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To what extent cardiac renewal occurs in adulthood and can be stimulated by therapeutic interventions is still a matter of investigation. In particular, over the last few years, significant effort has been made into awakening the endogenous regenerative potential of the adult mammalian heart after injury using different strategies. However, these studies are significantly hindered by the lack of tools to properly assess cardiomyocyte renewal over time. We have developed a genetic strategy, which we named CycleTrack, allowing a cumulative and accurate estimate of cardiomyocyte divisions in vivo. This method is based on the expression, in cardiomyocytes, of the Cre recombinase under the control of a 312-bp fragment of the Cyclin B2 promoter, which is specifically sensitive to cell proliferation. Replication-activated Cre acts on floxed GFP that is either present in the genome of transgenic mice or is exogenously delivered using Adeno-Associated Viral (AAV) vectors. As a result, cardiomyocytes traversing G2/M become irreversibly labeled. Using CycleTrack, we could monitor cardiomyocyte turnover in several physiological and pathological conditions. These included the measurement of the rate of proliferation of cardiomyocytes after apical resection in neonatal mice, the pro-regenerative effect of AAV9-mediated delivery of miRNA-199a and miRNA-590 after myocardial infarction in adult mice and the effect of pregnancy on myocardial hyperplasia. We also delivered the CycleTrack vectors into pig hearts to visualize cardiomyocyte turnover after myocardial infarction. Considering the large availability of Cre reporter mouse lines and the efficacy of AAVs to transfer genes into cardiomyocytes, we propose CycleTrack as a robust and straightforward tool to trace mitotic events for cardiac regeneration studies in small and large animal models.

Despite the development of new pharmacological and device-based therapies, no approaches currently exist to compensate for the irreversible loss of cardiomyocytes occurring after myocardial infarction. In a search for novel treatments that induce cardiomyocyte proliferation, we performed a high-content, fluorescence-microscopy-based, high-throughput screening in neonatal rat cardiomyocytes, using a library of FDA/EMA-approved drug library compounds. The screening, which was based on EdU incorporation as an S-phase marker, identified harmine as the most powerful drug enhancing proliferation in combination with miR-199a-3p and miR-590-3p, the most effective microRNAs that the laboratory had previously identified. Harmine treatment of CD1 mice showed an improved cardiac outcome upon harmine treatment compared to controls. Harmine is a potent inhibitor of the Dyrk1 family of protein kinases, with high specificity for the Dyrk1a isoform. Dyrk1a downregulation experiments in neonatal rat ventricular cardiomyocytes showed increased proliferation, consistent with a negative role of this kinase. The simultaneous knockdown of YAP1 blunted the effect of Dyrk1a downregulation. Of note, the YAP total protein levels were unchanged upon either Dyrk1a silencing or overexpression, but there was a marked modification of YAP subcellular localisation, being YAP1 nuclear upon knockdown and mostly cytoplasmic upon overexpression. Dyrk1a downregulation and harmine treatment activated YAP-controlled gene expression. In the course of these experiments, we also noticed a reciprocal regulation of YAP onto DYRK1a, as YAP downregulation markedly decreased Dyrk1a protein levels. This finding was reinforced by the observations that, in cardiomyocytes, the YAP-TEAD inhibitor verteporfin also decreased Dyrk1a protein levels. Of note, this downregulation was not rescued by the proteasome inhibitor MG132, thus indicating that the level of regulation is most likely transcriptional. These results support the role of Dyrk1a as an anti-proliferative factor in cardiomyocytes and highlight a novel feedback connection between Dyrk1a and YAP.

Rationale: In the microenvironment of atherosclerotic lesions, vascular smooth muscle cells (vSMCs) switch to a dedifferentiated state but the underlying molecular mechanisms driving this switch are not fully understood. Long noncoding RNAs (lncRNAs) are dysregulated during vascular pathology, but relatively little is known about their involvement in controlling vSMCs function. Cardiac mesoderm enhancer-associated noncoding RNA (CARMN) is a lncRNA located immediately upstream of the microRNAs-143 and -145 (miR-143 and miR-145 ), both involved in vSMCs function. Objective: We investigated the role of the lncRNA CARMN, independent from miR-143 and miR-145, as a potential regulator of vSMC phenotypes in vitro and the consequences of its loss during the development of atherosclerosis in vivo. We hypothesized that loss of CARMN is a primary event controlling the functional switch towards proatherogenic vSMC phenotype and accelerates the development of the plaques in vivo. Method and Results: Expression of CARMN lncRNA was silenced using locked nucleic acids antisense oligonucleotides (GapmeRs) in human coronary arterial smooth muscle cells, revealing that GapmeR-mediated loss of CARMN negatively affects miR-143 and miR-145 microRNA expression. RNA sequencing of CARMN-depleted human coronary arterial smooth muscle cells revealed large transcriptomic changes, associated with vSMC proliferation, migration, inflammation, lipid metabolism, and dedifferentiation. The use of miR-143 and miR-145 mimics revealed that CARMN regulates human coronary arterial smooth muscle cell proliferation in a microRNA-independent manner. In humans and mice, CARMN and associated microRNAs were downregulated in advanced versus early atherosclerotic lesions. Using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) knockout approach, we explored the implications of CARMN depletion during atherosclerosis in vivo. Consistent with in vitro results, the knockout of CARMN impaired the expression of miR-143 and miR-145 under homeostatic conditions. Importantly, when atherosclerosis was induced in these mice, CARMN knockout increased the volume, size, proinflammatory Lgals3 (galectin 3)-expressing cells content, and altered plaque composition, yielding an advanced phenotype. Conclusions: We identified the early loss of CARMN lncRNA as critical event which primes vSMCs towards a proatherogenic phenotype in vitro and accelerates the development of atherosclerosis in vivo.

Rationale: CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)–based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. Objective: To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. Methods and Results: We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6 , Sav1 , and Tbx20 , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1 , which increased the editing efficiency. Conclusions: Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo.

Rationale: Recent evidence indicates that a few human microRNAs (miRNAs), in particular hsa-miR-199a-3p and hsa-miR-590-3p, stimulate proliferation of cardiomyocytes and, once expressed in the mouse heart using viral vectors, induce cardiac regeneration after myocardial infarction. Viral vectors, however, are not devoid of safety issues and, more notably, drive expression of the encoded miRNAs for indefinite periods of time, which might not be desirable in light of human therapeutic application. Objective: As an alternative to the use of viral vectors, we wanted to assess the efficacy of synthetic miRNA mimics in inducing myocardial repair after single intracardiac injection using synthetic lipid formulations. Methods and Results: We comparatively analyzed the efficacy of different lipid formulations in delivering hsa-miR-199a-3p and hsa-miR-590-3p both in primary neonatal mouse cardiomyocytes and in vivo. We established a transfection protocol allowing persistence of these 2 mimics for at least 12 days after a single intracardiac injection, with minimal dispersion to other organs and long-term preservation of miRNA functional activity, as assessed by monitoring the expression of 2 mRNA targets. Administration of this synthetic formulation immediately after myocardial infarction in mice resulted in marked reduction of infarct size and persistent recovery of cardiac function. Conclusions: A single administration of synthetic miRNA–lipid formulations is sufficient to stimulate cardiac repair and restoration of cardiac function.

We have previously shown that VEGF165 gene delivery into ischemic skeletal muscle exerts not only proangiogenic, but also remarkable antiapoptotic and proregenerative activity. The aim of this study was to determine whether recombinant adeno-associated virus (rAAV)-mediated gene delivery of VEGF165 into cardiac muscle, during acute myocardial infarction, exerts a protective effect to promote long-term functional recovery. Acute infarction of the anterior LV wall was induced in 12 chronically instrumented dogs by permanent occlusion of the LAD coronary artery. Four hours after occlusion, rAAV-VEGF165 or rAAV-LacZ (n=6 each; 5×10 12 viral particles per animal) was directly injected with an echo-guided needle into the dysfunctional cardiac wall. LV and arterial pressure, dP/dt max , and ejection fraction were not significantly different between the two groups over time. In contrast, in the infarcted region, at four weeks after infarction, fractional shortening was 75±18% and −3±15% of baseline and length-pressure area was 54±15% and 0.8±15% of baseline in VEGF165 versus LacZ, respectively ( P P

Rationale: The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury. Objective: We wanted to assess efficacy of Notch pathway stimulation in neonatal and adult hearts as a means to induce cardiac regeneration after myocardial infarction in mice. Methods and Results: In early postnatal life, cardiomyocyte exit from the cell cycle was paralleled by decreased Notch signaling and the establishment of a repressive chromatin environment at Notch-responsive genes, characterized by recruitment of the polycomb group enhancer of zeste homolog 2 methyltransferase and the acquisition of the histone 3 Lysine 27 trimethylation histone mark, as detected by chromatin immunoprecipitation. Forced Notch pathway activation by adenoassociated virus gene transfer of activated Notch1 or its ligand Jagged1 expanded the proliferative capacity of neonatal cardiomyocytes; this correlated with increased transcription of Notch target genes and maintenance of an open chromatin conformation at their promoters. The same adenoassociated virus vectors, however, were largely ineffective in stimulating cardiac repair after myocardial infarction in adult mice, despite optimal and long-lasting transgene expression. Analysis of Notch-responsive promoters in adult cardiomyocytes showed marks of repressed chromatin and irreversible CpG DNA methylation. Induction of adult cardiomyocyte re-entry into the cell cycle with microRNAs was independent from Notch pathway reactivation. Conclusions: Notch pathway activation is crucial in regulating cardiomyocyte proliferation during the early postnatal life, but it is largely ineffective in driving cardiac regeneration in adults, because of permanent epigenetic modification at Notch-responsive promoters.

See related article, pages 928–936 Heart failure is characterized by sympathetic nervous system activation with consequent increase in cardiac and systemic norepinephrine (NE) release. Sympathetic hyperactivity preferentially targets the heart during early stages of failure and subsequently involves other organs. A landmark study published in 1984 by Cohn et al showed that plasma NE concentration in venous blood sampled at rest provides a better guide to the prognosis of chronic congestive heart failure than other indexes of cardiac performance.1 Although the augmented adrenergic drive in the failing heart is initially a compensatory mechanism to preserve contractility and cardiac output, in the long term it leads to very complex maladaptive phenomena (Figure). In particular, two deleterious effects must be mentioned, both caused by excessive catecholamine release: cardiomyocyte injury and functional denervation. NE is indeed markedly toxic for cardiomyocytes at concentrations typically found in the failing heart. Sustained β-adrenergic stimulation leads to cAMP-mediated calcium overload of the cardiac cell, with a resulting decrease in synthetic activity and/or viability2 and contractile impairment. It can also trigger myocyte apoptosis mediated by protein kinase A and calcium entry via voltage-dependent calcium channels.3 On the other hand, functional denervation of the failing heart is responsible for reduced inotropic support, especially during exercise, and can be attributed to at least three mechanisms: depletion of NE stores in cardiac nerve endings,4–5 selective downregulation of β1-adrenoceptors,6 and desensitization of the intracellular β-adrenergic signaling cascade, mainly mediated by β-adrenergic receptor kinase and β-arrestin.7 The excessive NE release in myocardial interstitial space is not counterbalanced by adequate sympathetic nerve ending reuptake, because the principal NE carrier, namely uptake-1, is downregulated8–10 …