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.

Pulse Duration Dependent Asymmetry in Molecular Transmembrane Transport Due to Electroporation in H9c2 Rat Cardiac Myoblast Cells In Vitro.

Electroporation (EP) is one of the successful physical methods for intracellular drug delivery, which temporarily permeabilizes plasma membrane by exposing cells to electric pulses. Orientation of cells in electric field is important for electroporation and, consequently, for transport of molecules through permeabilized plasma membrane. Uptake of molecules after electroporation are the greatest at poles of cells facing electrodes and is often asymmetrical. However, asymmetry reported was inconsistent and inconclusive-in different reports it was either preferentially anodal or cathodal. We investigated the asymmetry of polar uptake of calcium ions after electroporation with electric pulses of different durations, as the orientation of elongated cells affects electroporation to a different extent when using electric pulses of different durations in the range of 100 ns to 100 µs. The results show that with 1, 10, and 100 µs pulses, the uptake of calcium ions is greater at the pole closer to the cathode than at the pole closer to the anode. With shorter 100 ns pulses, the asymmetry is not observed. A different extent of electroporation at different parts of elongated cells, such as muscle or cardiac cells, may have an impact on electroporation-based treatments such as drug delivery, pulse-field ablation, and gene electrotransfection.

Spatiotemporal expression of long noncoding RNA Moshe modulates heart cell lineage commitment.

The roles of long non-coding RNA (LncRNA) have been highlighted in various development processes including congenital heart defects (CHD). Here, we characterized the molecular function of LncRNA, Moshe (1010001N08ik-203), one of the Gata6 antisense transcripts located upstream of Gata6, which is involved in both heart development and the most common type of congenital heart defect, atrial septal defect (ASD). During mouse embryonic development, Moshe was first detected during the cardiac mesoderm stage (E8.5 to E9.5) where Gata6 is expressed and continues to increase at the atrioventricular septum (E12.5), which is involved in ASD. Functionally, the knock-down of Moshe during cardiogenesis caused significant repression of Nkx2.5 in cardiac progenitor stages and resulted in the increase in major SHF lineage genes, such as cardiac transcriptional factors (Isl1, Hand2, Tbx2), endothelial-specific genes (Cd31, Flk1, Tie1, vWF), a smooth muscle actin (a-Sma) and sinoatrial node-specific genes (Shox2, Tbx18). Chromatin Isolation by RNA Purification showed Moshe activates Nkx2.5 gene expression via direct binding to its promoter region. Of note, Moshe was conserved across species, including human, pig and mouse. Altogether, this study suggests that Moshe is a heart-enriched lncRNA that controls a sophisticated network of cardiogenesis by repressing genes in SHF via Nkx2.5 during cardiac development and may play an important role in ASD.

Patterning of vertebrate cardiac progenitor fields by retinoic acid signaling.

The influence of retinoic acid (RA) signaling on vertebrate development has a well-studied history. Cumulatively, we now understand that RA signaling has a conserved requirement early in development restricting cardiac progenitors within the anterior lateral plate mesoderm of vertebrate embryos. Moreover, genetic and pharmacological manipulations of RA signaling in vertebrate models have shown that proper heart development is achieved through the deployment of positive and negative feedback mechanisms, which maintain appropriate RA levels. In this brief review, we present a chronological overview of key work that has led to a current model of the critical role for early RA signaling in limiting the generation of cardiac progenitors within vertebrate embryos. Furthermore, we integrate the previous work in mice and our recent findings using zebrafish, which together show that RA signaling has remarkably conserved influences on the later-differentiating progenitor populations at the arterial and venous poles. We discuss how recognizing the significant conservation of RA signaling on the differentiation of these progenitor populations offers new perspectives and may impact future work dedicated to examining vertebrate heart development.

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.

Rutin Modulates MAPK Pathway Differently from Quercetin in Angiotensin II-Induced H9c2 Cardiomyocyte Hypertrophy.

Rutin is a flavonoid with antioxidant property. It has been shown to exert cardioprotection against cardiomyocyte hypertrophy. However, studies regarding its antihypertrophic property are still lacking, whether it demonstrates similar antihypertrophic effect to its metabolite, quercetin. Hence, this study aimed to investigate the effects of both flavonoids on oxidative stress and mitogen-activated protein kinase (MAPK) pathway in H9c2 cardiomyocytes that were exposed to angiotensin II (Ang II) to induce hypertrophy. Cardiomyocytes were exposed to Ang II (600 nM) with or without quercetin (331 µM) or rutin (50 µM) for 24 h. A group given vehicle served as the control. The concentration of the flavonoids was chosen based on the reported effective concentration to reduce cell hypertrophy or cardiac injury in H9c2 cells. Exposure to Ang II increased cell surface area, intracellular superoxide anion level, NADPH oxidase and inducible nitric oxide synthase activities, and reduced cellular superoxide dismutase activity and nitrite level, which were similarly reversed by both rutin and quercetin. Rutin had no significant effects on phosphorylated proteins of extracellular signal-related kinases (ERK1/2) and p38 but downregulated phosphorylated c-Jun N-terminal kinases (JNK1/2), which were induced by Ang II. Quercetin, on the other hand, had significantly downregulated the phosphorylated proteins of ERK1/2, p38, and JNK1/2. The quercetin inhibitory effect on JNK1/2 was stronger than the rutin. In conclusion, both flavonoids afford similar protective effects against Ang II-induced cardiomyocyte hypertrophy, but they differently modulate MAPK pathway.

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.

Characterization of a common progenitor pool of the epicardium and myocardium.

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.

Metabolic and Redox Regulation of Cardiovascular Stem Cell Biology and Pathology.

Significance: Cardiovascular stem cells are important for regeneration and repair of damaged tissue. Recent Advances: Pluripotent stem cells have a unique metabolism, which is adopted for their energetic and biosynthetic demand as rapidly proliferating cells. Stem cell differentiation requires an exceptional metabolic flexibility allowing for metabolic remodeling between glycolysis and oxidative phosphorylation. Critical Issues: Respiration is associated with the generation of reactive oxygen species (ROS) by the mitochondrial respiratory chain. But also the membrane-bound protein nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) contributes to ROS levels. ROS not only play a significant role in stem cell differentiation and tissue renewal but also cause senescence and contribute to tissue aging. Future Directions: For utilization of stem cells in therapeutic approaches, a deep understanding of the molecular mechanisms how metabolism and the cellular redox state regulate stem cell differentiation is required. Modulating the redox state of stem cells using antioxidative agents may be suitable to enhance activity of endothelial progenitor cells. Antioxid. Redox Signal. 35, 163-181.

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.

Fetal Hypoxia Impacts on Proliferation and Differentiation of Sca-1+ Cardiac Progenitor Cells and Maturation of Cardiomyocytes: A Role of MicroRNA-210.

Hypoxia is one of the most frequent and severe stresses to an organism's homeostatic mechanisms, and hypoxia during gestation has profound adverse effects on the heart development increasing the occurrence of congenital heart defects (CHDs). Cardiac progenitor cells (CPCs) are responsible for early heart development and the later occurrence of heart disease. However, the mechanism of how hypoxic stress affects CPC fate decisions and contributes to CHDs remains a topic of debate. Here we examined the effect of hypoxic stress on the regulations of CPC fate decisions and the potential mechanism. We found that experimental induction of hypoxic responses compromised CPC function by regulating CPC proliferation and differentiation and restraining cardiomyocyte maturation. In addition, echocardiography indicated that fetal hypoxia reduced interventricular septum thickness at diastole and the ejection time, but increased the heart rate, in mouse young adult offspring with a gender-related difference. Further study revealed that hypoxia upregulated microRNA-210 expression in Sca-1+ CPCs and impeded the cell differentiation. Blockage of microRNA-210 with LNA-anti-microRNA-210 significantly promoted differentiation of Sca-1+ CPCs into cardiomyocytes. Thus, the present findings provide clear evidence that hypoxia alters CPC fate decisions and reveal a novel mechanism of microRNA-210 in the hypoxic effect, raising the possibility of microRNA-210 as a potential therapeutic target for heart disease.

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats.

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05-5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients.

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.

Matrix-guided control of mitochondrial function in cardiac myocytes.

In ventricular myocardium, extracellular matrix (ECM) remodeling is a hallmark of physiological and pathological growth, coincident with metabolic rewiring of cardiac myocytes. However, the direct impact of the biochemical and mechanical properties of the ECM on the metabolic function of cardiac myocytes is mostly unknown. Furthermore, understanding the impact of distinct biomaterials on cardiac myocyte metabolism is critical for engineering physiologically-relevant models of healthy and diseased myocardium. For these reasons, we systematically measured morphological and metabolic responses of neonatal rat ventricular myocytes cultured on fibronectin- or gelatin-coated polydimethylsiloxane (PDMS) of three elastic moduli and gelatin hydrogels with four elastic moduli. On all substrates, total protein content, cell morphology, and the ratio of mitochondrial DNA to nuclear DNA were preserved. Cytotoxicity was low on all substrates, although slightly higher on PDMS compared to gelatin hydrogels. We also quantified oxygen consumption rates and extracellular acidification rates using a Seahorse extracellular flux analyzer. Our data indicate that several metrics associated with baseline glycolysis and baseline and maximum mitochondrial function are enhanced when cardiac myocytes are cultured on gelatin hydrogels compared to all PDMS substrates, irrespective of substrate rigidity. These results yield new insights into how mechanical and biochemical cues provided by the ECM impact mitochondrial function in cardiac myocytes. STATEMENT OF SIGNIFICANCE: Cardiac development and disease are associated with remodeling of the extracellular matrix coincident with metabolic rewiring of cardiac myocytes. However, little is known about the direct impact of the biochemical and mechanical properties of the extracellular matrix on the metabolic function of cardiac myocytes. In this study, oxygen consumption rates were measured in neonatal rat ventricular myocytes maintained on several commonly-used biomaterial substrates to reveal new relationships between the extracellular matrix and cardiac myocyte metabolism. Several mitochondrial parameters were enhanced on gelatin hydrogels compared to synthetic PDMS substrates. These data are important for comprehensively understanding matrix-regulation of cardiac myocyte physiology. Additionally, these data should be considered when selecting scaffolds for engineering in vitro cardiac tissue models.

Cited2 regulates proliferation and survival in young and old mouse cardiac stem cells.

BACKGROUND: Cardiac stem cells (CSCs) exhibit age-dependent characteristics. Cited2 has been implicated in the regulation of heart development; however, there is little known about how Cited2 affects CSC aging. RESULTS: Cited2 mRNA and protein level was downregulated in aging heart tissue and CSCs. Old (O)-CSCs showed decreased differentiation and proliferation capacities as compared to Young (Y)-CSCs, the decrease in cell proliferation, increase in apoptosis, and cell cycle arrest in G0/G1 phase in CSCs are mediated by knocdown CITED2expression in (Y)-CSCs. CONCLUSIONS: Cited2 plays an important role in cell cycle progression and in maintaining the balance between CSC proliferation and apoptosis in the process of aging without influencing cell fate decisions. These findings have important implications for cell-based therapies for heart repair.