Regulation of Cardiomyocyte Proliferation by microRNAs and Small Molecules

Understanding the molecular mechanisms regulating cardiac cell proliferation during the embryonic, fetal and adult life holds a paramount importance in view of developing innovative strategies aimed at inducing myocardial regeneration after cardiac damage. Previous high throughput screening studies in our laboratory identified a series of microRNAs able to trigger cardiomyocyte proliferation and stimulate cardiac regeneration after myocardial infarction. In the first part of this project, we investigated the mechanism of action of the top ten most effective of these miRNAs, revealing an involvement of the Hippo-YAP pathway in their action. We found that all the investigated miRNAs activated YAP-mediated transcription, nuclear localization of active YAP and increased expression of YAP responsive genes. Of notice, miR-199a-3p, one of the most effective miRNAs exerted its direct effect on two mRNA targets impinging on the Hippo pathway, the inhibitory kinase TAOK1 and the E3 ubiquitin ligase, β−TrCP. Most of the miRNAs inducing proliferation (including miR-199a-3p) also modulated the dynamics of the actin cytoskeleton in the treated cardiomyocytes, which displayed a rounded shape and gross bundles of actin fibers at the cytoplasm periphery. Consistent with these observations, we found that the Cofilin2 mRNA was a direct target of four of the investigated miRNAs and that downregulation of Cofilin2 itself was sufficient to promote cardiomyocyte proliferation, activate nuclear translocation of YAP and stimulate transcription of TEAD-responsive genes. The second part of the project was aimed at identifying small molecules exerting a mitogenic effect on neonatal cardiomyocytes through an unbiased high-throughput screening (HTS) of a library of 780 FDA-registered drugs. The neuroactive alkaloid harmine was identified as the most powerful molecule at inducing cardiomyocyte proliferation in vitro and heart regeneration after myocardial infarction in vivo. Harmine exerted its activity through the inhibition of the dual specificity phosphorylation-regulated tyrosine kinase, Dyrk1a and, again, the activation of YAP nuclear translocation. Collectively, these results identify both YAP activation and actin cytoskeleton remodelling as major determinants of cardiomyocyte proliferation and establish the molecular basis for the development of pharmacological therapies to promote heart regeneration through the stimulation of the endogenous capacity of cardiomyocytes to proliferate

A–C Estrogens as Potent and Selective Estrogen Receptor-Beta Agonists (SERBAs) to Enhance Memory Consolidation under Low-Estrogen Conditions

Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs) with in vivo efficacy that are A–C estrogens, lacking the B and D estrogen rings. The most potent and selective A–C estrogen is selective for activating ER relative to seven other nuclear hormone receptors, with a surprising 750-fold selectivity for the β over α isoform and with EC50s of 20–30 nM in cell-based and direct binding assays. Comparison of potency in different assays suggests that the ER isoform selectivity is related to the compound’s ability to drive the productive conformational change needed to activate transcription. The compound also shows in vivo efficacy after microinfusion into the dorsal hippocampus and after intraperitoneal injection (0.5 mg/kg) or oral gavage (0.5 mg/kg). This simple yet novel A–C estrogen is selective, brain penetrant, and facilitates memory consolidation

Association between Endothelial Selectin (E-selectin) gene polymorphisms and E-selectin level with visceral leishmaniais, in an ARMS-PCR based study

Background: In the visceral leishmaniasis (VL), parasites reside in reticuluendothelial system, mainly in macrophages. Endothelial Selectin (E-selectin) might play an important role in leukocyte-endothelium interactions and inflammatory cell recruitment. The aim of this study was determining E-selectin level and its polymorphism in three groups, patients, seropositive and healthy individuals. Methods: Serum soluble E-selectin levels as well as 2 polymorphisms of E-selectin (Ser128Arg and Leu554Phe) were measured in a cohort of patients with documented VL (n=64), a healthy control group (n=74) and a seropositive for VL but without any symptoms (n=81). Circulation concentration of E-selectin levels was measured by ELIS. The amplification refractory mutation system (ARMS)-PCR procedure was used for detecting polymorphisms. Results: The mean of E-selectin levels significantly differed between three groups (P<0.026), and were increased in patients in comparison with other groups. Difference was more considerable between two groups of patients and healthy ones (patients 92.8 ng/ml; healthy individuals 71.9 ng/ml). Polymorphisms were associated with soluble E-selectin levels and altogether explained 14.4%, 7.2%, and 8.7% in patients, seropositive and seronegative healthy individuals, respectively. Distribution of polymorphisms of 128Ser/Arg and 554Leu/Phe among three groups was not different significantly; however, there was a considerable arrangement in distribution of Ser128Arg polymorphism and 128Arg allele in healthy group was more than two fold of patients (55% against 20%). Conclusion: The association between soluble E-selectin levels and visceral leishmaniasis suggests that this molecule might have significant role in the inflammatory process in VL. Moreover, frequency of 128Arg allele in healthy group was higher than patients

A biomaterials approach for therapeutic angiogenesis

Peripheral arterial disease (PAD) affects over 200 million people worldwide and can lead to limb ischaemia, amputation and death. Therapeutic angiogenesis aims to promote the formation of new blood vessels in order to treat ischaemia. The programming inherent within cells can be utilised to treat diseases at the cellular level. Adipose derived mesenchymal stem cells (ADMSCs) have been shown to secrete pro-angiogenic proteins, thus could have great potential as a therapy for ischemic disease. In addition, biomaterials can effectively deliver therapeutics to a target site and utilise physical characteristics to influence cell behaviour. Surface topography is known to influence cell alignment, morphology and affect cellular expression of growth factors. This work investigated the effect of surface topography on the secretion of angiogenic growth factors from ADMSCs. Hierarchically structured substrate materials were prepared from poly-DL-lactide-co-glycolide (PLGA) using a thermally induced phase separation (TIPS) process. TIPS materials were characterised using atomic force microscopy to quantify roughness and stiffness, as well as scanning electron microscopy techniques where PLGA processed with TIPS were shown to have higher surface roughness and porosity values. ADMSC proliferation increased on the TIPS-processed substrates compared with the control substrates and the effect of surface topography on the angiogenic secretome of ADMSCs was measured using an in vitro model of angiogenesis, proteomic analysis and measurement of vascular endothelial growth factor (VEGF165). VEGF165 was significantly increased in the supernatants collected from ADMSCs cultured on the TIPS substrate compared with control substrates when normalised for the number of cells. The collected supernatants resulted in increased capillary tubule length, number of capillary junctions and capillary branches in the in vitro angiogenesis assay compared with supernatants collected from control substrates. 5 This work also investigated the effects of TIPS-processed materials implanted in a pre-clinical model of PAD. Laser Doppler imaging revealed an increase in revascularisation in the ischeamic limbs treated TIPS processed materials compared with control materials. Histology and von Willebrand factor staining revealed evidence of blood vessel formation around the implanted TIPS processed materials. This study has shown that ADMSCs seeded onto 2D and 3D TIPS-processed PLGA secreted increased quantities of pro-angiogenic factors in vitro, and when implanted in vivo, TIPS-processed biomaterials improved reperfusion in a pre-clinical model of PAD. These findings open up the opportunity for utilising a unique biomaterial for the treatment of ischemic disease through the promotion of angiogenesis

Investigating the delivery of IGF-1 with in vitro and in vivo model systems of myocardial infarction

Myocardial infarction (MI) is characterised by the irreversible death of cardiac muscle with loss of up to 1 billion cardiomyocytes (CM). Despite survival post-MI dramatically improving in the last two decades, more than 20% of patients suffering MI will still develop heart failure (HF), an incurable condition where the heart is no longer able to meet the body’s needs for blood supply. Amongst novel therapeutic avenues currently being explored, intramyocardial delivery of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) holds great promise to replace the lost functional tissue. However, the effects of the ischemic microenvironment on these cells still need to be investigated, and protective strategies need to be developed. This thesis examines the delivery of the pro-survival growth factor Insulin like Growth Factor-1 (IGF-1) in the settings of hiPSC-CMs exposed to acidic pH and through a hydrogel-based approach in an in vivo model of MI. Following MI, the heart switches from aerobic metabolism to anaerobic glycolysis, causing a pH drop to 6.5-6.8. The aim of the first part of this thesis was to mitigate the effects of acidic pH on hiPSC-CMs using the pro-survival growth factor IGF-1. It was shown that acidic pH negatively affects hiPSC-CMs in terms of viability, metabolic activity, cardiac gene expression and CMs yield obtained through differentiation. IGF-1 was able to recover the effects of acidic pH, and it could, therefore, be used as a protective strategy for in vivo cell therapy approaches. Another promising strategy for preventing HF progression following MI is the minimally invasive delivery of injectable hydrogels, which can provide mechanical support to damaged tissue and deliver bioactive factors with pro-survival actions. Here, a thermoresponsive injectable hydrogel composed of a triblock copolymer of polyethylene glycol (PEG) and polycaprolactone (PCL) was synthesised and characterised in vitro and in vivo. The hydrogel was prepared with or without insulin-like growth factor-1 (IGF-1) and injected intramyocardially in a mouse MI model. Echocardiography, strain analysis and histological assessments showed that the injection of the biodegradable thermoresponsive hydrogel was effective in ameliorating pathological remodelling, improving overall cardiac function and myocardial mechanics. In the future, implementing novel therapeutic approaches like the ones presented in this thesis could prevent the progression to HF, improving the quality of life of patients affected by myocardial infarction and limiting the socio-economic burden of the disease.Open Acces

Myocardial Viability Imaging using Manganese-Enhanced MRI in the First Hours after Myocardial Infarction

Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning but are challenging to obtain. Here, manganese, a calcium analogue and clinically approved magnetic resonance imaging (MRI) contrast agent, is used as an imaging biomarker of myocardial viability in the first hours after experimental MI. Safe Mn dosing is confirmed by measuring in vitro beating rates, calcium transients, and action potentials in cardiomyocytes, and in vivo heart rates and cardiac contractility in mice. Quantitative T1 mapping-manganese-enhanced MRI (MEMRI) reveals elevated and increasing Mn uptake in viable myocardium remote from the infarct, suggesting MEMRI offers a quantitative biomarker of cardiac inotropy. MEMRI evaluation of infarct size at 1 h, 1 and 14 days after MI quantifies myocardial viability earlier than the current gold-standard technique, late-gadolinium-enhanced MRI. These data, coupled with the re-emergence of clinical Mn -based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients

Heart rate responses to autonomic challenges in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is accompanied by structural alterations and dysfunction in central autonomic regulatory regions, which may impair dynamic and static cardiovascular regulation, and contribute to other syndrome pathologies. Characterizing cardiovascular responses to autonomic challenges may provide insights into central nervous system impairments, including contributions by sex, since structural alterations are enhanced in OSA females over males. The objective was to assess heart rate responses in OSA versus healthy control subjects to autonomic challenges, and, separately, characterize female and male patterns. We studied 94 subjects, including 37 newly-diagnosed, untreated OSA patients (6 female, age mean ± std: 52.1 ± 8.1 years; 31 male aged 54.3 ± 8.4 years), and 57 healthy control subjects (20 female, 50.5 ± 8.1 years; 37 male, 45.6 ± 9.2 years). We measured instantaneous heart rate with pulse oximetry during cold pressor, hand grip, and Valsalva maneuver challenges. All challenges elicited significant heart rate differences between OSA and control groups during and after challenges (repeated measures ANOVA, p&lt;0.05). In post-hoc analyses, OSA females showed greater impairments than OSA males, which included: for cold pressor, lower initial increase (OSA vs. control: 9.5 vs. 7.3 bpm in females, 7.6 vs. 3.7 bpm in males), OSA delay to initial peak (2.5 s females/0.9 s males), slower mid-challenge rate-of-increase (OSA vs. control: -0.11 vs. 0.09 bpm/s in females, 0.03 vs. 0.06 bpm/s in males); for hand grip, lower initial peak (OSA vs. control: 2.6 vs. 4.6 bpm in females, 5.3 vs. 6.0 bpm in males); for Valsalva maneuver, lower Valsalva ratio (OSA vs. control: 1.14 vs. 1.30 in females, 1.29 vs. 1.34 in males), and OSA delay during phase II (0.68 s females/1.31 s males). Heart rate responses showed lower amplitude, delayed onset, and slower rate changes in OSA patients over healthy controls, and impairments may be more pronounced in females. The dysfunctions may reflect central injury in the syndrome, and suggest autonomic deficiencies that may contribute to further tissue and functional pathologies

Development of in vitro Drug Screening Platforms Using Human Induced Pluripotent Stem Cell-Derived Cardiovascular Cells

Drug-induced cardiotoxicity is a critical challenge in the development of new drugs. Since the advent of human pluripotent stem cell-derived cardiomyocytes (CMs), researchers have explored ways to utilize these cells for in vitro preclinical drug screening applications. One area of interest is microphysiological systems (i.e. organ-on-a-chip), which aims to create more complex in vitro models of human organ systems, thus improving drug response predictions. In this dissertation, we investigated novel analysis methods and model platforms for detecting drug-induced cardiotoxicity using human induced pluripotent stem cell (iPSC)-derived cardiovascular cells. First, we utilized human iPSC-derived CMs (iPS-CMs) to establish optical methods of detecting cardioactive compounds. We utilized optical flow to assess the iPS-CM contractions captured using brightfield microscopy. The parameters were then analyzed using a machine learning algorithm to determine the accuracy of detection that can be obtained by the model for a given drug concentration. This result was compared to the analysis of the calcium transients measured using a genetically encoded calcium indicator (GCaMP6). The brightfield contraction analysis matched the detection sensitivity of fluorescent calcium transient analysis, while also being able to detect the effects of excitation-contraction decoupler (blebbistatin), which was not detected using calcium transient analysis. Second, we utilize iPS-CMs to model trastuzumab-related cardiotoxicity. Trastuzumab, a monoclonal antibody against ErbB2 (Her2), is used to treat Her2+ breast cancer and has known clinical cardiotoxicity. We demonstrated that an active ErbB2 signaling via binding of neuregulin-1 (NRG-1) to ErbB4 is necessary to detect the cardiotoxic effects of trastuzumab. Activation of ErbB2/4 pathway via NRG-1 is cardioprotective, and we also demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) similarly activates the ErbB2/4 pathway. Finally, we established a co-culture platform of iPS-CMs and endothelial cells (ECs), which recapitulated the physiological phenomenon of EC-secreted NRG-1 activating the ErbB2/4 pathway on the CMs. Third, we demonstrated the use of human iPSC-derived ECs (iPS-ECs) for creating 3-dimensionial vascular networks inside microfluidic devices. The iPS-ECs were characterized for EC markers and physiological functions. We utilized a CDH5-mCherry iPSC line to create iPS-ECs that expressed VE-cadherin fused to mCherry. The vascular networks formed by the iPS-ECs were patent and perfusable, retaining 70 kDa dextran within the lumen of the vessels. The vasculature responded to small molecule inhibitors, showing increased vessel formation in response to TGF-β inhibitor SB431542 and decreased vessel formation in response to multi-targeted receptor tyrosine kinase inhibitor sunitinib. Taken together, our findings advance the current understanding and utility of iPS-CMs for drug screening applications, while establishing platforms for creating microphysiological systems that incorporate iPS-EC co-culture. The use of iPSC-derived cells opens possibilities for disease-specific and patient-specific drug screening applications in the future

The development of a platform to manipulate cardiomyocyte structure and function

Cardiac tissue engineering to replace damaged areas of the postmitotic heart is still presented with significant challenges, due to the complex and dynamic interplay of electrical, mechanical and biochemical signals involved in the myocardium. The advancement of regenerative approaches is focussed on understanding the underlying regulatory mechanisms involved throughout cardiac development. However, current knowledge of how biophysical cues in the stem cell niche can modulate cell behaviour is limited. Firstly, polyacrylamide-co-acrylic acid was used as an in vitro stiffness-tuneable platform to test the effect of substrate mechanics on human induced pluripotent stem cell (hiPSC) differentiation into cardiomyocytes (CM). The results showed that the optimum differentiation efficiency level peaked at the embryonic-like stiffness of 560 Pa, with increased upregulation of cardiac genes. Functionally, hiPSC-CMs showed a biphasic relationship with a faster calcium transient and higher force generation at cardiac physiological stiffness. Next, shape was incorporated into the experimental design via CardioArray, a custom-built platform which mimics both the stiffness and shape of an adult human CM. This system can accommodate individual hiPSC-CMs to adopt the 3D geometry of an adult CM, while at the same time providing the relevant stiffness cues from the underlying substrate. The results highlighted the specific contribution of stiffness and 3D shape to α-sarcomeric structure, cell membrane stiffness, single cell gene expression and intracellular calcium cycling. Finally, the electrical microenvironment was investigated as a third infleuncing factor on hiPSC-CM development. A hybrid conductive polyaniline-Scl2 scaffold was fabricated, showing long term electronic stability and no cell toxicity when interfaced with electrosensitive hiPSC-CMs. This could provide electromechanical stability in model studies. Improvement of conduction velocity was observed in an in vitro myocardial slice model. As a whole, this thesis demonstrates the differential effects of substrate mechanics on hiPSC cardiac differentiation, providing a novel crucial understanding of how biophysical cues modulate the stem cell niche during differentiation and in vitro culture.Open Acces