The Implication of Low Dose Dimethyl Sulfoxide on Mitochondrial Function and Oxidative Damage in Cultured Cardiac and Cancer Cells.

Although numerous studies have demonstrated the biological and multifaceted nature of dimethyl sulfoxide (DMSO) across different in vitro models, the direct effect of "non-toxic" low DMSO doses on cardiac and cancer cells has not been clearly explored. In the present study, H9c2 cardiomyoblasts and MCF-7 breast cancer cells were treated with varying concentrations of DMSO (0.001-3.7%) for 6 days. Here, DMSO doses < 0.5% enhanced the cardiomyoblasts respiratory control ratio and cellular viability relative to the control cells. However, 3.7% DMSO exposure enhanced the rate of apoptosis, which was driven by mitochondrial dysfunction and oxidative stress in the cardiomyoblasts. Additionally, in the cancer cells, DMSO (≥0.009) led to a reduction in the cell's maximal respiratory capacity and ATP-linked respiration and turnover. As a result, the reduced bioenergetics accelerated ROS production whilst increasing early and late apoptosis in these cells. Surprisingly, 0.001% DMSO exposure led to a significant increase in the cancer cells proliferative activity. The latter, therefore, suggests that the use of DMSO, as a solvent or therapeutic compound, should be applied with caution in the cancer cells. Paradoxically, in the cardiomyoblasts, the application of DMSO (≤0.5%) demonstrated no cytotoxic or overt therapeutic benefits.

Non-Coding RNAs in Stem Cell Regulation and Cardiac Regeneration: Current Problems and Future Perspectives.

Although advances in rapid revascularization strategies following acute myocardial infarction (AMI) have led to improved short and long-term outcomes, the associated loss of cardiomyocytes and the subsequent remodeling result in an impaired ventricular function that can lead to heart failure or death. The poor regenerative capacity of the myocardium and the current lack of effective regenerative therapies have driven stem cell research in search of a possible solution. One approach involves the delivery of stem cells to the site of injury in order to stimulate repair response. Although animal studies initially delivered promising results, the application of similar techniques in humans has been hampered by poor target site retention and oncogenic considerations. In response, several alternative strategies, including the use of non-coding RNAs (ncRNAs), have been introduced with the aim of activating and regulating stem cells or inducing stem cell status in resident cells. Circular RNAs (circRNAs) and microRNAs (miRNAs) are ncRNAs with pivotal functions in cell proliferation and differentiation, whose role in stem cell regulation and potential significance for the field of cardiac regeneration is the primary focus of this review. We also address the general advantages of ncRNAs as promising drivers of cardiac regeneration and potent stem cell regulators.

Human cardiac stem cells rejuvenated by modulating autophagy with MHY-1685 enhance the therapeutic potential for cardiac repair.

Stem cell-based therapies with clinical applications require millions of cells. Therefore, repeated subculture is essential for cellular expansion, which is often complicated by replicative senescence. Cellular senescence contributes to reduced stem cell regenerative potential as it inhibits stem cell proliferation and differentiation as well as the activation of the senescence-associated secretory phenotype (SASP). In this study, we employed MHY-1685, a novel mammalian target of rapamycin (mTOR) inhibitor, and examined its long-term priming effect on the activities of senile human cardiac stem cells (hCSCs) and the functional benefits of primed hCSCs after transplantation. In vitro experiments showed that the MHY-1685‒primed hCSCs exhibited higher viability in response to oxidative stress and an enhanced proliferation potential compared to that of the unprimed senile hCSCs. Interestingly, priming MHY-1685 enhanced the expression of stemness-related markers in senile hCSCs and provided the differentiation potential of hCSCs into vascular lineages. In vivo experiment with echocardiography showed that transplantation of MHY-1685‒primed hCSCs improved cardiac function than that of the unprimed senile hCSCs at 4 weeks post-MI. In addition, hearts transplanted with MHY-1685-primed hCSCs exhibited significantly lower cardiac fibrosis and higher capillary density than that of the unprimed senile hCSCs. In confocal fluorescence imaging, MHY-1685‒primed hCSCs survived for longer durations than that of the unprimed senile hCSCs and had a higher potential to differentiate into endothelial cells (ECs) within the infarcted hearts. These findings suggest that MHY-1685 can rejuvenate senile hCSCs by modulating autophagy and that as a senescence inhibitor, MHY-1685 can provide opportunities to improve hCSC-based myocardial regeneration.

Extracellular vesicles from thalassemia patients carry iron-containing ferritin and hemichrome that promote cardiac cell proliferation.

Extracellular vesicles (EVs) are bioactive, submicron-sized membrane vesicles released from all cell types upon activation or apoptosis. EVs including microparticles (MPs) and exosomes have emerged as important mediators of cell-to-cell communication in both normal and pathological states including thalassemia (thal). However, the role of EVs derived from ß-thal patients with iron overload (+ IO) and without iron overload (-IO) on cardiac cells is unclear. We hypothesized plasma EVs in thal patients containing ferritin (iron storage protein) and a denaturated hemoglobin-hemichrome that induce cardiac cell proliferation. The origins and numbers of EVs isolated from plasma of normal, thal (+ IO), and (- IO) patients were compared and determined for their iron and iron-containing proteins along with their effects on cardiac and endothelial cells. Data shows that MPs were originated from many cell sources with marked numbers of platelet origin. Only the number of RBC-derived MPs in thal (+ IO) patients was significantly high when compared to normal controls. Although MPs derived from both normal and thal patients promoted cardiac cell proliferation in a dose-dependent manner, only exosomes from thal patients promoted cardiac cell proliferation compared to the untreated. Moreover, the exosomes from thal (+ IO) potentially induce higher cardiac cell proliferation and angiogenesis in terms of tube number than thal (- IO) and normal controls. Interestingly, ferritin content in the exosomes isolated from thal (+ IO) was higher than that found in the MPs isolated from the same patient. The exosomes of thal patients with higher serum ferritin level also contained greater level of ferritin inside the exosomes. Apart from ferritin, there were trends of increasing hemichrome and iron presented in the plasma EVs and EV-treated H9C2 cells. Findings from this study support the hypothesis that EVs from ß-thal patients carry iron-load proteins that leads to the induction of cardiac cell proliferation.

Cardiac Cell Therapy: Insights into the Mechanisms of Tissue Repair.

Stem cell-based cardiac therapies have been extensively studied in recent years. However, the efficacy of cell delivery, engraftment, and differentiation post-transplant remain continuous challenges and represent opportunities to further refine our current strategies. Despite limited long-term cardiac retention, stem cell treatment leads to sustained cardiac benefit following myocardial infarction (MI). This review summarizes the current knowledge on stem cell based cardiac immunomodulation by highlighting the cellular and molecular mechanisms of different immune responses to mesenchymal stem cells (MSCs) and their secretory factors. This review also addresses the clinical evidence in the field.

Unravelling the Biology of Adult Cardiac Stem Cell-Derived Exosomes to Foster Endogenous Cardiac Regeneration and Repair.

Cardiac remuscularization has been the stated goal of the field of regenerative cardiology since its inception. Along with the refreshment of lost and dysfunctional cardiac muscle cells, the field of cell therapy has expanded in scope encompassing also the potential of the injected cells as cardioprotective and cardio-reparative agents for cardiovascular diseases. The latter has been the result of the findings that cell therapies so far tested in clinical trials exert their beneficial effects through paracrine mechanisms acting on the endogenous myocardial reparative/regenerative potential. The endogenous regenerative potential of the adult heart is still highly debated. While it has been widely accepted that adult cardiomyocytes (CMs) are renewed throughout life either in response to wear and tear and after injury, the rate and origin of this phenomenon are yet to be clarified. The adult heart harbors resident cardiac/stem progenitor cells (CSCs/CPCs), whose discovery and characterization were initially sufficient to explain CM renewal in response to physiological and pathological stresses, when also considering that adult CMs are terminally differentiated cells. The role of CSCs in CM formation in the adult heart has been however questioned by some recent genetic fate map studies, which have been proved to have serious limitations. Nevertheless, uncontested evidence shows that clonal CSCs are effective transplantable regenerative agents either for their direct myogenic differentiation and for their paracrine effects in the allogeneic setting. In particular, the paracrine potential of CSCs has been the focus of the recent investigation, whereby CSC-derived exosomes appear to harbor relevant regenerative and reparative signals underlying the beneficial effects of CSC transplantation. This review focuses on recent advances in our knowledge about the biological role of exosomes in heart tissue homeostasis and repair with the idea to use them as tools for new therapeutic biotechnologies for "cell-less" effective cardiac regeneration approaches.

Cardiac Progenitor Cells from Stem Cells: Learning from Genetics and Biomaterials.

Cardiac Progenitor Cells (CPCs) show great potential as a cell resource for restoring cardiac function in patients affected by heart disease or heart failure. CPCs are proliferative and committed to cardiac fate, capable of generating cells of all the cardiac lineages. These cells offer a significant shift in paradigm over the use of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes owing to the latter's inability to recapitulate mature features of a native myocardium, limiting their translational applications. The iPSCs and direct reprogramming of somatic cells have been attempted to produce CPCs and, in this process, a variety of chemical and/or genetic factors have been evaluated for their ability to generate, expand, and maintain CPCs in vitro. However, the precise stoichiometry and spatiotemporal activity of these factors and the genetic interplay during embryonic CPC development remain challenging to reproduce in culture, in terms of efficiency, numbers, and translational potential. Recent advances in biomaterials to mimic the native cardiac microenvironment have shown promise to influence CPC regenerative functions, while being capable of integrating with host tissue. This review highlights recent developments and limitations in the generation and use of CPCs from stem cells, and the trends that influence the direction of research to promote better application of CPCs.

Pluripotent Stem Cell-Based Therapeutics for Muscular Dystrophies.

Pluripotent stem cells (PSCs) represent an attractive cell source for treating muscular dystrophies (MDs) since they easily allow for the generation of large numbers of highly regenerative myogenic progenitors. Using reprogramming technology, patient-specific PSCs have been derived for several types of MDs, and genome editing has allowed correction of mutations, opening the opportunity for their therapeutic application in an autologous transplantation setting. However, there has been limited progress on preclinical studies that validate the therapeutic potential of these gene corrected PSC-derived myogenic progenitors. In this review, we highlight the major research advances, challenges, and future prospects towards the development of PSC-based therapeutics for MDs.

Effects of Spaceflight and Simulated Microgravity on YAP1 Expression in Cardiovascular Progenitors: Implications for Cell-Based Repair.

Spaceflight alters many processes of the human body including cardiac function and cardiac progenitor cell behavior. The mechanism behind these changes remains largely unknown; however, simulated microgravity devices are making it easier for researchers to study the effects of microgravity. To study the changes that take place in cardiac progenitor cells in microgravity environments, adult cardiac progenitor cells were cultured aboard the International Space Station (ISS) as well as on a clinostat and examined for changes in Hippo signaling, a pathway known to regulate cardiac development. Cells cultured under microgravity conditions, spaceflight-induced or simulated, displayed upregulation of downstream genes involved in the Hippo pathway such as YAP1 and SOD2. YAP1 is known to play a role in cardiac regeneration which led us to investigate YAP1 expression in a sheep model of cardiovascular repair. Additionally, to mimic the effects of microgravity, drug treatment was used to induce Hippo related genes as well as a regulator of the Hippo pathway, miRNA-302a. These studies provide insight into the changes that occur in space and how the effects of these changes relate to cardiac regeneration studies.

Isodunnianol alleviates doxorubicin-induced myocardial injury by activating protective autophagy.

Recurrent cardiotoxicity limits the clinical application of doxorubicin (DOX); however the detailed molecular mechanism of DOX cardiotoxicity remains unclear. In the current study, we found that a natural product extracted from Illicium verum, isodunnianol (IDN), mitigates DOX-induced cardiotoxicity by regulating autophagy and apoptosis both in vitro and in vivo. DOX suppressed protective autophagy and induced apoptosis in H9C2 cardiac myoblasts. Additionally, IDN demonstrated up-regulated autophagy and reduced apoptosis through the activation of the AMPK-ULK1 pathway. In addition, the beneficial effects of IDN on DOX which induced myocardial injury were dependent on AMPK and ULK1 phosphorylation. Similar results were also observed in a DOX-induced cardiotoxicity rat model. The combination of IDN and DOX resulted in decreased apoptosis and inflammatory myocardial fibrosis compared to the DOX mono-treatment group. In summary, our findings provide novel insights into the prevention of DOX-related toxicity by isodunnianol, a food source natural product, warranting further investigation.

Column-Free Method for Isolation and Culture of C-Kit Positive Stem Cells from Atrial Explants.

Ever since the discovery of stem cells, their isolation from tissues and expansion in culture has been extensively studied due to its potential for therapeutic application. The magnetic-assisted cell sorting (MACS) method is the most widely used technique for the sorting of cells based on their cell surface markers. Though effective, the major drawbacks are high cost and the requirement for the frequent replacement of the columns. In the column-free method, the cells are sorted using the same principle of immune-magnetic isolation but does not require magnetic columns, making it cost-effective. The isolation of c-kit+ stem cells from atrial explants using column-free magnet is found to be efficient and yields homogenous population of stem cells. This method saves time and labor and is economical when working with large sample sizes.

The mechanistic role of oxidative stress in cigarette smoke-induced cardiac stem cell dysfunction and prevention by ascorbic acid.

Cigarette smoking causes a vast array of diseases including cardiovascular diseases. Our laboratory focuses on investigating cigarette smoke (CS)-induced cardiovascular malfunction and the responsible mechanisms utilizing the model, c-kit-positive cardiac stem cells (CSCs). The main objective of our study is to investigate whether CS extracts (CSEs) cause impairment of CSC functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of CSC proteins and antioxidant enzymes, can modulate CSC functions and these modifications can be attenuated by ascorbate treatment. Our specific aims are (1) to investigate CSE-induced oxidative modification of CSC proteins via carbonylation, and prevention by ascorbic acid; (2) to investigate CSE-induced oxidative modification of antioxidant enzymes and ascorbic acid-mediated modulations; and (3) to investigate CSE-induced changes in CSC functions and protection by ascorbic acid. CSCs were cultured, and the aqueous extracts of CSE were prepared. CSE-induced modulations of CSC viability, oxidative modification of proteins, and antioxidant enzyme activities were detected using standard assays including Apostain, bromodeoxyuridine, and Oxiblot. CSE caused oxidative modification of CSC proteins, changed antioxidant enzyme levels, attenuated CSC proliferation, and accelerated CSC apoptosis. Ascorbic acid prevented CSE-induced CSC malfunctions, and ascorbic acid therapy might be useful in smoker CSC recipients and to condition CSCs prior to the transplant in the future. Cardiac stem cell therapy is currently undergoing in clinical trials.

Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential.

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

Cardiac Stem Cell Characteristics in Physiological and Pathological Conditions.

For nearly a century, the adult heart was considered as a post-mitotic organ. The discovery of a resident cardiac stem cell (CSC) population in the heart has dramatically undermined this notion with the support of encouraging preclinical and clinical studies aiming to regenerate the damaged heart after a myocardial infarction (MI). There are two ways to obtain CSCs for transplantation: Allogeneic and autologous sources. Autologous cells may be obtained from the patients' own tissue. Obtaining cells from diseased patients may contain a risk for altered stem cell characteristics. In addition to MI, these patients may also suffer from pathological conditions such as hypertension, diabetes mellitus, heart failure, congenital heart disease or cancer, which are known to alter CSC characteristics. It is also known that physiological conditions such as aging and death affect CSC functions in the heart. Our knowledge about the CSC characteristics in various physiological and pathological conditions may shed light on our opinion about the regenerative capacity and biological activity of these cells in these situations. Defining these properties may guide the researchers and clinicians in choosing and obtaining the most qualified CSC populations for cardiac regenerative medicine therapies. The purpose of this review is to describe the alterations in CSC characteristics in various physiological and pathological conditions.

Preventive effect of apigenin against isoproterenol-induced apoptosis in cardiomyoblasts.

We investigated the effect of apigenin, a dietary flavonoid, on isoproterenol hydrochloride (ISO)-induced apoptotic signaling in cardiomyoblast H9C2 cells. The results showed that apigenin treatment (10 µM) prevented ISO (31.25 µM)-induced lipid peroxidative levels and antioxidants status in H9C2 cells. Furthermore, apigenin inhibited expression of inflammatory markers in ISO-treated cells. In addition, apigenin prevented ISO-induced DNA damage and apoptotic signaling through modulating the expression of Bax, caspase-3, -8 and -9, cytochrome c, and Fas proteins in H9C2 cells. It is concluded that apigenin prevents ISO-induced antioxidants depletion, oxidative DNA damage, inflammatory, and apoptotic signaling in H9C2 cells. Thus, the present results demonstrated that apigenin has a cardioprotective effect on cardiomyoblasts cells.

ERß targets ZAK and attenuates cellular hypertrophy via SUMO-1 modification in H9c2 cells.

Aberrant expression of leucine zipper- and sterile ɑ motif-containing kinase (ZAK) observed in pathological human myocardial tissue is associated with the progression and elevation of hypertrophy. Our previous reports have correlated high levels of estrogen (E2) and abundant estrogen receptor (ER) α with a low incidence of pathological cardiac-hypertrophy and heart failure in the premenopause female population. However, the effect of elevated ERß expression is not well known yet. Therefore, in this study, we have analyzed the cardioprotective effects and mechanisms of E2 and/or ERß against ZAK overexpression-induced cellular hypertrophy. We have used transient transfection to overexpress ERß into the ZAK tet-on H9c2 cells that harbor the doxycycline-inducible ZAK plasmid. The results show that ZAK overexpression in H9c2 cells resulted in hypertrophic effects, which was correlated with the upregulation of p-JNK and p-p38 MAPKs and their downstream transcription factors c-Jun and GATA-4. However, ERß and E2 with ERß overexpressions totally suppressed the effects of ZAK overexpression and inhibited the levels of p-JNK, p-p38, c-Jun, and GATA-4 effectively. Our results further reveal that ERß directly binds with ZAK under normal conditions; however, ZAK overexpression reduced the association of ZAK-ERß. Interestingly, increase in ERß and E2 along with ERß overexpression both enhanced the binding strengths of ERß and ZAK and reduced the ZAK protein level. ERß overexpression also suppressed the E3 ligase-casitas B-lineage lymphoma (CBL) and attenuated CBL-phosphoinositide 3-kinase (PI3K) protein association to prevent PI3K protein degradation. Moreover, ERß and/or E2 blocked ZAK nuclear translocation via the inhibition of small ubiquitin-like modifier (SUMO)-1 modification. Taken together, our results further suggest that ERß overexpression strongly suppresses ZAK-induced cellular hypertrophy and myocardial damage.

High vulnerability of the heart and liver to 3-hydroxypalmitic acid-induced disruption of mitochondrial functions in intact cell systems.

Patients affected by long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency predominantly present severe liver and cardiac dysfunction, as well as neurological symptoms during metabolic crises, whose pathogenesis is still poorly known. In this study, we demonstrate for the first time that pathological concentrations of 3-hydroxypalmitic acid (3HPA), the long-chain hydroxyl fatty acid (LCHFA) that most accumulates in LCHAD deficiency, significantly decreased adenosine triphosphate-linked and uncoupled mitochondrial respiration in intact cell systems consisting of heart fibers, cardiomyocytes, and hepatocytes, but less intense in diced forebrain. 3HPA also significantly reduced mitochondrial Ca2+ retention capacity and membrane potential in Ca2+ -loaded mitochondria more markedly in the heart and the liver, with mild or no effects in the brain, supporting a higher susceptibility of the heart and the liver to the toxic effects of this fatty acid. It is postulated that disruption of mitochondrial energy and Ca2+ homeostasis caused by the accumulation of LCHFA may contribute toward the severe cardiac and hepatic clinical manifestations observed in the affected patients.

Safety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With ST-Segment Elevation Myocardial Infarction and Left Ventricular Dysfunction.

RATIONALE: Allogeneic cardiac stem cells (AlloCSC-01) have shown protective, immunoregulatory, and regenerative properties with a robust safety profile in large animal models of heart disease. OBJECTIVE: To investigate the safety and feasibility of early administration of AlloCSC-01 in patients with ST-segment-elevation myocardial infarction. METHODS AND RESULTS: CAREMI (Safety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With STEMI and Left Ventricular Dysfunction) was a phase I/II multicenter, randomized, double-blind, placebo-controlled trial in patients with ST-segment-elevation myocardial infarction, left ventricular ejection fraction ≤45%, and infarct size ≥25% of left ventricular mass by cardiac magnetic resonance, who were randomized (2:1) to receive AlloCSC-01 or placebo through the intracoronary route at days 5 to 7. The primary end point was safety and included all-cause death and major adverse cardiac events at 30 days (all-cause death, reinfarction, hospitalization because of heart failure, sustained ventricular tachycardia, ventricular fibrillation, and stroke). Secondary safety end points included major adverse cardiac events at 6 and 12 months, adverse events, and immunologic surveillance. Secondary exploratory efficacy end points were changes in infarct size (percentage of left ventricular mass) and indices of ventricular remodeling by magnetic resonance at 12 months. Forty-nine patients were included (92% male, 55±11 years), 33 randomized to AlloCSC-01 and 16 to placebo. No deaths or major adverse cardiac events were reported at 12 months. One severe adverse events in each group was considered possibly related to study treatment (allergic dermatitis and rash). AlloCSC-01 elicited low levels of donor-specific antibodies in 2 patients. No immune-related adverse events were found, and no differences between groups were observed in magnetic resonance-based efficacy parameters at 12 months. The estimated treatment effect of AlloCSC-01 on the absolute change from baseline in infarct size was -2.3% (95% confidence interval, -6.5% to 1.9%). CONCLUSIONS: AlloCSC-01 can be safely administered in ST-segment-elevation myocardial infarction patients with left ventricular dysfunction early after revascularization. Low immunogenicity and absence of immune-mediated events will facilitate adequately powered studies to demonstrate their clinical efficacy in this setting. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT02439398.