Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterised by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing. TRANSLATIONAL PERSPECTIVE: There are currently no specific therapies for improving diastolic function in patients with HCM. We show for the first time that myeloperoxidase (MPO) is present in and is up-regulated in cardiomyocytes derived from human iPSCs obtained from HCM patients, where it impairs cardiomyocyte relaxation by reducing phosphorylation of cardiac MYBPC3. Treatment with the MPO inhibitor, AZD5904, restored MYBPC3 phosphorylation and alleviated the relaxation defect, demonstrating cardiomyocyte MPO to be a novel therapeutic target for improving diastolic function in HCM, a treatment strategy which can be evaluated in HCM patients given that MPO inhibitors are already available for clinical testing

Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome

Functional odontoblastic-like cells derived from human iPSCs

10.1177/0022034517730026Journal of Dental Research97177-8

Fatty acid metabolism driven mitochondrial bioenergetics promotes advanced developmental phenotypes in human induced pluripotent stem cell derived cardiomyocytes

Background: Preferential utilization of fatty acids for ATP production represents an advanced metabolic phenotype in developing cardiomyocytes. We investigated whether this phenotype could be attained in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and assessed its influence on mitochondrial morphology, bioenergetics, respiratory capacity and ultra-structural architecture. Methods and results: Whole-cell proteome analysis of day 14 and day 30-CMs maintained in glucose media revealed a positive influence of extended culture on mitochondria-related processes that primed the day 30-CMs for fatty acid metabolism. Supplementing the day 30-CMs with palmitate/oleate (fatty acids) significantly enhanced mitochondrial remodeling, oxygen consumption rates and ATP production. Metabolomic analysis upon fatty acid supplementation revealed a β-oxidation fueled ATP elevation that coincided with presence of junctional complexes, intercalated discs, t-tubule-like structures and adult isoform of cardiac troponin T. In contrast, glucose-maintained day 30-CMs continued to harbor underdeveloped ultra-structural architecture and more subdued bioenergetics, constrained by suboptimal mitochondria development. Conclusion: The advanced metabolic phenotype of preferential fatty acid utilization was attained in hiPSC-CMs, whereby fatty acid driven β-oxidation sustained cardiac bioenergetics and respiratory capacity resulting in ultra-structural and functional characteristics similar to those of developmentally advanced cardiomyocytes. Better understanding of mitochondrial bioenergetics and ultra-structural adaptation associated with fatty acid metabolism has important implications in the study of cardiac physiology that are associated with late-onset mitochondrial and metabolic adaptations

Phosphatidylserine supplementation as a novel strategy for reducing myocardial infarct size and preventing adverse left ventricular remodeling

10.3390/ijms22094401International Journal of Molecular Sciences229440

Metabolic reprogramming of immune cells by mitochondrial division inhibitor-1 to prevent post-vascular injury neointimal hyperplasia

Background and aims: New treatments are needed to prevent neointimal hyperplasia that contributes to postangioplasty and stent restenosis in patients with coronary artery disease (CAD) and peripheral arterial disease (PAD). We investigated whether modulating mitochondrial function using mitochondrial division inhibitor-1 (Mdivi-1) could reduce post-vascular injury neointimal hyperplasia by metabolic reprogramming of macrophages from a pro-inflammatory to anti-inflammatory phenotype. Methods and Results: In vivo Mdivi-1 treatment of Apoe /- mice fed a high-fat diet and subjected to carotid-wire injury decreased neointimal hyperplasia by 68%, reduced numbers of plaque vascular smooth muscle cells and pro-inflammatory M1-like macrophages, and decreased plaque inflammation, endothelial activation, and apoptosis, when compared to control. Mdivi-1 treatment of human THP-1 macrophages shifted polarization from a pro-inflammatory M1-like to an anti-inflammatory M2-like phenotype, reduced monocyte chemotaxis and migration to CCL2 and macrophage colony stimulating factor (M-CSF) and decreased secretion of proinflammatory mediators. Finally, treatment of pro-inflammatory M1-type-macrophages with Mdivi-1 metabolically reprogrammed them to an anti-inflammatory M2-like phenotype by inhibiting oxidative phosphorylation and attenuating the increase in succinate levels and correcting the decreased levels of arginine and citrulline. Conclusions: We report that treatment with Mdivi-1 inhibits post-vascular injury neointimal hyperplasia by metabolic reprogramming macrophages towards an anti-inflammatory phenotype thereby highlighting the therapeutic potential of Mdivi-1 for preventing neointimal hyperplasia and restenosis following angioplasty and stenting in CAD and PAD patients

A Systemic Evaluation of Cardiac Differentiation from mRNA Reprogrammed Human Induced Pluripotent Stem Cells

<div><p>Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by “foot-print free” reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.</p></div

Effects to adrenergic stimulation on cardiomyocytes.

<p>A, Multielectrode array (MEA) tracing of dose dependent effects isoproterenol (1–1000 nM) on beating frequency (Hz) on hiPSC derived cardiomyocytes. Note a significant increase in beat rates with increasing dose. *p<0.05 vs control (baseline). Data represented as mean ± SEM of three independent experiments. B, β-adrenergic (propanolol) inhibition of beating frequency under adrenergic stimulation (isoproterenol). Note propranolol (2000 nM) significantly reduced beating frequency post isoproterenol (100 nM) stimulation. C, Alteration in corrected field potential durations (cFPD) in propranolol inhibitor under isoproterenol stimulation. Note isoproterenol reduced cFPDs that reverse with propranolol. *p<0.05 vs control (baseline) and ^#p<0.05 vs Isoproterenol group. Data represented as mean ± SEM of three independent experiments. The dotted line (red) shows field potential durations in each trace. Abbreviations: I- Isoproterenol; P- Propranolol; B- Baseline; W- Washout.</p

Characterization of transgene free iPSC.

<p>A, Micrographs showing morphological changes during mRNA reprograming on day 6 (i), 8 (ii), 15 (iii) and 20 (iv) with live Tra-1-60 (v) staining (day 20). vi, image shows hiPSCs positively stained with alkaline phosphatase. B, Immunostaining of the undifferentiated hiPSC colonies with Oct-4, Sox2, Nanog, SSEA-4, Tra-1-60 and Tra-1-81 antibodies followed by counterstaining with DAPI. Scale bar –200 µm. C, Semi-quantitative gene expression levels of pluripotency associated genes in two hiPSC clones (lane 2 and 3) in comparison with hESC line (lane 1, positive control) with GAPDH as internal loading control. D, A typical normal karyogram of hiPSC clone 1 and Hematoxylin and eosin (H&E) staining of teratoma sections of clone 1 showing the presence of ectoderm (neural rosettes), mesoderm (cartilage) and endoderm (secretory tubule). Scale bar –200 µm.</p

Structural and molecular characterization of iPSC-CM.

<p>A, Immunohistochemisty staining of α-actinin, MHC, troponin T and MLC2v on day 18 EB. B, Immunofluorescence images of dissociated EBs for transcriptional factor, Nkx2.5, structural proteins, cardiac troponin-T (cTnT), titin, myosin light chain 2a (MLC2a), sarcomeric α-actinin, ion channel, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) and gap junction connexin 45 (Cx45). Nuclei were counterstained with DAPI in all images. Scale bar: 50 µm.</p