different experiments. In this review, we summarize the knowledge obtained by trials in which mesenchymal cell-based therapy has been practiced. Furthermore, we accentuate the developments in the purification and lineage specification of MSCs as well as BMMNCs that have influenced the progress of future stem cell-based therapies with special attention on cardiovascular disease.

Loss of long non-coding RNA CRRL promotes cardiomyocyte regeneration and improves cardiac repair by functioning as a competing endogenous RNA.

Long noncoding RNAs (lncRNAs) play critical roles in the development of myocardial hypertrophy and may stimulate endogenous myocardial regeneration to prevent heart failure after myocardial infarction (MI). However, whether lncRNAs are involved in regulating myocardial regeneration after MI remains unclear. The present study aimed to identify human-derived lncRNAs that are involved in endogenous cardiomyocyte (CM) regeneration. By analyzing publicly available RNA-seq data of human fetal and normal adult cardiac tissues, we identified a novel human-derived adult upregulated lncRNA designated cardiomyocyte regeneration-related lncRNA (CRRL). Bioinformatics analysis indicated that CRRL is involved in the negative regulation of CM proliferation. First, we observed that the loss of CRRL attenuates post-MI remodeling and preserves cardiac function in adult rats. Through loss-of-function approaches, we found that CRRL knockdown promotes neonatal rat CM proliferation both in vivo and in vitro. Furthermore, we demonstrated that CRRL acts as a competing endogenous RNA (ceRNA) by directly binding to miR-199a-3p and thereby increasing the expression of Hopx, a target gene of miR-199a-3p and a critical negative regulatory factor of CM proliferation. Thus, CRRL suppresses cardiomyocyte regeneration by directly binding to miR-199a-3p, indicating that loss of CRRL facilitates myocardial regeneration and may be a new potential therapeutic strategy for heart failure.

Shockwaves prevent from heart failure after acute myocardial ischaemia via RNA/protein complexes.

Shock wave treatment (SWT) was shown to induce regeneration of ischaemic myocardium via Toll-like receptor 3 (TLR3). The antimicrobial peptide LL37 gets released by mechanical stress and is known to form complexes with nucleic acids thus activating Toll-like receptors. We suggested that SWT in the acute setting prevents from the development of heart failure via RNA/protein release. Myocardial infarction in mice was induced followed by subsequent SWT. Heart function was assessed 4 weeks later via transthoracic echocardiography and pressure-volume measurements. Human umbilical vein endothelial cells (HUVECs) were treated with SWT in the presence of RNase and proteinase and analysed for proliferation, tube formation and LL37 expression. RNA release and uptake after SWT was evaluated. We found significantly improved cardiac function after SWT. SWT resulted in significantly higher numbers of capillaries and arterioles and less left ventricular fibrosis. Supernatants of treated cells activated TLR3 reporter cells. Analysis of the supernatant revealed increased RNA levels. The effect could not be abolished by pre-treatment of the supernatant with RNase, but only by a sequential digestion with proteinase and RNase hinting strongly towards the involvement of RNA/protein complexes. Indeed, LL37 expression as well as cellular RNA uptake were significantly increased after SWT. We show for the first time that SWT prevents from left ventricular remodelling and cardiac dysfunction via RNA/protein complex release and subsequent induction of angiogenesis. It might therefore develop a potent regenerative treatment alternative for ischaemic heart disease.