Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work ...was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the I
inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of I
on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.
The mechanism by which stem cell-based therapy improves heart function is still unknown, but paracrine mechanisms seem to be involved. Adipose-derived stem cells (ADSCs) secrete several factors, ...including insulin-like growth factor-1 (IGF-1), which may contribute to myocardial regeneration. Our aim was to investigate whether the overexpression of IGF-1 in ADSCs (IGF-1-ADSCs) improves treatment of chronically infarcted rat hearts. ADSCs were transduced with a lentiviral vector to induce IGF-1 overexpression. IGF-1-ADSCs transcribe100- to 200-fold more IGF-1 mRNA levels compared to nontransduced ADSCs. IGF-1 transduction did not alter ADSC immunophenotypic characteristics even under hypoxic conditions. However, IGF-1-ADSCs proliferate at higher rates and release greater amounts of growth factors such as IGF-1, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) under normoxic and hypoxic conditions. Importantly, IGF-1 secreted by IGF-1-ADSCs is functional given that Akt-1 phosphorylation was remarkably induced in neonatal cardiomyocytes cocultured with IGF-1-ADSCs, and this increase was prevented with phosphatidylinositol 3-kinase (PI3K) inhibitor treatment. Next, we tested IGF-1-ADSCs in a rat myocardial infarction (MI) model. MI was performed by coronary ligation, and 4 weeks after MI, animals received intramyocardial injections of either ADSCs (n = 7), IGF-1-ADSCs (n = 7), or vehicle (n = 7) into the infarcted border zone. Left ventricular function was evaluated by echocardiography before and after 6 weeks of treatment, and left ventricular hemodynamics were assessed 7 weeks after cell injection. Notably, IGF-1-ADSCs improved left ventricular ejection fraction and cardiac contractility index, but did not reduce scar size when compared to the ADSC-treated group. In summary, transplantation of ADSCs transduced with IGF-1 is a superior therapeutic approach to treat MI compared to nontransduced ADSCs, suggesting that gene and cell therapy may bring additional benefits to the treatment of MI.
Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and ...detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb‐iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb‐iPSC were 10‐fold less resistant to H2O2, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb‐iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb‐iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb‐iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb‐iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.
Chagas disease is caused by the parasite Trypanosoma cruzi . It is a common cause of heart disease in endemic areas of Latin America. The year 2009 marks the 100th anniversary of the discovery of T ...cruzi infection and Chagas disease by the Brazilian physician Carlos Chagas. Chagasic cardiomyopathy develops in from 10% to 30% of persons who are chronically infected with this parasite. Echocardiography and magnetic resonance imaging (MRI) are important modalities in the evaluation and prognostication of individuals with chagasic heart disease. The etiology of chagasic heart disease likely is multifactorial. Parasite persistence, autoimmunity, and microvascular abnormalities have been studied extensively as possible pathogenic mechanisms. Experimental studies suggest that alterations in cardiac gap junctions may be etiologic in the pathogenesis of conduction abnormalities. The diagnosis of chronic Chagas disease is made by serology. The treatment of this infection has shortcomings that need to be addressed. Cardiac transplantation and bone marrow stem cell therapy for persons with Chagas disease have received increasing research attention in recent years.
The failure to translate preclinical results to the clinical setting is the rule, not the exception. One reason that is frequently overlooked is whether the animal model reproduces distinctive ...features of human disease. Another is the reproducibility of the method used to measure treatment effects in preclinical studies. Left ventricular (LV) function improvement is the most common endpoint in preclinical cardiovascular disease studies, while echocardiography is the most frequently used method to evaluate LV function. In this work, we conducted a robust echocardiographic evaluation of LV size and function in dogs chronically infected by
.
Echocardiography was performed blindly by two distinct observers in mongrel dogs before and between 6 and 9 months post infection. Parameters analyzed included end-systolic volume (ESV), end-diastolic volume (EDV), ejection fraction (EF), and fractional shortening (FS). We observed a significant LVEF and FS reduction in infected animals compared to controls, with no significant variation in volumes. However, the effect of chronic infection in systolic function was quite variable, with EF ranging from 17 to 66%. Using the cut-off value of EF ≤ 40%, established for dilated cardiomyopathy (DCM) in dogs, only 28% of the infected dogs were affected by the chronic infection.
The canine model of CCC mimics human disease, reproducing the percentage of individuals that develop heart failure during the chronic infection. It is thus mandatory to establish inclusion criteria in the experimental design of canine preclinical studies to account for the variable effect that chronic infection has on systolic function.
During the course of Chagas disease, infectious forms of
are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) ...demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells
toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses
cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells
interdependent activation of B2R and endothelin receptors endothelin A receptor (ET
R)/endothelin B receptor (ET
R) led us to hypothesize that
might reciprocally benefit from the formation of infection-associated edema
activation of kallikrein-kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent "contact" activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation
generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to "leaky" HCP-forged by low dose histamine application-and found that the proinflammatory phenotype of TCTs was boosted by BK generated
the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ET
R/ET
R. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated
activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues.
Chagas disease, resulting from infection with the parasite Trypanosoma cruzi (T. cruzi), is a major cause of cardiomyopathy in Latin America. Drug therapy for acute and chronic disease is limited. ...Stem cell therapy with bone marrow mesenchymal cells (MSCs) has emerged as a novel therapeutic option for cell death-related heart diseases, but efficacy of MSC has not been tested in Chagas disease.
We now report the use of cell-tracking strategies with nanoparticle labeled MSC to investigate migration of transplanted MSC in a murine model of Chagas disease, and correlate MSC biodistribution with glucose metabolism and morphology of heart in chagasic mice by small animal positron emission tomography (microPET). Mice were infected intraperitoneally with trypomastigotes of the Brazil strain of T. cruzi and treated by tail vein injection with MSC one month after infection. MSCs were labeled with near infrared fluorescent nanoparticles and tracked by an in vivo imaging system (IVIS). Our IVIS results two days after transplant revealed that a small, but significant, number of cells migrated to chagasic hearts when compared with control animals, whereas the vast majority of labeled MSC migrated to liver, lungs and spleen. Additionally, the microPET technique demonstrated that therapy with MSC reduced right ventricular dilation, a phenotype of the chagasic mouse model.
We conclude that the beneficial effects of MSC therapy in chagasic mice arise from an indirect action of the cells in the heart rather than a direct action due to incorporation of large numbers of transplanted MSC into working myocardium.
Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is ...currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model.
Anthracyclines are chemotherapeutic drugs widely used in the frontline of cancer treatment. The therapeutic mechanisms involve the stabilization of topoisomerase IIα, DNA, and the anthracycline ...molecule in a ternary complex that is recognized as DNA damage. Redox imbalance is another vital source of oxidative DNA damage. Together, these mechanisms lead to cytotoxic effects in neoplastic cells. However, anthracycline treatment can elicit cardiotoxicity and heart failure despite the therapeutic benefits. Topoisomerase IIβ and oxidative damage in cardiac cells have been the most reported pathophysiological mechanisms. Alternatively, cardiac cells can undergo stress-induced senescence when exposed to anthracyclines, a state primarily characterized by cell cycle arrest, organelle dysfunction, and a shift to senescence-associated secretory phenotype (SASP). The SASP can propagate senescence to neighboring cells in an ongoing process that leads to the accumulation of senescent cells, promoting cellular dysfunction and extracellular matrix remodeling. Therefore, the accumulation of senescent cardiac cells is an emerging pathophysiological mechanism associated with anthracycline-induced cardiotoxicity. This paradigm also raises the potential for therapeutic approaches to clear senescent cells in treating anthracycline-induced cardiotoxicity (i,e, senolytic therapies).
•TST promoted cardiac hypertrophy in aged rats.•LV contractile function during IR was increased in aged rats treated with TST.•Infarct size of aged hearts was increased in TST-treated aged rat ...hearts.•p-Akt levels of aged hearts were decreased by TST.•Susceptibility to IR injury of adult hearts was not significantly affected by TST.
Aging is followed by numerous physiological limitations that reduce health span, particularly cardiovascular and metabolic disorders. Testosterone supplementation therapy (TST) has been widely used in the treatment of aging dysfunctions in either adult or aged patients, although recent evidence have suggested that the incidence of myocardial infarction might be increased in elderly patients. So far, though, the effects of TST in the progression of cardiac ischemia/reperfusion (IR) injury in aged hearts remain unclear. Male aged (23–24 months old) and adult (6 months old) Wistar rats were treated with placebo (Old + Placebo n = 5 / Adult + Placebo n = 5) or TST (Old + TST n = 7 / Adult + TST n = 5) for 30 days. After euthanasia, artificially-perfused isolated rat hearts were submitted to IR. Cardiac expression levels of genes encoding α and β myosin heavy chain (MHC), ryanodine receptor (RyR), brain-natriuretic peptide (BNP), sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), glucose-regulated protein 78 kDa (GRP78), eukaryotic initiation factor 2α (eIF2α), C/EBP-homologous protein (CHOP), caspase 3 and B cell lymphoma 2 (Bcl-2) were accessed by qRT-PCR. Protein levels of CHOP, p-Akt, and p-glycogen synthase kinase 3β (p-GSK-3β) were measured by Western Blot. Compared to placebo-treated aged rats, Old + TST group exhibited increased heart weight and up-regulation of αMHC mRNA expression levels, whereas βMHC mRNA expression (p < 0.05). During reperfusion, left ventricular developed pressure, dP/dt+, dP/dt-, and cardiac contractile function index were increased in Old + TST rat hearts (p < 0.05), whereas infarct size was increased (p < 0.05) in comparison with Old + Placebo group. p-Akt levels of Old + TST rat hearts were decreased when compared to Old + Placebo group. Conversely, TST did not promote significant effects in adult rat hearts. Taken together, these findings suggest that myocardial stunning and infarct size of aged hearts were distinctly affected by TST.
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