Timon Seeger

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

MicroRNA-29 in Aortic Dilation: Implications for Aneurysm Formation

Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

MicroRNA-29 in aortic dilation: implications for aneurysm formation [Molecular Medicine]

Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

Immunosenescence-associated microRNAs in age and heart failure

Ageing of the immune system, immunosenescence, is characterized by impaired lymphopoiesis, especially B‐lymphocyte maturation, and is a hallmark of chronic heart failure (CHF). MicroRNAs (miRNAs) are non‐coding, small RNAs, which post‐transcriptionally control gene expression of multiple target genes. The miR‐181 family is known to control haematopoietic lineage differentiation. Here, we study the role of the miR‐181 family in immunosenescence and CHF.

Long‐term inhibition of miR‐21 leads to reduction of obesity in db/db mice

To assess the effect of long‐term pharmacological inhibition of miR‐21 in a model of metabolic syndrome and obesity.

Elevated Levels of the Mediator of Catabolic Bone Remodeling RANKL in the Bone Marrow Environment Link Chronic Heart Failure with Osteoporosis

Background—Chronic heart failure (CHF) is associated with a 4-fold increased risk for osteoporotic fractures. Therefore, we sought to identify the pathophysiological link between chronic heart failure and catabolic bone remodeling. Methods and Results—In a total cohort of 153 subjects (123 patients with CHF, 30 patients with coronary artery disease and preserved cardiac function) as well as mice with heart failure, bone marrow (BM) plasma levels of the catabolic receptor activator of NF-&kgr;B ligand (RANKL), and its antagonist, osteoprotegerin were measured. The osteoclast inducing activity of BM plasma was tested in cell culture. BM plasma levels of RANKL and of the ratio RANKL/osteoprotegerin were significantly elevated in patients with CHF. On multivariate regression analysis, parameters of severity and duration of heart failure were independent determinants of elevated BM plasma RANKL levels. BM plasma levels of RANKL were directly correlated with the systemic marker of bone turnover C-telopeptide of type 1 collagen (r=0.6; P<0.001). Alterations in BM plasma levels of RANKL/osteoprotegerin were confirmed in a mouse model of postinfarction heart failure. Stimulation of human mesenchymal cells with BM plasma obtained from CHF patients increased the formation of osteoclasts, and this effect was blocked by the RANKL inhibition. Conclusions—CHF is associated with a profound and selective elevation of the bone resorption stimulating RANKL within the BM microenvironment. These data for the first time disclose a direct pathophysiological pathway linking CHF with catabolic bone remodeling associated with an increased osteoporotic fracture risk. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT 00289822, NCT 00284713, NCT 00326989, NCT 00962364.

MicroRNAs in cardiovascular ageing

MicroRNAs (miRs) have emerged as potent regulators of pathways in physiological and disease contexts. This review focuses on the role of miRs in ageing of the cardiovascular system. Several miRs have been described to be regulated during ageing and some of these miRs are involved in the regulation of ageing‐related processes. We discuss the roles of miR‐34, miR‐217 and miR‐29, which are induced during ageing in the vasculature. The roles of miR‐34, miR‐29 (age‐induced) and miR‐18/19, which are decreased during ageing in the heart, are discussed as well. Furthermore, numerous miRs that play a role in diseases associated with ageing, like diabetes, atherosclerosis, hypertension, cardiac hypertrophy and atrial fibrillation, are also briefly discussed. miRs also serve as circulating biomarkers for cardiovascular ageing or ageing‐associated diseases. Finally, pharmacological modulation of ageing‐related miRs might become a promising strategy to combat cardiovascular ageing in a clinical setting.

A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell–Based Models for Cardiovascular Diseases

Rationale: Targeted genetic engineering using programmable nucleases such as transcription activator–like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. Objective: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. Methods and Results: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN–mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt–Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. Conclusions: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

Genome Editing in Cardiovascular Biology

Genome editing has emerged as a powerful tool in research and is entering the stage of therapeutic applications. In the cardiovascular field, its role in basic and translational research is well established. However, biological and technical barriers currently hamper the therapeutic potential of genome editing for cardiovascular diseases. This viewpoint discusses possible routes for promoting therapeutic use of genome editing in the cardiovascular system. Genome editing has rapidly emerged as a powerful tool in basic and translational research. Zinc finger nucleases and TALENs (transcription activator-like effector nucleases) catalyzed the field initially. With the development of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system, the field is expanding even more rapidly because of its efficacy, specificity, and ease of use (Figure [A]). Generally, genome editing tools induce site-specific DNA double-strand breaks at a specific genomic site, resulting in the activation of the nonhomologous end-joining (NHEJ) and homologous recombination (HR) cellular endogenous double-strand break repair machinery (Figure [B]). Recent advances of the CRISPR technology also allow for RNA recognition, making it possible to cleave RNA, enhance or inhibit translation, support isolation of specific RNA:protein complexes, or induce specific post-transcriptional modifications.1 Genome editing tools can also be used to control gene expression on the transcriptional level. Deactivation of the catalytic site of CRISPR/Cas9 (dCas9) results in specific binding to the DNA without inducing double-strand breaks. Fusion of dCas9 to DNA-binding domains, such as activator or repressor domains, results in transcriptional activation or inhibition of a specific gene, respectively.2 Despite the major impact genome editing already has on basic and translational research, regulatory processes have delayed therapeutic applications from reaching the clinic. Nevertheless, effective new therapies are anticipated in the near future for various disease conditions, especially if major issues around safety and toxicity are resolved. Figure. Genome editing …

Inhibition of let-7 augments the recruitment of epicardial cells and improves cardiac function after myocardial infarction

Heart failure due to myocardial infarction is a major cause of mortality. The microRNA (miR) family let-7 is expressed during embryonic development and is up-regulated in differentiated cells. The aim of this study was to study the role of let-7 after acute myocardial infarction (AMI). We designed an antimiR to inhibit the highest expressed members of the let-7 family, let-7 a, b and c. Administration at day 0 and day 2 after AMI resulted in sustained knockdown of let-7 after 28days. Let-7 inhibition prevented deterioration of cardiac functions compared to control treatment which was especially due to improvements in the infarcted, apical cardiac segments. We observed higher contents of fibrosis in the border zone as well as increased numbers of cells positive for TCF21, which is also expressed in epicardial cells. Markers were augmented after let-7 inhibition and let-7 blocked EMT in epicardial cells in vitro. Lineage tracing in TCF21(iCre/+):R26R(tdT) mice showed abundant tomato positive cells in the infarct and border zone. In conclusion, let-7 inhibition resulted in functional benefits due to an increase in recruitment of epicardial cells and EMT.