Non-human primate studies for cardiomyocyte transplantation-ready for translation?

Non-human primates (NHP) are valuable models for late translational pre-clinical studies, often seen as a last step before clinical application. The unique similarity between NHPs and humans is often the subject of ethical concerns. However, it is precisely this analogy in anatomy, physiology, and the immune system that narrows the translational gap to other animal models in the cardiovascular field. Cell and gene therapy approaches are two dominant strategies investigated in the research field of cardiac regeneration. Focusing on the cell therapy approach, several xeno- and allogeneic cell transplantation studies with a translational motivation have been realized in macaque species. This is based on the pressing need for novel therapeutic options for heart failure patients. Stem cell-based remuscularization of the injured heart can be achieved via direct injection of cardiomyocytes (CMs) or patch application. Both CM delivery approaches are in the late preclinical stage, and the first clinical trials have started. However, are we already ready for the clinical area? The present review concentrates on CM transplantation studies conducted in NHPs, discusses the main sources and discoveries, and provides a perspective about human translation.

Functions and Clinical Significance of Myocardial Cell-Derived Immunoglobulins.

Immunoglobulins (Igs) have been widely accepted to be exclusively expressed by B cells. Nonetheless, this theory is challenged by mounting evidence which suggests that Igs can also be generated by non B cells (non B-Ig), including cardiomyocytes (CM). Non B-Ig exhibits unique physical and chemical characteristics, unique variable region sequences and functions, which diverge from those of B-Ig. For instance, non B-Ig demonstrates hydrophobicity, limited diversity in the variable region, and extracellular matrix protein activity. Likewise, cardiomyocytes can express different classes of Igs, including IgM, IgG, and free Igκ light chains (cardiomyocyte derived-Igs, CM-Igs). In particular, CM-Igs can be secreted into the extracellular space in various cardiovascular diseases, such as myocardial ischaemia and myocardial fibrosis where they might be involved in complement activation and direct damage to cardiomyocytes. Nevertheless, the precise pathological activity of CM-Igs remains unclear. Recently, Zhu et al. focused on studying the sequence characteristics and functions of CM-Igκ; they discovered that the CM-Igκ exhibits a unique VJ recombination pattern, high hydrophobicity, and is principally located on the intercalated discs and cross striations of the cardiomyocytes. Interestingly, loss of Igκ in cardiomyocytes results in structural disorders in intercalated discs and dysfunction in myocardial contraction and conduction. Mechanically, Igκ promotes the stabilisation of plectin, a cytoskeleton cross-linker protein that connects desmin to desomsome, to maintain the normal structure of the intercalated disc. This finding indicates that CM-Igκ plays an integral role in maintaining cytoskeleton structure. Consequently, it is imperative to reveal the physiological functions and mechanisms of pathological injury associated with CM-Igs.

SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

The ischemia-enhanced myocardial infarction protection-related lncRNA protects against acute myocardial infarction.

Long non-coding RNA RP11-64B16.4 (myocardial infarction protection-related lncRNA [MIPRL]) is among the most abundant and the most upregulated lncRNAs in ischemic human hearts. However, its role in ischemic heart disease is unknown. We found MIPRL was conserved between human and mouse and its expression was increased in mouse hearts after acute myocardial infarction (AMI) and in cultured human and mouse cardiomyocytes after hypoxia. The infarcted size, cardiac cell apoptosis, cardiac dysfunction, and cardiac fibrosis were aggravated in MIPRL knockout mice after AMI. The above adverse results could be reversed by re-expression of MIPRL via adenovirus expressing MIPRL. Both in vitro and in vivo, we identified that heat shock protein beta-8 (HSPB8) was a target gene of MIPRL, which was involved in MIPRL-mediated anti-apoptotic effects on cardiomyocytes. We further discovered that MIPRL could combine with the messenger RNA (mRNA) of HSPB8 and increase its expression in cardiomyocytes by enhancing the stability of HSPB8 mRNA. In summary, we have found for the first time that the ischemia-enhanced lncRNA MIPRL protects against AMI via its target gene HSPB8. MIPRL might be a novel promising therapeutic target for ischemic heart diseases such as AMI.

Generation of cardiomyocytes from stem cells cultured on nanofibrous scaffold: Experimental approach for attenuation of myocardial infarction.

The current study was constructed to fabricate polyamide based nanofibrous scaffolds (NS) and to define the most promising one for the generation of cardiomyocytes from adipose tissue derived mesenchymal stem cells (ADMSCs). This purpose was extended to assess the potentiality of the generated cardiomyocytes in relieving myocardial infarction (MI) in rats. Production and characterization of NSs were carried out. ADMSCs were cultured on NS and induced to differentiate into cardiomyocytes by specific growth factors. Molecular analysis for myocyte-specific enhancer factor 2 C (MEF2C) and alpha sarcomeric actin (α-SCA) expression was done to confirm the differentiation of ADMSCs into cardiomyocytes for further transplantation into MI induced rats. Implantation of cells in MI afflicted rats boosted heart rate, ST height and PR interval and lessened P duration, RR, QTc and QRS intervals. Also, this type of medication minified serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) enzymes activity as well as serum and cardiac troponin T (Tn-T) levels and upraised serum and cardiac α-SCA and cardiac connexin 43 (CX 43) levels. Microscopic feature of cardiac tissue sections of rats in the treated groups revealed great renovation in the cardiac microarchitecture. Conclusively, this attempt gains insight into a realistic strategy for recovery of MI through systemic employment of in vitro generated cardiomyocytes.

SENP1-Mediated HSP90ab1 DeSUMOylation in Cardiomyocytes Prevents Myocardial Fibrosis by Paracrine Signaling.

Myocardial infarction (MI) triggers a poor ventricular remodeling response, but the underlying mechanisms remain unclear. Here, the authors show that sentrin-specific protease 1 (SENP1) is downregulated in post-MI mice and in patients with severe heart failure. By generating cardiomyocyte-specific SENP1 knockout and overexpression mice to assess cardiac function and ventricular remodeling responses under physiological and pathological conditions. Increased cardiac fibrosis in the cardiomyocyte-specific SENP1 deletion mice, associated with increased fibronectin (Fn) expression and secretion in cardiomyocytes, promotes fibroblast activation in response to myocardial injury. Mechanistically, SENP1 deletion in mouse cardiomyocytes increases heat shock protein 90 alpha family class B member 1 (HSP90ab1) SUMOylation with (STAT3) activation and Fn secretion after ventricular remodeling initiated. Overexpression of SENP1 or mutation of the HSP90ab1 Lys72 ameliorates adverse ventricular remodeling and dysfunction after MI. Taken together, this study identifies SENP1 as a positive regulator of cardiac repair and a potential drug target for the treatment of MI. Inhibition of HSP90ab1 SUMOylation stabilizes STAT3 to inhibit the adverse ventricular remodeling response.

Deep Learning Resolves Myovascular Dynamics in the Failing Human Heart.

The adult mammalian heart harbors minute levels of cycling cardiomyocytes (CMs). Large numbers of images are needed to accurately quantify cycling events using microscopy-based methods. CardioCount is a new deep learning-based pipeline to rigorously score nuclei in microscopic images. When applied to a repository of 368,434 human microscopic images, we found evidence of coupled growth between CMs and cardiac endothelial cells in the adult human heart. Additionally, we found that vascular rarefaction and CM hypertrophy are interrelated in end-stage heart failure. CardioCount is available for use via GitHub and via Google Colab for users with minimal machine learning experience.

Glycation in the cardiomyocyte.

Glycation is a protein post-translational modification that can occur on lysine and arginine residues as a result of a non-enzymatic process known as the Maillard reaction. This modification is irreversible, so the only way it can be removed is by protein degradation and replacement. Small reactive carbonyl species, glyoxal and methylglyoxal, are the primary glycating agents and are elevated in several conditions associated with an increased risk of cardiovascular disease, including diabetes, rheumatoid arthritis, smoking, and aging. Thus, how protein glycation impacts the cardiomyocyte is of particular interest, to both understand how these conditions increase the risk of cardiovascular disease and how glycation might be targeted therapeutically. Glycation can affect the cardiomyocyte through extracellular mechanisms, including RAGE-based signaling, glycation of the extracellular matrix that modifies the mechanical environment, and signaling from the vasculature. Intracellular glycation of the cardiomyocyte can impact calcium handling, protein quality control and cell death pathways, as well as the cytoskeleton, resulting in a blunted contractility. While reducing protein glycation and its impact on the heart has been an active area of drug development, multiple clinical trials have had mixed results and these compounds have not been translated to the clinic-highlighting the challenges of modulating myocyte glycation. Here we will review protein glycation and its effects on the cardiomyocyte, therapeutic attempts to reverse these, and offer insight as to the future of glycation studies and patient treatment.

Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling.

BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection. METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385. RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs. CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

Improving iPSC Differentiation Using a Nanodot Platform.

Differentiation of induced pluripotent stem cells (iPSCs) is an extremely complex process that has proven difficult to study. In this research, we utilized nanotopography to elucidate details regarding iPSC differentiation by developing a nanodot platform consisting of nanodot arrays of increasing diameter. Subjecting iPSCs cultured on the nanodot platform to a cardiomyocyte (CM) differentiation protocol revealed several significant gene expression profiles that were associated with poor differentiation. The observed expression trends were used to select existing small-molecule drugs capable of modulating differentiation efficiency. BRD K98 was repurposed to inhibit CM differentiation, while iPSCs treated with NSC-663284, carmofur, and KPT-330 all exhibited significant increases in not only CM marker expression but also spontaneous beating, suggesting improved CM differentiation. In addition, quantitative polymerase chain reaction was performed to determine the gene regulation responsible for modulating differentiation efficiency. Multiple genes involved in extracellular matrix remodeling were correlated with a CM differentiation efficiency, while genes involved in the cell cycle exhibited contrasting expression trends that warrant further studies. The results suggest that expression profiles determined via short time-series expression miner analysis of nanodot-cultured iPSC differentiation can not only reveal drugs capable of enhancing differentiation efficiency but also highlight crucial sets of genes related to processes such as extracellular matrix remodeling and the cell cycle that can be targeted for further investigation. Our findings confirm that the nanodot platform can be used to reveal complex mechanisms behind iPSC differentiation and could be an indispensable tool for optimizing iPSC technology for clinical applications.

Near-infrared-triggered plasmonic regulation and cardiomyocyte-based biosensing system for in vitro bradyarrhythmia treatment.

Bradyarrhythmia, a life-threatening cardiovascular disease, is an increasing burden for the healthcare system. Currently, surgery, implanted device, and drug are introduced to treat the bradyarrhythmia in clinical practice. However, these conventional therapeutic strategies suffer from the invasive surgery, power supply, or drug side effect, respectively, hence developing the alternative therapeutic strategy is necessarily imperative. Here, a convenient and effective strategy to treat the bradyarrhythmia is proposed using near-infrared-triggered Au nanorod (NR) based plasmonic photothermal effect (PPE). Moreover, electrophysiology of cardiomyocytes is dynamically monitored by the integrated biosensing-regulating system during and after the treatment. Cardiomyocyte-based bradyarrhythmia recover rhythmic for a long time by regulating plasmonic photothermal effect. Furthermore, the regulatory mechanism is qualitatively investigated to verify the significant thermal stimulation in the recovery process. This study establishes a reliable platform for long-term recording and evaluation of mild photothermal therapy for bradyarrhythmia in vitro, offering an efficient and non-invasive strategy for the potential clinical applications.

Transfer of cardiomyocyte-derived extracellular vesicles to neighboring cardiac cells requires tunneling nanotubes during heart development.

Rationale: Extracellular vesicles (EVs) are thought to mediate intercellular communication during development and disease. Yet, biological insight to intercellular EV transfer remains elusive, also in the heart, and is technically challenging to demonstrate. Here, we aimed to investigate biological transfer of cardiomyocyte-derived EVs in the neonatal heart. Methods: We exploited CD9 as a marker of EVs, and generated two lines of cardiomyocyte specific EV reporter mice: Tnnt2-Cre; double-floxed inverted CD9/EGFP and αMHC-MerCreMer; double-floxed inverted CD9/EGFP. The two mouse lines were utilized to determine whether developing cardiomyocytes transfer EVs to other cardiac cells (non-myocytes and cardiomyocytes) in vitro and in vivo and investigate the intercellular transport pathway of cardiomyocyte-derived EVs. Results: Genetic tagging of cardiomyocytes was confirmed in both reporter mouse lines and proof of concept in the postnatal heart showed that, a fraction of EGFP+/MYH1- non-myocytes exist firmly demonstrating in vivo cardiomyocyte-derived EV transfer. However, two sets of direct and indirect EGFP +/- cardiac cell co-cultures showed that cardiomyocyte-derived EGFP+ EV transfer requires cell-cell contact and that uptake of EGFP+ EVs from the medium is limited. The same was observed when co-cultiring with mouse macrophages. Further mechanistic insight showed that cardiomyocyte EV transfer occurs through type I tunneling nanotubes. Conclusion: While the current notion assumes that EVs are transferred through secretion to the surroundings, our data show that cardiomyocyte-derived EV transfer in the developing heart occurs through nanotubes between neighboring cells. Whether these data are fundamental and relate to adult hearts and other organs remains to be determined, but they imply that the normal developmental process of EV transfer goes through cell-cell contact rather than through the extracellular compartment.

Unravelling the Impact of RNA Methylation Genetic and Epigenetic Machinery in the Treatment of Cardiomyopathy.

Cardiomyopathy (CM) represents a heterogeneous group of diseases primarily affecting cardiac structure and function, with genetic and epigenetic dysregulation playing a pivotal role in its pathogenesis. Emerging evidence from the burgeoning field of epitranscriptomics has brought to light the significant impact of various RNA modifications, notably N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N1-methyladenosine (m1A), 2'-O-methylation (Nm), and 6,2'-O-dimethyladenosine (m6Am), on cardiomyocyte function and the broader processes of cardiac and vascular remodelling. These modifications have been shown to influence key pathological mechanisms including mitochondrial dysfunction, oxidative stress, cardiomyocyte apoptosis, inflammation, immune response, and myocardial fibrosis. Importantly, aberrations in the RNA methylation machinery have been observed in human CM cases and animal models, highlighting the critical role of RNA methylating enzymes and their potential as therapeutic targets or biomarkers for CM. This review underscores the necessity for a deeper understanding of RNA methylation processes in the context of CM, to illuminate novel therapeutic avenues and diagnostic tools, thereby addressing a significant gap in the current management strategies for this complex disease.

M1 macrophage-derived exosomes inhibit cardiomyocyte proliferation through delivering miR-155.

BACKGROUND: M1 macrophages are closely associated with cardiac injury after myocardial infarction (MI). Increasing evidence shows that exosomes play a key role in pathophysiological regulation after MI, but the role of M1 macrophage-derived exosomes (M1-Exos) in myocardial regeneration remains unclear. In this study, we explored the impact of M1 macrophage-derived exosomes on cardiomyocytes regeneration in vitro and in vivo. METHODS: M0 macrophages were induced to differentiate into M1 macrophages with GM-CSF (50 ng/mL) and IFN-γ (20 ng/mL). Then M1-Exos were isolated and co-incubated with cardiomyocytes. Cardiomyocyte proliferation was detected by pH3 or ki67 staining. Quantitative real-time PCR (qPCR) was used to test the level of miR-155 in macrophages, macrophage-derived exosomes and exosome-treated cardiomyocytes. MI model was constructed and LV-miR-155 was injected around the infarct area, the proliferation of cardiomyocytes was counted by pH3 or ki67 staining. The downstream gene and pathway of miR-155 were predicted and verified by dual-luciferase reporter gene assay, qPCR and immunoblotting analysis. IL-6 (50 ng/mL) was added to cardiomyocytes transfected with miR-155 mimics, and the proliferation of cardiomyocytes was calculated by immunofluorescence. The protein expressions of IL-6R, p-JAK2 and p-STAT3 were detected by Western blot. RESULTS: The results showed that M1-Exos suppressed cardiomyocytes proliferation. Meanwhile, miR-155 was highly expressed in M1-Exos and transferred to cardiomyocytes. miR-155 inhibited the proliferation of cardiomyocytes and antagonized the pro-proliferation effect of interleukin 6 (IL-6). Furthermore, miR-155 targeted gene IL-6 receptor (IL-6R) and inhibited the Janus kinase 2(JAK)/Signal transducer and activator of transcription (STAT3) signaling pathway. CONCLUSION: M1-Exos inhibited cardiomyocyte proliferation by delivering miR-155 and inhibiting the IL-6R/JAK/STAT3 signaling pathway. This study provided new insight and potential treatment strategy for the regulation of myocardial regeneration and cardiac repair by macrophages.

Runx1 is sufficient but not required for cardiomyocyte cell cycle activation.

Factors responsible for cardiomyocyte proliferation could serve as potential therapeutics to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach on cardiomyocyte cell cycle and ploidy led us to the transcription factor, RUNX1. Here, we examine the effect of Runx1 on cardiomyocyte cell cycle during postnatal development and cardiac regeneration using cardiomyocyte-specific gain- and loss-of-function mouse models. RUNX1 is expressed in cardiomyocytes during early postnatal life, decreases to negligible levels by 3 weeks of age, and increases upon myocardial injury, all consistent with observed rates of cardiomyocyte cell cycle activity. Loss of Runx1 transiently stymied cardiomyocyte cell cycle activity during normal postnatal development, a result that corrected itself and did not extend to the context of neonatal heart regeneration. On the other hand, cardiomyocyte-specific Runx1 overexpression resulted in an expansion of diploid cardiomyocytes in uninjured hearts and expansion of 4N cardiomyocytes in the context of neonatal cardiac injury, suggesting Runx1 overexpression is sufficient to induce cardiomyocyte cell cycle responses. Persistent overexpression of Runx1 for >1 month continued to promote cardiomyocyte cell cycle activity resulting in substantial hyperpolyploidization (≥8N DNA content). This persistent cell cycle activation was accompanied by ventricular dilation and adverse remodeling, raising the concern that continued cardiomyocyte cell cycling can have detrimental effects.

Programmed death of cardiomyocytes in cardiovascular disease and new therapeutic approaches.

Cardiovascular diseases (CVDs) have a complex pathogenesis and pose a major threat to human health. Cardiomyocytes have a low regenerative capacity, and their death is a key factor in the morbidity and mortality of many CVDs. Cardiomyocyte death can be regulated by specific signaling pathways known as programmed cell death (PCD), including apoptosis, necroptosis, autophagy, pyroptosis, and ferroptosis, etc. Abnormalities in PCD can lead to the development of a variety of cardiovascular diseases, and there are also molecular-level interconnections between different PCD pathways under the same cardiovascular disease model. Currently, the link between programmed cell death in cardiomyocytes and cardiovascular disease is not fully understood. This review describes the molecular mechanisms of programmed death and the impact of cardiomyocyte death on cardiovascular disease development. Emphasis is placed on a summary of drugs and potential therapeutic approaches that can be used to treat cardiovascular disease by targeting and blocking programmed cell death in cardiomyocytes.

Fabrication and Optimization of Poly(ε-caprolactone) Microspheres Loaded with S-Nitroso-N-Acetylpenicillamine for Nitric Oxide Delivery.

Heart disease is one of the leading causes of death in the United States and throughout the world. While there are different techniques for reducing or preventing the impact of heart disease, nitric oxide (NO) is administered as nitroglycerin for reversing angina or chest pain. Unfortunately, due to its gaseous and short-lived half-life, NO can be difficult to study or even administer. Therefore, controlled delivery of NO is desirable for therapeutic use. In the current study, the goal was to fabricate NO-releasing microspheres (MSs) using a donor molecule, S-Nitroso-N-Acetyl penicillamine, (SNAP), and encapsulating it in poly(ε-caprolactone) (PCL) using a single-emulsion technique that can provide sustained delivery of NO to cells over time without posing any toxicity risks. Optimization of the fabrication process was performed by varying the duration of homogenization (5, 10, and 20 min) and its effect on entrapment efficiency and size. The optimized SNAP-MS had an entrapment efficiency of ˃50%. Furthermore, we developed a modified method for NO detection by using NO microsensors to detect the NO release from SNAP-MSs in real time, showing sustained release behavior. The fabricated SNAP-MSs were tested for biocompatibility with HUVECs (human umbilical vein endothelial cells), which were found to be biocompatible. Lastly, we tested the effect of controlled NO delivery to human induced pluripotent stem-derived cardiomyocytes (hiPSC-CMs) via SNAP-MSs, which showed a significant improvement in the electrophysiological parameters and alleviated anoxic stress.

A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

Contractility assessment using aligned human iPSC-derived cardiomyocytes.

INTRODUCTION: Cardiac safety assessment, such as lethal arrhythmias and contractility dysfunction, is critical during drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been shown to be useful in predicting drug-induced proarrhythmic risk through international validation studies. Although cardiac contractility is another key function, fit-for-purpose hiPSC-CMs in evaluating drug-induced contractile dysfunction remain poorly understood. In this study, we investigated whether alignment of hiPSC-CMs on nanopatterned culture plates can assess drug-induced contractile changes more efficiently than non-aligned monolayer culture. METHODS: Aligned hiPSC-CMs were obtained by culturing on 96-well culture plates with a ridge-groove-ridge nanopattern on the bottom surface, while non-aligned hiPSC-CMs were cultured on regular 96-well plates. Next-generation sequencing and qPCR experiments were performed for gene expression analysis. Contractility of the hiPSC-CMs was assessed using an imaging-based motion analysis system. RESULTS: When cultured on nanopatterned plates, hiPSC-CMs exhibited an aligned morphology and enhanced expression of genes encoding proteins that regulate contractility, including myosin heavy chain, calcium channel, and ryanodine receptor. Compared to cultures on regular plates, the aligned hiPSC-CMs also showed both enhanced contraction and relaxation velocity. In addition, the aligned hiPSC-CMs showed a more physiological response to positive and negative inotropic agents, such as isoproterenol and verapamil. DISCUSSION: Taken together, the aligned hiPSC-CMs exhibited enhanced structural and functional properties, leading to an improved capacity for contractility assessment compared to the non-aligned cells. These findings suggest that the aligned hiPSC-CMs can be used to evaluate drug-induced cardiac contractile changes.