MicroRNA-216a is essential for cardiac angiogenesis.

While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.

Intercellular transfer of miR-200c-3p impairs the angiogenic capacity of cardiac endothelial cells.

As mediators of intercellular communication, extracellular vesicles containing molecular cargo, such as microRNAs, are secreted by cells and taken up by recipient cells to influence their cellular phenotype and function. Here we report that cardiac stress-induced differential microRNA content, with miR-200c-3p being one of the most enriched, in cardiomyocyte-derived extracellular vesicles mediates functional cross-talk with endothelial cells. Silencing of miR-200c-3p in mice subjected to chronic increased cardiac pressure overload resulted in attenuated hypertrophy, smaller fibrotic areas, higher capillary density, and preserved cardiac ejection fraction. We were able to maximally rescue microvascular and cardiac function with very low doses of antagomir, which specifically silences miR-200c-3p expression in non-myocyte cells. Our results reveal vesicle transfer of miR-200c-3p from cardiomyocytes to cardiac endothelial cells, underlining the importance of cardiac intercellular communication in the pathophysiology of heart failure.

The adult heart requires baseline expression of the transcription factor Hand2 to withstand right ventricular pressure overload.

AIMS: Research on the pathophysiology of right ventricular (RV) failure has, in spite of the associated high mortality and morbidity, lagged behind compared to the left ventricle (LV). Previous work from our lab revealed that the embryonic basic helix-loop-helix transcription factor heart and neural crest derivatives expressed-2 (Hand2) is re-expressed in the adult heart and activates a 'foetal gene programme' contributing to pathological cardiac remodelling under conditions of LV pressure overload. As such, ablation of cardiac expression of Hand2 conferred protection to cardiac stress and abrogated the maladaptive effects that were observed upon increased expression levels. In this study, we aimed to understand the contribution of Hand2 to RV remodelling in response to pressure overload induced by pulmonary artery banding (PAB). METHODS AND RESULTS: In this study, Hand2F/F and MCM- Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic- and MRI-derived haemodynamic parameters as well as molecular remodelling were assessed for all experimental groups and compared to sham-operated controls. Six weeks after PAB, levels of Hand2 expression increased in the control-banded animals but, as expected, remained absent in the knockout hearts. Despite the dramatic differences in Hand2 expression, pressure overload resulted in impaired cardiac function independently of the genotype. In fact, Hand2 depletion seems to sensitize the RV to pressure overload as these mice develop more hypertrophy and more severe cardiac dysfunction. Higher expression levels of HAND2 were also observed in RV samples of human hearts from patients with pulmonary hypertension. In turn, the LV of RV pressure-overloaded hearts was also dramatically affected as reflected by changes in shape, decreased LV mass, and impaired cardiac function. RNA-sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV. CONCLUSION: Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressu re overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodelling of the RV in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure.

A microRNA program regulates the balance between cardiomyocyte hyperplasia and hypertrophy and stimulates cardiac regeneration.

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b~25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b~25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b~25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b~25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b~25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart.

Comparison of different chemically modified inhibitors of miR-199b in vivo.

MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3' to the 5' end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5' end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.

Molecular signature of progenitor cells isolated from young and adult human hearts.

The loss of endogenous cardiac regenerative capacity within the first week of postnatal life has intensified clinical trials to induce cardiac regeneration in the adult mammalian heart using different progenitor cell types. We hypothesized that donor age-related phenotypic and functional characteristics of cardiac progenitor cells (CPC) account for mixed results of cell-based cardiac repair. We compared expression profiles and cell turnover rates of human heart-derived c-kitpos progenitors (c-kitpos CPC) and cardiosphere-derived cells (CDC) from young and adult donor origin and studied their in vitro angiogenic and cardiac differentiation potential, which can be relevant for cardiac repair. We report that 3-dimensional CDC expansion recapitulates a conducive environment for growth factor and cytokine release from adult donor cells (aCDC) that optimally supports vascular tube formation and vessel sprouting. Transdifferentiation capacity of c-kitpos CPCs and CDCs towards cardiomyocyte-like cells was modest, however, most notable in young c-kitpos cells and adult CDCs. Progenitors isolated with different methods thus show cell- and donor-specific characteristics that may account for variable contributions in functional myocardial recovery.

Non-coding RNAs in cardiac hypertrophy.

Heart failure is one of the largest contributors to disease burden and healthcare outflow in the Western world. Despite significant progress in the treatment of heart failure, disease prognosis remains very poor, with the only curative therapy still being heart transplantation. To counteract the current situation, efforts have been made to better understand the underlying molecular pathways in the progression of cardiac disease towards heart failure, and to link the disease to novel therapeutic targets such as non-coding RNAs. The non-coding part of the genome has gained prominence over the last couple of decades, opening a completely new research field and establishing different non-coding RNAs species as fundamental regulators of cellular functions. Not surprisingly, their dysregulation is increasingly being linked to pathology, including to cardiac disease. Pre-clinically, non-coding RNAs have been shown to be of great value as therapeutic targets in pathological cardiac remodelling and also as diagnostic/prognostic biomarkers for heart failure. Therefore, it is to be expected that non-coding RNA-based therapeutic strategies will reach the bedside in the future and provide new and more efficient treatments for heart failure. Here, we review recent discoveries linking the function and molecular interactions of non-coding RNAs with the pathophysiology of cardiac hypertrophy and heart failure.

miR-199b-5p is a regulator of left ventricular remodeling following myocardial infarction.

Myocardial infarction (MI), the globally leading cause of heart failure, morbidity and mortality, involves post-MI ventricular remodeling, a complex process including acute injury healing, scar formation and global changes in the surviving myocardium. The molecular mechanisms involved in adverse post-infarct left ventricular remodeling still remain poorly defined. Recently, microRNAs have been implicated in the development and progression of various cardiac diseases as crucial regulators of gene expression. We previously demonstrated that in a murine model of pressure overload, a model of heart failure secondary to aortic stenosis or chronic high blood pressure, elevated myocardial expression of miR-199b-5p is sufficient to activate calcineurin/NFAT signaling, leading to exaggerated cardiac pathological remodeling and dysfunction. Given the differences in left ventricular remodeling secondary to post-infarct healing and pressure overload, we evaluated miR-199b function in post-MI remodeling. We confirmed that the expression of miR-199b is elevated in the post-infarcted heart. Transgenic animals with cardiomyocyte-restricted overexpression of miR-199b-5p displayed exaggerated pathological remodeling after MI, reflected by severe systolic and diastolic dysfunction and fibrosis deposition. Conversely, therapeutic silencing of miR-199b-5p in MI-induced cardiac remodeling by using an antagomir to specifically inhibit endogenous miR-199b-5p in vivo, resulted in efficient suppression of cardiac miR-199b-5p expression and attenuated cardiac dysfunction and dilation following MI. Mechanistically, miR-199b-5p influenced the expression of three predicted target genes in post-infarcted hearts, dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a), the notch1 receptor and its ligand jagged1. In conclusion, here we provide evidence supporting that stress-induced miR-199b-5p participates in post-infarct remodeling by simultaneous regulation of distinct target genes.

Growth properties of cardiac stem cells are a novel biomarker of patients' outcome after coronary bypass surgery.

BACKGROUND: The efficacy of bypass surgery in patients with ischemic cardiomyopathy is not easily predictable; preoperative clinical conditions may be similar, but the outcome may differ significantly. We hypothesized that the growth reserve of cardiac stem cells (CSCs) and circulating cytokines promoting CSC activation are critical determinants of ventricular remodeling in this patient population. METHODS AND RESULTS: To document the growth kinetics of CSCs, population-doubling time, telomere length, telomerase activity, and insulin-like growth factor-1 receptor expression were measured in CSCs isolated from 38 patients undergoing bypass surgery. Additionally, the blood levels of insulin-like growth factor-1, hepatocyte growth factor, and vascular endothelial growth factor were evaluated. The variables of CSC growth were expressed as a function of the changes in wall thickness, chamber diameter and volume, ventricular mass-to-chamber volume ratio, and ejection fraction, before and 12 months after surgery. A high correlation was found between indices of CSC function and cardiac anatomy. Negative ventricular remodeling was not observed if CSCs retained a significant growth reserve. The high concentration of insulin-like growth factor-1 systemically pointed to the insulin-like growth factor-1-insulin-like growth factor-1 receptor system as a major player in the adaptive response of the myocardium. hepatocyte growth factor, a mediator of CSC migration, was also high in these patients preoperatively, as was vascular endothelial growth factor, possibly reflecting the vascular growth needed before bypass surgery. Conversely, a decline in CSC growth was coupled with wall thinning, chamber dilation, and depressed ejection fraction. CONCLUSIONS: The telomere-telomerase axis, population-doubling time, and insulin-like growth factor-1 receptor expression in CSCs, together with a high circulating level of insulin-like growth factor-1, represent a novel biomarker able to predict the evolution of ischemic cardiomyopathy following revascularization.