Human Induced Pluripotent Stem Cells: From Cell Origin, Genomic Stability, and Epigenetic Memory to Translational Medicine.

The potential of human induced pluripotent stem cells (iPSCs) to self-renew indefinitely and to differentiate virtually into any cell type in unlimited quantities makes them attractive for in vitro disease modeling, drug screening, personalized medicine, and regenerative therapies. As the genome of iPSCs thoroughly reproduces that of the somatic cells from which they are derived, they may possess genetic abnormalities, which would seriously compromise their utility and safety. Genetic aberrations could be present in donor somatic cells and then transferred during iPSC generation, or they could occur as de novo mutations during reprogramming or prolonged cell culture. Therefore, to warrant the safety of human iPSCs for clinical applications, analysis of genetic integrity, particularly during iPSC generation and differentiation, should be carried out on a regular basis. On the other hand, reprogramming of somatic cells to iPSCs requires profound modifications in the epigenetic landscape. Changes in chromatin structure by DNA methylations and histone tail modifications aim to reset the gene expression pattern of somatic cells to facilitate and establish self-renewal and pluripotency. However, residual epigenetic memory influences the iPSC phenotype, which may affect their application in disease therapeutics. The present review discusses the somatic cell origin, genetic stability, and epigenetic memory of iPSCs and their impact on basic and translational research.

The small molecule ACM-001 improves cardiac function in a rat model of severe cancer cachexia.

AIMS: Cachexia, a common manifestation of malignant cancer, is not only associated with weight loss, but also with severe cardiac atrophy and impaired cardiac function. Here, we investigated the effects of ACM-001 (0.3 or 3 mg/kg/day) in comparison to carvedilol (3 or 30 mg/kg/day), metropolol (50 or 100 mg/kg/day), nebivolol (1 or 10 mg/kg/day) and tertatolol (0.5 or 5 mg/kg/day) on cardiac mass and function in a rat cancer cachexia model. METHODS AND RESULTS: Young male Wistar Han rats were inoculated i.p. with 108 Yoshida hepatoma AH-130 cells and treated once daily with verum or placebo by gavage. Cardiac function (echocardiography), body weight and body composition (nuclear magnetic resonance scans) were assessed. The hearts of animals were euthanized on day 11 (placebo and 3 mg/kg/day ACM-001) were used for signalling studies. Beta-blockers had no effect on tumour burden. ACM-001 reduced body weight loss (placebo: -34 ± 2.4 g vs. 3 mg/kg/day ACM-001: -14.8 ± 8.4 g, p = 0.033). Lean mass wasting was attenuated (placebo: -16.5 ± 2.34 g vs. 3 mg/kg/day ACM-001: -2.4 ± 6.7 g, p = 0.037), while fat loss was similar (p = 0.4) on day 11. Placebo animals lost left ventricular mass (-101 ± 14 mg), which was prevented only by 3 mg/kg/day ACM-001 (7 ± 25 mg, p < 0.01 vs. placebo). ACM-001 improved the ejection fraction (EF) (ΔEF: placebo: -24.3 ± 2.6 vs. 3 mg/kg/day ACM-001: 0.1 ± 2.9, p < 0.001). Cardiac output was 50% lower in the placebo group (-41 ± 4 ml/min) compared to baseline, while 3 mg/kg/day ACM-001 preserved cardiac output (-5 ± 8 ml/min, p < 0.01). The molecular mechanisms involved inhibition of protein degradation and activation of protein synthesis pathways. CONCLUSION: This study shows that 3 mg/kg/day ACM-001 restores the anabolic/catabolic balance in cardiac muscle leading to improved function. Moreover, not all beta-blockers have similar effects.

Synergistic Adverse Effects of Azithromycin and Hydroxychloroquine on Human Cardiomyocytes at a Clinically Relevant Treatment Duration.

Adverse effects of drug combinations and their underlying mechanisms are highly relevant for safety evaluation, but often not fully studied. Hydroxychloroquine (HCQ) and azithromycin (AZM) were used as a combination therapy in the treatment of COVID-19 patients at the beginning of the pandemic, leading to higher complication rates in comparison to respective monotherapies. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ, AZM, and their combination on the structure and functionality of cardiomyocytes, and to better understand the underlying mechanisms. Our results demonstrate synergistic adverse effects of AZM and HCQ on electrophysiological and contractile function of iPSC-CMs. HCQ-induced prolongation of field potential duration (FPDc) was gradually increased during 7-day treatment period and was strongly enhanced by combination with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, more pronounced contractile dysfunctions, and more elevated conduction velocity, compared to respective monotreatments. Mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased cellular accumulation of HCQ and AZM as well as increased Cx43- and Nav1.5-protein levels.

Role of Leptin in Cardiovascular Diseases.

The adipocyte-derived adipokine leptin exerts pleiotropic effects, which are essential for the regulation of energy balance and cell metabolism, for controlling inflammatory and immune responses, and for the maintenance of homeostasis of the cardiovascular system. Leptin resistance in obese or type 2 diabetes mellitus (T2DM) patients is defined as a decrease in tissue response to leptin. In the cardiovascular system, leptin resistance exhibits the adverse effect on the heart's response to stress conditions and promoting cardiac remodeling due to impaired cardiac metabolism, increased fibrosis, vascular dysfunction, and enhanced inflammation. Leptin resistance or leptin signaling deficiency results in the risk increase of cardiac dysfunction and heart failure, which is a leading cause of obesity- and T2DM-related morbidity and mortality. Animal studies using leptin- and leptin receptor- (Lepr) deficient rodents have provided many useful insights into the underlying molecular and pathophysiological mechanisms of obese- and T2DM-associated metabolic and cardiovascular diseases. However, none of the animal models used so far can fully recapitulate the phenotypes of patients with obese or T2DM. Therefore, the role of leptin in the human cardiovascular system, and whether leptin affects cardiac function directly or acts through a leptin-regulated neurohumoral pathway, remain elusive. As the prevalence of obesity and diabetes is continuously increasing, strategies are needed to develop and apply human cell-based models to better understand the precise role of leptin directly in different cardiac cell types and to overcome the existing translational barriers. The purpose of this review is to discuss the mechanisms associated with leptin signaling deficiency or leptin resistance in the development of metabolic and cardiovascular diseases. We analyzed and comprehensively addressed substantial findings in pathophysiological mechanisms in commonly used leptin- or Lepr-deficient rodent models and highlighted the differences between rodents and humans. This may open up new strategies to develop directly and reliably applicable models, which resemble the human pathophysiology in order to advance health care management of obesity- and T2DM-related cardiovascular complications.

IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency.

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2-/-) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2-/--iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca2+ transients in RYR2-/--iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca2+ ([Ca2+ ]o) and caffeine in a concentration-dependent manner, while RYR2-/--iPSC-CMs showed inconsistent reactions to [Ca2+ ]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2-/--iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2-/--iPSC-CMs is mediated by SERCA. Additionally, RYR2-/--iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency.