scholarly journals RBM20-mutations induce disturbed splicing of calcium relevant genes and guides clinically therapy in different cardiomyopathies

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
S Rebs ◽  
F Sedaghat-Hamedani ◽  
E Kayvanpour ◽  
D Huebscher ◽  
A Wagdi ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Model ◽  
Left Ventricular ◽  
Patient Specific ◽  
Adrenergic Stimulation ◽  
Cardiovascular Research ◽  
National Budget ◽  
Molecular Events ◽  
The Impact

Abstract Background Mutations in the splice factor RBM20 account for ∼3% of genetic cardiomyopathies. Mutations at position R634 in the hotspot RS-domain were found to cause dilative cardiomyopathy (DCM) (R634W) or left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remain unknown. Purpose We aimed here to identify the molecular events caused by the distinct RBM20 mutations from DCM and LVNC patients using a patient-specific induced stem cell model (iPSC) and test if the currently clinically used β-blockers (Metroprolol) are suitable for different RBM20-dependent cardiomyopathies. Methods We generated iPSC-cardiomyocytes of 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC). We investigated alternative splicing, sarcomeric regularity, cAMP-level, kinase-specific phosphorylation of Ca2+ players and Ca2+ handling. To investigate the impact of the genetic background, isogenic rescue lines were generated by CRISPR/Cas9. Different clinical drugs as Metoprolol and Verapamil were used to analyze the pharmacological improvement in vitro. Results We investigated the splicing pattern of the 2 RBM20 mutations in DCM and LVNC iPSC-CMs and observed common isoform changes in titin and a 24bp-insertion in the gene RYR2. The Ca2+ handling gene triadin is misspliced in LVNC-CMs, whereas the structural gene LDB3 is misspliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes, both DCM and LVNC-CMs exhibited an irregular sarcomeric structure. The Ca2+ handling gene CAMK2δ was predominantly misspliced in LVNC-CMs leading to CAMK2δ-dependent hyperphosphorylation of its target PLN-Thr17 and subsequently to shortened Ca2+ elimination time and weakened response to β-adrenergic stimulation. By contrast, DCM-CMs exhibited increased Ca2+ sparks and decreased systolic and diastolic Ca2+ levels. RBM20 expression itself was decreased in LVNC-CMs, but not in DCM-CMs. This highlights that 2 distinct RBM20 mutations can lead to different pathological Ca2+ phenotypes. Isogenic CRISPR/Cas9 repair of both RBM20 mutations in LVNC and DCM demonstrated a rescue in gene missplicing, sarcomeric regularity and the Ca2+ handling aberrations and underscored the causative nature of the 2 mutations and their diverging effects. Ca2+ channel blockage with Verapamil showed a significant improvement of some of the LVNC disease characteristics compared to commonly clinically used β-blocker Metoprolol and underpins the potential clinical use of this drug in patients with LVNC. Conclusion We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC and DCM. In summary, our results suggest that the molecular aberrations in alternative splicing differ depending on the distinct mutation in RBM20 and lead to shared and differential pathologies. Verapamil could be a good candidate in the treatment of RBM20-dependent LVNC. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Bunderministerium für Bildung und Forschung BMBFGerman Center for Cardiovascular Research DZHK

RBM20-mutations induce disturbed splicing of calcium relevant genes in patient-specific stem cell models of cardiomyopathies

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Rebs ◽  
F Sedaghat-Hamedani ◽  
E Kayvanpour ◽  
D Huebscher ◽  
H Katus ◽  
...  
Keyword(s):  
Stem Cell ◽  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Left Ventricular ◽  
Calcium Handling ◽  
Patient Specific ◽  
Funding Source ◽  
Cardiac Phenotype

Abstract Background and aim Mutations in the splice factor RBM20 have been identified to account for ∼3% of cardiomyopathies. In particular, the highly conserved RS-domain is a hotspot for disease-associated mutations. Distinct mutations at position 634 in the RS-domain were already described to be associated to dilative cardiomyopathy (DCM) (R634W) or to left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the molecular mechanisms that govern the heterogenic entity of DCM and LVNC remain largely unknown. We aimed to analyze the molecular driver behind the RBM20 mutation-based DCM and LVNC in a patient-specific stem cell model. Methods Human somatic cells from 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC) were reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into functional cardiomyocytes (CM). Gene expression, alternative splicing activity, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of important Ca2+ players, and physiological cardiac functions as Ca2+ homeostasis were analyzed (Fluo3 and Fura4). Isogenic rescue lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations to the cardiac phenotype. Results We investigated the role of RBM20 mutations in DCM and LVNC-iPSC-CMs RBM20-splicing and observed common splice defects in titin-isoform-switch or a 24bp insertion in the gene ryanodine receptor 2 (RYR2).. In contrast, the calcium-handling gene Camk2δ was predominantly mis-spliced in LVNC-CMs, whereas the structural gene LDB3 was mis-spliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes both DCM and LVNC-CMs exhibited an irregular sarcomeric structure at the Z-disk and M-line. Interestingly, the LVNC-CMs showed faster Ca2+ transient decay time and weakened response to β-adrenergic stimulation. In contrast, the DCM-CMs did exhibit increased Ca2+-sparks and decreased systolic and diastolic Ca2+ highlighting that two distinct missense mutations can lead to different pathological Ca2+ phenotypes. Ca2+ kinetic defects in LVNC-iPSC-CMs were independent of cAMP, but in line with Camk2δ-dependent hyperphosphorylation of the specific target PLN. Isogenic WT-iPSC lines were generated using CRISPR/Cas9 technology and underscored the role of RBM20-mutations in cardiomyopathies as the sarcomeric defects, Ca2+ cycling and leakage were rescued for both LVNC-CMs and DCM-CMs. Conclusion We show the first iPSC-model of splice-defect-associated RBM20-dependent LVNC and DCM. Our data demonstrate that RBM20-R634L induce mis-splicing of Camk2δ leading to hyperphosphorylation of PLN-Thr17 along with increased Ca2+ kinetics in LVNC, whereas RBM20-R634W induced RYR2-dependent Ca2+ leak with disturbed systolic and diastolic Ca2+in DCM. Taken together these results suggest that the molecular aberrations in alternative splicing differ depending on the distinct missense mutation in RBM20. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): BMBF, DZHK German Center for Cardiovascular research

β-blockade abolishes the augmented cardiac tPA release induced by transactivation of heterodimerised bradykinin receptor-2 and β2-adrenergic receptor in vivo

2014 ◽  
Vol 112 (11) ◽  
pp. 951-959 ◽  
Author(s):  
Morten Eriksen ◽  
Arnfinn Ilebekk ◽  
Alessandro Cataliotti ◽  
Cathrine Rein Carlson ◽  
Torstein Lyberg ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Sympathetic Nerves ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Adrenergic Stimulation ◽  
Β2 Adrenergic Receptor ◽  
Β Blocker

SummaryBradykinin (BK) receptor-2 (B2R) and β2-adrenergic receptor (β2AR) have been shown to form heterodimers in vitro. However, in vivo proofs of the functional effects of B2R-β2AR heterodimerisation are missing. Both BK and adrenergic stimulation are known inducers of tPA release. Our goal was to demonstrate the existence of B2R-β2AR heterodimerisation in myocardium and to define its functional effect on cardiac release of tPA in vivo. We further investigated the effects of a non-selective β-blocker on this receptor interplay. To investigate functional effects of B2R-β2AR heterodimerisation (i. e. BK transactivation of β2AR) in vivo, we induced serial electrical stimulation of cardiac sympathetic nerves (SS) in normal pigs that underwent concomitant BK infusion. Both SS and BK alone induced increases in cardiac tPA release. Importantly, despite B2R desensitisation, simultaneous BK infusion and SS (BK+SS) was characterised by 2.3 ± 0.3-fold enhanced tPA release compared to SS alone. When β-blockade (propranolol) was introduced prior to BK+SS, tPA release was inhibited. A persistent B2R-β2AR heterodimer was confirmed in BK-stimulated and nonstimulated left ventricular myocardium by immunoprecipitation studies and under non-reducing gel conditions. All together, these results strongly suggest BK transactivation of β2AR leading to enhanced β2AR-mediated release of tPA. Importantly, non-selective β-blockade inhibits both SS-induced release of tPA and the functional effects of B2R-β2AR heterodimerisation in vivo, which may have important clinical implications.

Protective Effect of Butylphthalide on Neuronal Apoptosis in Parkinson Rats and Its Effect on miR-146a-5p Expression and Phosphatidylinositide 3-Kinases/Protein Kinase B Pathway

2021 ◽  
Vol 11 (10) ◽  
pp. 1908-1917
Author(s):  
Rongkang Mai ◽  
Yiyao Cao ◽  
Huitian Yu ◽  
Yong Zheng ◽  
Juke Huang
Keyword(s):  
Cell Model ◽  
Morphological Changes ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Vehicle Group ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Oil Emulsion ◽  
The Impact

80 male Wistar rats were stochastically assigned to Sham + Vehicle group, Sham + BUT group, PD + Vehicle group and PD + BUT group. Rotenone PD model rats were prepared by subcutaneous injection of rotenone sunflower oil emulsion 2 mg/(kg · d) for 5 consecutive weeks. Butylphthalide 80 mg/(kg · d) were given to the rats in Sham + BUT group and PD + BUT group by gavage from the first day of rotenone injection for 5 weeks. Subsequently, the motor retardation ability and the morphological changes of the substantia nigra (SN) of each group were evaluated. Meanwhile, the levels of neuronal injury, apoptosis, inflammation and oxidative stress in each group of rats were assayed. The impact of BUT treatment on miR-146a-5p expression and PI3K/AKT signal pathway in rat brain tissue was assayed. Finally, by constructing a PD cell model of the neurotoxin 6-hydroxydopamine (6-OHDA)-treated human neuroblastoma cell line SH-SY5Y, the in vitro anti-PD pharmacological effect of BUT was further verified.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy

2019 ◽  
Vol 56 (8) ◽  
pp. 548-556 ◽  
Author(s):  
Malte Lenders ◽  
Franciska Stappers ◽  
Christoph Niemietz ◽  
Boris Schmitz ◽  
Michel Boutin ◽  
...  
Keyword(s):  
Fabry Disease ◽  
Cell Model ◽  
White Blood Cells ◽  
Disease Patient ◽  
Patient Specific ◽  
Specific Cell ◽  
Missense Mutations ◽  
Activity Measurements

BackgroundPatients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD.MethodsSince current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy.ResultsUnder treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells.ConclusionWe conclude that repeated AGAL activity measurements in patients’ white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

scholarly journals Cardiomyocyte deletion of mitofusin-1 leads to mitochondrial fragmentation and improves tolerance to ROS-induced mitochondrial dysfunction and cell death

2012 ◽  
Vol 302 (1) ◽  
pp. H167-H179 ◽  
Author(s):  
Kyriakos N. Papanicolaou ◽  
Gladys A. Ngoh ◽  
Erinne R. Dabkowski ◽  
Kelly A. O'Connell ◽  
Rogerio F. Ribeiro ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Mitochondrial Permeability Transition ◽  
Microscopic Analysis ◽  
Left Ventricular ◽  
Mitochondrial Morphology ◽  
Mitochondrial Fragmentation ◽  
Electron Microscopic ◽  
Mitofusin 2 ◽  
The Impact

Molecular studies examining the impact of mitochondrial morphology on the mammalian heart have previously focused on dynamin related protein-1 (Drp-1) and mitofusin-2 (Mfn-2), while the role of the other mitofusin isoform, Mfn-1, has remained largely unexplored. In the present study, we report the generation and initial characterization of cardiomyocyte-specific Mfn-1 knockout (Mfn-1 KO) mice. Using electron microscopic analysis, we detect a greater prevalence of small, spherical mitochondria in Mfn-1 KO hearts, indicating that the absence of Mfn-1 causes a profound shift in the mitochondrial fusion/fission balance. Nevertheless, Mfn-1 KO mice exhibit normal left-ventricular function, and isolated Mfn-1 KO heart mitochondria display a normal respiratory repertoire. Mfn-1 KO myocytes are protected from mitochondrial depolarization and exhibit improved viability when challenged with reactive oxygen species (ROS) in the form of hydrogen peroxide (H2O2). Furthermore, in vitro studies detect a blunted response of KO mitochondria to undergo peroxide-induced mitochondrial permeability transition pore opening. These data suggest that Mfn-1 deletion confers protection against ROS-induced mitochondrial dysfunction. Collectively, we suggest that mitochondrial fragmentation in myocytes is not sufficient to induce heart dysfunction or trigger cardiomyocyte death. Additionally, our data suggest that endogenous levels of Mfn-1 can attenuate myocyte viability in the face of an imminent ROS overload, an effect that could be associated with the ability of Mfn-1 to remodel the outer mitochondrial membrane.

scholarly journals TSH Combined with TSHR Aggravates Diabetic Peripheral Neuropathy by Promoting Oxidative Stress and Apoptosis in Schwann Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-23
Author(s):  
Jingwen Fan ◽  
Qi Pan ◽  
Qun Gao ◽  
Wenqing Li ◽  
Fei Xiao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Peripheral Neuropathy ◽  
Diabetic Peripheral Neuropathy ◽  
Cell Model ◽  
Cell Damage ◽  
Motor Nerve ◽  
Thyroid Stimulating Hormone ◽  
Motor Nerve Conduction Velocity ◽  
The Impact

Subclinical hypothyroidism (SCH) is associated with diabetic peripheral neuropathy (DPN); however, the mechanism underlying this association remains unknown. This study is aimed at examining neurofunctional and histopathological alterations in a type 2 diabetes (T2DM) mouse model of SCH and investigating the impact of thyroid-stimulating hormone (TSH) in an in vitro DPN cell model established using RSC96 cells under high glucose (HG) and palmitic acid (PA) stimulation. Our results indicated that T2DM, in combination with SCH, aggravated abnormal glucose and lipid metabolism in T2DM and dramatically destroyed the peripheral nervous system by increasing paw withdrawal latency, decreasing motor nerve conduction velocity, and exacerbating ultrastructural deterioration of the damaged sciatic nerve caused by diabetes. Furthermore, the results of our in vitro experiments showed that TSH intensified HG/PA-induced RSC96 cell damage by inducing oxidative stress, mitochondrial dysfunction, and apoptosis. More importantly, TSHR knockout or inhibition of PA-induced TSHR palmitoylation could alleviate the apoptosis induced by TSH. Overall, in this study, the novel mechanisms by which TSH, as an independent risk factor for DPN progression, aggravating Schwann cell apoptosis and demyelination, are elucidated. These findings indicate that TSHR could be a potential target for both the prevention and treatment of DPN and, possibly, other microvascular diseases, and have implication in the clinical management of patients with DPN.

scholarly journals Characterization of Hematopoietic Differentiation Profiles of MPN Patient-Derived Inducible Pluripotent Stem Cells Harboring Homozygous Vs Heterozygous Calreticulin Mutations

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3065-3065
Author(s):  
Lijuan Han ◽  
Marcelo A. Szymanski Toledo ◽  
Alexandre Theocharides ◽  
Angela Maurer ◽  
Tim H. Brümmendorf ◽  
...  
Keyword(s):  
Ips Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
In Vitro Differentiation ◽  
Wild Type ◽  
Ips Cell ◽  
Cell Clones ◽  
Calr Mutations ◽  
The Impact

Abstract Introduction: Somatic calreticulin (CALR) mutations were discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) and have been shown to be mutually exclusive with Janus kinase 2 (JAK2) and thrombopoietin receptor (MPL) mutations. Recent studies demonstrated that the binding of CALR mutant proteins to MPL induces constitutive activation of the JAK/STAT pathway, thus causing cellular transformation and abnormal megakaryopoiesis. Additionally, it has been reported that patients carrying homozygously mutated CALR ins5 exhibit myeloperoxidase (MPO) deficiency as a result of the absence of CALR chaperone function. However, the impact of CALR mutant homozygosity vs. heterozygosity in CALR del52 mutations as well as on hematopoietic differentiation has not yet been studied. Furthermore, clonal heterogeneity of hematopoietic stem/progenitor cell (HSPC) populations in a patient, together with technical limitations isolating single clones, are major challenges, when determining the impact of CALR mutant zygosity on clonal composition and diversity in MPN. To overcome these limitations, we generated patient-specific iPS cells carrying homozygous or heterozygous CALR mutations or their wild-type counterparts to study their roles in hematopoietic differentiation. Methods: iPS cells were generated by reprogramming peripheral blood-derived mononuclear cells from three patients carrying CALR del52, ins5, or del31 mutations using a CytoTune iPS 2.0 Sendai Reprogramming Kit. Individual colonies were picked and screened for CALR genotypes by PCR. Pluripotency of iPS cells was confirmed by immunofluorescences, and the clones were screened for additional mutations using panel-based next generation sequencing (NGS). Subsequently, CALR iPS cells were subjected to embryonic body formation, mesoderm commitment, and hematopoietic differentiation using our standard in vitro differentiation protocol. CD34+ HSPCs were MACS-sorted and characterized by flow cytometry, cytospins and RNA expression analysis on days 10, 15, and 20 during differentiation. Hematopoietic progenitors, erythrocytes, granulocytes, and megakaryocytes were identified by defined lineage markers. MPO expression was assessed by flow cytometry and cytochemical staining. Results: We established patient-specific iPS cells carrying CALR del52, ins5 or del31 mutation after written informed consent (Table 1). Pluripotency markers OCT4, Tra-1-60 and Tra-1-81 expression were confirmed in all iPS cell clones. In accordance with findings in peripheral blood cells, we detected MPO deficiency in homozygous iPS cell-derived CD15+ cells from CALRins5- and, in addition, also from CALRdel52-mutated patients (pMFI=0.0106 and pMFI=0.0187, resp.). Intriguingly, in vitro hematopoietic differentiation assays revealed additional abnormalities, such as decreased CD66b+ granulocytes derived from homozygous CALR del52 or ins5 iPS cells vs. heterozygous iPS cells on day 10 (pdel52=0.0303 and pins5=0.0253, resp.) and a trend towards increased KIThigh+CD45+ cells. Megakaryopoiesis, defined by CD41+CD42b+ cells, was increased in CALRins5 homozygous vs. heterozygous clones (p=0.0031). However, this bias was not observed in all clones, indicating clone-specific megakaryocytic differentiation potential. No phenotypic differences during hematopoietic differentiation were observed in iPS cell-derived progenitors carrying heterozygous CALRdel31 mutation and its isogenic unmutated CALR controls. Furthermore, our NGS data revealed patient-specific sets of co-occurring mutations in iPS cell clones, which may have contributed to the observed patient-specific phenotypes. As an example, the IDH2 R140Q mutation, reported to block cell differentiation, was found in approximately half of the CALRdel52 iPS clones, and these clones failed to differentiate into the hematopoietic lineage in vitro. Conclusions: We successfully generated patient-specific CALR mutant iPS cells. Upon in vitro differentiation, we detected MPO deficiency and aberrant granulocytic differentiation in CALR homozygous but not heterozygous or wild-type clones. Thus, it is now possible at the single stem cell level to further analyze the molecular mechanisms of CALR-mutant induced MPO deficiency and altered hematopoietic differentiation, in order to better understand disease biology in ET and PMF. Disclosures Brümmendorf: Merck: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Takeda: Consultancy.

scholarly journals Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow

2020 ◽  
Author(s):  
Patrick A. Murphy ◽  
Noor Jailkhani ◽  
Sarah-Anne Nicholas ◽  
Amanda M. Del Rosario ◽  
Jeremy L. Balsbaugh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Alternative Splicing ◽  
Low Flow ◽  
Matrix Composition ◽  
Splice Isoforms ◽  
Vitro Assay ◽  
Disturbed Flow ◽  
The Impact

Objective: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. Conclusions: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

scholarly journals Dysfunctional crosstalk of cardiomyocytes and cardiac fibroblasts in a pluripotent stem cell model of dilated cardiomyopathy

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
W Maurer ◽  
S Koehne ◽  
A Maus ◽  
M Tiburcy ◽  
S Schlick ◽  
...  
Keyword(s):  
Stem Cell ◽  
Dilated Cardiomyopathy ◽  
Pluripotent Stem Cell ◽  
Cell Model ◽  
Cardiac Fibroblasts ◽  
Left Ventricular ◽  
Patient Specific ◽  
Funding Source ◽  
New Variant ◽  
Healthy Control

Abstract Background/Purpose Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and contractile dysfunction. Fibrosis is one major phenotypic result in DCM, pointing to the contribution of both, cardiomyocytes (CM) and cardiac fibroblasts (cFB) to DCM. The molecular basis of most DCM cases remains unknown. Nevertheless, it is known that up to 35% of all cases have a family history, linked to mutations in more than 30 gene loci. The aim of this study is to analyse the crosstalk of iPSC-CM and cFB and the underlying genetic and molecular causes in a patient-specific induced pluripotent stem cell (iPSC) model of DCM. Methods and results For this purpose a 4-member family was recruited containing 2 patients (father and daughter) with severe DCM and heart transplantation. iPSCs of all family members were generated and differentiated into iPSC-CMs. All iPSC-CMs express general cardiac markers, e.g. βMHC, α-actinin. Interestingly, αMHC expression was decreased in diseased iPSC-CMs in comparison to control cells. Additionally, the sarcomeric regularity was decreased in diseased iPSC-CMs. As we found significantly increased fibrosis (22%) in explanted myocardium of the diseased father compared to healthy myocardium (8%), both cFB and CM seem to play an important role. From the same myocardium primary cFBs were isolated and shown to express typical cFB markers clearly distinguishing these cells from non-fibroblasts as well as from fibroblasts with different origin. To analyse the contribution of cFBs and CMs to DCM on a functional level, 3D engineered heart muscles (EHMs) were generated in different diseased/healthy cell combinations. EHMs composed of both or either one affected DCM-iPSC-CMs and/or DCM-cFBs in comparison to healthy control EHMs did not produce any measurable force, indicating that the DCM-EHM phenotype is clearly diseased. Evaluation of tissues' viscoelasticity showed that DCM-cFB, DCM-iPSC-CMs and DCM-EHMs were stiffer than healthy control EHMs. Thus these data suggest that apart from the obvious dysfunction of DCM CMs, DCM cFBs clearly contribute to the contractile pathophysiology in DCM EHMs. Furthermore, whole exome sequencing of iPSCs was conducted to identify disease-causing variants. This analyses point towards a new genetic variant in the FLNc gene coding for a protein important in development, stabilization and maintenance of myofibrils. Rescue of this variant by CRISPR Cas9 genome editing will shed more light onto the role of this variant during DCM development in the future. Conclusion Using a ps-iPSC-CM model of a 4-member family with two severe DCM patients, we could demonstrate a clear contribution of both cell types, iPSC-CMs and cFB, to the contractile pathophysiology of DCM. We identified a potentially disease-causing new variant in the FLNc gene, which may contribute to the impaired functionality within the diseased iPSC-CMs and EHM. This makes FLNc a new therapeutic target for DCM. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): IRTG1816

Abstract 146: Glucagon-like Peptide-1 Receptor Activation Promotes Dual Benefits For Reversing Microvasculopathy And Mitochondrial Damage In Diabetic Heart.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Akio Monji ◽  
Yasuko K Bando ◽  
Toko Mitsui ◽  
Morihiko Aoyama ◽  
Hiroya Kawase ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Receptor Activation ◽  
Left Ventricular ◽  
Mitochondrial Damage ◽  
Wall Thickening ◽  
Mouse Heart ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Impact

PURPOSE: Glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) is a remedy for type 2 diabetes mellitus (T2DM). Ex4 ameliorates cardiac dysfunction in preclinical and clinical settings. However, it remains unclear whether the impact of Ex4 on cardiac remodeling in diabetic cardiomyopathy (DMC), of which primary characteristics are microvasculopathy and mitochondrial damage. Methods and Results: Diet-induced T2DM (DIO) mice and age- and gender-matched lean control mice were allocated into EX4 (24 nmole/kg/day for 40 days; DIO-Ex4 and LEAN-Ex4) and vehicle groups (DIO-veh and LEAN-veh). We first confirmed the GLP-1R expression in every single chamber of mouse heart by immunoblotting and PCR. Ex4 treatment ameliorated both systemic and cardiac insulin resistance without affecting body weight in DIO. Cardiac capillary density of DIO-veh was reduced compared to those LEAN-veh, which were reversed by Ex4 treatment. Tube formation assay and immunoblot analysis using culture endothelial cells revealed that Ex4 directly enhanced in vitro angiogenesis in a PKA/eNOS-dependent fashion. Systolic and diastolic left-ventricular (LV) dysfunctions observed in DIO-veh were restored by Ex4 with decline in LV wall thickening. Myocardial fibrosis detected using sirius-red staining and tissue oxidative stress detected by a fluorescence indicator DHE were attenuated in DIO-Ex4. Of note, analyses using transmission electron microscopy and a fluorescence indicator for damaged mitochondria (mitotracker red) revealed that Ex4 treatment reversed cardiac mitochondrial remodeling and increased healthy mitochondria. Ex4 treatment modulated cardiac oxidative stress balance by upregulating antioxidative molecules (SOD, thioredoxin, glutathione peroxidase) and reduction of NOX4 level; whereas it had no influence on NOX2 level. Conclusions: Ex4 enhances cardiac angiogenesis via GLP-1R-mediated activation of PKA/eNOS axis and accelerates reverses remodeling of myocardial mitochondria, at least in part, via its facilitating effects on antioxidative defense.

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