Disruption of TUFT1, a Desmosome-Associated Protein, Causes Skin Fragility, Woolly Hair, and Palmoplantar Keratoderma.

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.

Evaluation of Senescence and Its Prevention in Doxorubicin-Induced Cardiotoxicity Using Dynamic Engineered Heart Tissues.

Background: Doxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence. Objectives: The aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target. Methods: Biopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell-derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol. Results: Senescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function. Conclusions: Senescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.

Iron deficiency is related to lower muscle mass in community-dwelling individuals and impairs myoblast proliferation.

BACKGROUND: Loss of muscle mass is linked with impaired quality of life and an increased risk of morbidity and premature mortality. Iron is essential for cellular processes such as energy metabolism, nucleotide synthesis and numerous enzymatic reactions. As the effects of iron deficiency (ID) on muscle mass and function are largely unknown, we aimed to assess the relation between ID and muscle mass in a large population-based cohort, and subsequently studied effects of ID on cultured skeletal myoblasts and differentiated myocytes. METHODS: In a population-based cohort of 8592 adults, iron status was assessed by plasma ferritin and transferrin saturation, and muscle mass was estimated using 24-h urinary creatinine excretion rate (CER). The relationships of ferritin and transferrin saturation with CER were assessed by multivariable logistic regression. Furthermore, mouse C2C12 skeletal myoblasts and differentiated myocytes were subjected to deferoxamine with or without ferric citrate. Myoblast proliferation was measured with a colorimetric 5-bromo-2'-deoxy-uridine ELISA assay. Myocyte differentiation was assessed using Myh7-stainings. Myocyte energy metabolism, oxygen consumption rate and extracellular acidification rate were assessed using Seahorse mitochondrial flux analysis, and apoptosis rate with fluorescence-activated cell sorting. RNA sequencing (RNAseq) was used to identify ID-related gene and pathway enrichment in myoblasts and myocytes. RESULTS: Participants in the lowest age- and sex-specific quintile of plasma ferritin (OR vs middle quintile 1.62, 95% CI 1.25-2.10, P < 0.001) or transferrin saturation (OR 1.34, 95% CI 1.03-1.75, P = 0.03) had a significantly higher risk of being in the lowest age- and sex-specific quintile of CER, independent of body mass index, estimated GFR, haemoglobin, hs-CRP, urinary urea excretion, alcohol consumption and smoking status. In C2C12 myoblasts, deferoxamine-induced ID reduced myoblast proliferation rate (P-trend <0.001) but did not affect differentiation. In myocytes, deferoxamine reduced myoglobin protein expression (-52%, P < 0.001) and tended to reduce mitochondrial oxygen consumption capacity (-28%, P = 0.10). Deferoxamine induced gene expression of cellular atrophy markers Trim63 (+20%, P = 0.002) and Fbxo32 (+27%, P = 0.048), which was reversed by ferric citrate (-31%, P = 0.04 and -26%, P = 0.004, respectively). RNAseq indicated that both in myoblasts and myocytes, ID predominantly affected genes involved in glycolytic energy metabolism, cell cycle regulation and apoptosis; co-treatment with ferric citrate reversed these effects. CONCLUSIONS: In population-dwelling individuals, ID is related to lower muscle mass, independent of haemoglobin levels and potential confounders. ID impaired myoblast proliferation and aerobic glycolytic capacity, and induced markers of myocyte atrophy and apoptosis. These findings suggest that ID contributes to loss of muscle mass.

Multi-omics analyses identify molecular signatures with prognostic values in different heart failure aetiologies.

BACKGROUND: Heart failure (HF) is the leading cause of morbidity and mortality worldwide, and there is an urgent need for more global studies and data mining approaches to uncover its underlying mechanisms. Multiple omics techniques provide a more holistic molecular perspective to study pathophysiological events involved in the development of HF. METHODS: In this study, we used a label-free whole myocardium multi-omics characterization from three commonly used mouse HF models: transverse aortic constriction (TAC), myocardial infarction (MI), and homozygous Phospholamban-R14del (PLN-R14Δ/Δ). Genes, proteins, and metabolites were analysed for differential expression between each group and a corresponding control group. The core transcriptome and proteome datasets were used for enrichment analysis. For genes that were upregulated at both the RNA and protein levels in all models, clinical validation was performed by means of plasma level determination in patients with HF from the BIOSTAT-CHF cohort. RESULTS: Cell death and tissue repair-related pathways were upregulated in all preclinical models. Fatty acid oxidation, ATP metabolism, and Energy derivation processes were downregulated in all investigated HF aetiologies. Putrescine, a metabolite known for its role in cell survival and apoptosis, demonstrated a 4.9-fold (p < 0.02) increase in PLN-R14Δ/Δ, 2.7-fold (p < 0.005) increase in TAC mice, and 2.2-fold (p < 0.02) increase in MI mice. Four Biomarkers were associated with all-cause mortality (PRELP: Hazard ratio (95% confidence interval) 1.79(1.35, 2.39), p < 0.001; CKAP4: 1.38(1.21, 1.57), p < 0.001; S100A11: 1.37(1.13, 1.65), p = 0.001; Annexin A1 (ANXA1): 1.16(1.04, 1.29) p = 0.01), and three biomarkers were associated with HF-Related Rehospitalization, (PRELP: 1.88(1.4, 2.53), p < 0.001; CSTB: 1.15(1.05, 1.27), p = 0.003; CKAP4: 1.18(1.02, 1.35), P = 0.023). CONCLUSIONS: Cell death and tissue repair pathways were significantly upregulated, and ATP and energy derivation processes were significantly downregulated in all models. Common pathways and biomarkers with potential clinical and prognostic associations merit further investigation to develop optimal management and therapeutic strategies for all HF aetiologies.

Evaluation of the Interaction of Sex Hormones and Cardiovascular Function and Health.

PURPOSE OF REVIEW: Sex hormones drive development and function of reproductive organs or the development of secondary sex characteristics but their effects on the cardiovascular system are poorly understood. In this review, we identify the gaps in our understanding of the interaction between sex hormones and the cardiovascular system. RECENT FINDINGS: Studies are progressively elucidating molecular functions of sex hormones in specific cell types in parallel with the initiation of crucial large randomized controlled trials aimed at improving therapies for cardiovascular diseases (CVDs) associated with aberrant levels of sex hormones. In contrast with historical assumptions, we now understand that men and women show different symptoms and progression of CVDs. Abnormal levels of sex hormones pose an independent risk for CVD, which is apparent in conditions like Klinefelter syndrome, androgen insensitivity syndrome, and menopause. Moreover, sex hormone-based therapies remain understudied and may not be beneficial for cardiovascular health.

Pathophysiology and risk factors of peripartum cardiomyopathy.

Peripartum cardiomyopathy (PPCM) is a potentially fatal form of idiopathic heart failure with variable prevalence across different countries and ethnic groups. The cause of PPCM is unclear, but environmental and genetic factors and pregnancy-associated conditions such as pre-eclampsia can contribute to the development of PPCM. Furthermore, animal studies have shown that impaired vascular and metabolic function might be central to the development of PPCM. A better understanding of the pathogenic mechanisms involved in the development of PPCM is necessary to establish new therapies that can improve the outcomes of patients with PPCM. Pregnancy hormones tightly regulate a plethora of maternal adaptive responses, including haemodynamic, structural and metabolic changes in the cardiovascular system. In patients with PPCM, the peripartum period is associated with profound and rapid hormonal fluctuations that result in a brief period of disrupted cardiovascular (metabolic) homeostasis prone to secondary perturbations. In this Review, we discuss the latest studies on the potential pathophysiological mechanisms of and risk factors for PPCM, with a focus on maternal cardiovascular changes associated with pregnancy. We provide an updated framework to further our understanding of PPCM pathogenesis, which might lead to an improvement in disease definition.

Additional burden of iron deficiency in heart failure patients beyond the cardio-renal anaemia syndrome: findings from the BIOSTAT-CHF study.

AIMS: Whereas the combination of anaemia and chronic kidney disease (CKD) has been extensively studied in patients with heart failure (HF), the contribution of iron deficiency (ID) to this dysfunctional interplay is unknown. We aimed to assess clinical associates and pathophysiological pathways related to ID in this multimorbid syndrome. METHODS AND RESULTS: We studied 2151 patients with HF from the BIOSTAT-CHF cohort. Patients were stratified based on ID (transferrin saturation <20%), anaemia (World Health Organization definition) and/or CKD (estimated glomerular filtration rate <60 ml/min/1.73 m2 ). Patients were mainly men (73.3%), with a median age of 70.5 (interquartile range 61.4-78.1). ID was more prevalent than CKD and anaemia (63.3%, 47.2% and 35.6% respectively), with highest prevalence in those with concomitant CKD and anaemia (77.5% vs. 59.3%; p < 0.001). There was a considerable overlap in biomarkers and pathways between patients with isolated ID, anaemia or CKD, or in combination, with processes related to immunity, inflammation, cell survival and cancer amongst the common pathways. Key biomarkers shared between syndromes with ID included transferrin receptor, interleukin-6, fibroblast growth factor-23, and bone morphogenetic protein 6. Having ID, either alone or on top of anaemia and/or CKD, was associated with a lower overall summary Kansas City Cardiomyopathy Questionnaire score, an impaired 6-min walk test and increased incidence of hospitalizations and/or mortality in multivariable analyses (all p < 0.05). CONCLUSION: Iron deficiency, CKD and/or anaemia in patients with HF have great overlap in biomarker profiles, suggesting common pathways associated with these syndromes. ID either alone or on top of CKD and anaemia is associated with worse quality of life, exercise capacity and prognosis of patients with worsening HF.

Selenoprotein DIO2 Is a Regulator of Mitochondrial Function, Morphology and UPRmt in Human Cardiomyocytes.

Members of the fetal-gene-program may act as regulatory components to impede deleterious events occurring with cardiac remodeling, and constitute potential novel therapeutic heart failure (HF) targets. Mitochondrial energy derangements occur both during early fetal development and in patients with HF. Here we aim to elucidate the role of DIO2, a member of the fetal-gene-program, in pluripotent stem cell (PSC)-derived human cardiomyocytes and on mitochondrial dynamics and energetics, specifically. RNA sequencing and pathway enrichment analysis was performed on mouse cardiac tissue at different time points during development, adult age, and ischemia-induced HF. To determine the function of DIO2 in cardiomyocytes, a stable human hPSC-line with a DIO2 knockdown was made using a short harpin sequence. Firstly, we showed the selenoprotein, type II deiodinase (DIO2): the enzyme responsible for the tissue-specific conversion of inactive (T4) into active thyroid hormone (T3), to be a member of the fetal-gene-program. Secondly, silencing DIO2 resulted in an increased reactive oxygen species, impaired activation of the mitochondrial unfolded protein response, severely impaired mitochondrial respiration and reduced cellular viability. Microscopical 3D reconstruction of the mitochondrial network displayed substantial mitochondrial fragmentation. Summarizing, we identified DIO2 to be a member of the fetal-gene-program and as a key regulator of mitochondrial performance in human cardiomyocytes. Our results suggest a key position of human DIO2 as a regulator of mitochondrial function in human cardiomyocytes.

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy.

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.

Iron Deficiency in Heart Failure: Mechanisms and Pathophysiology.

Iron is an essential micronutrient for a myriad of physiological processes in the body beyond erythropoiesis. Iron deficiency (ID) is a common comorbidity in patients with heart failure (HF), with a prevalence reaching up to 59% even in non-anaemic patients. ID impairs exercise capacity, reduces the quality of life, increases hospitalisation rate and mortality risk regardless of anaemia. Intravenously correcting ID has emerged as a promising treatment in HF as it has been shown to alleviate symptoms, improve quality of life and exercise capacity and reduce hospitalisations. However, the pathophysiology of ID in HF remains poorly characterised. Recognition of ID in HF triggered more research with the aim to explain how correcting ID improves HF status as well as the underlying causes of ID in the first place. In the past few years, significant progress has been made in understanding iron homeostasis by characterising the role of the iron-regulating hormone hepcidin, the effects of ID on skeletal and cardiac myocytes, kidneys and the immune system. In this review, we summarise the current knowledge and recent advances in the pathophysiology of ID in heart failure, the deleterious systemic and cellular consequences of ID.

Red-light-sensitive BODIPY photoprotecting groups for amines and their biological application in controlling heart rhythm.

Control of biological function by the use of photoremovable protecting groups (PPGs) is a gateway towards many new medical developments. Herein, we report the synthesis and application of efficient and biocompatible BODIPY-based photoprotecting groups for amines, which are cleavable with red light in the phototherapeutic window region (λ > 650 nm). We use the most promising PPG for the protection of dopamine and apply it to control the beating frequency of human cardiomyocytes.

In peripartum cardiomyopathy plasminogen activator inhibitor-1 is a potential new biomarker with controversial roles.

AIMS: Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease occurring in previously heart-healthy women. A common pathomechanism in PPCM involves the angiostatic 16 kDa-prolactin (16 kDa-PRL) fragment, which via NF-κB-mediated up-regulation of microRNA-(miR)-146a induces vascular damage and heart failure. We analyse whether the plasminogen activator inhibitor-1 (PAI-1) is involved in the pathophysiology of PPCM. METHODS AND RESULTS: In healthy age-matched postpartum women (PP-Ctrl, n = 53, left ventricular ejection fraction, LVEF > 55%), PAI-1 plasma levels were within the normal range (21 ± 10 ng/mL), but significantly elevated (64 ± 38 ng/mL, P < 0.01) in postpartum PPCM patients at baseline (BL, n = 64, mean LVEF: 23 ± 8%). At 6-month follow-up (n = 23), PAI-1 levels decreased (36 ± 14 ng/mL, P < 0.01 vs. BL) and LVEF (49 ± 11%) improved. Increased N-terminal pro-brain natriuretic peptide and Troponin T did not correlate with PAI-1. C-reactive protein, interleukin (IL)-6 and IL-1ß did not differ between PPCM patients and PP-Ctrl. MiR-146a was 3.6-fold (P < 0.001) higher in BL-PPCM plasma compared with PP-Ctrl and correlated positively with PAI-1. In BL-PPCM serum, 16 kDa-PRL coprecipitated with PAI-1, which was associated with higher (P < 0.05) uPAR-mediated NF-κB activation in endothelial cells compared with PP-Ctrl serum. Cardiac biopsies and dermal fibroblasts from PPCM patients displayed higher PAI-1 mRNA levels (P < 0.05) than healthy controls. In PPCM mice (due to a cardiomyocyte-specific-knockout for STAT3, CKO), cardiac PAI-1 expression was higher than in postpartum wild-type controls, whereas a systemic PAI-1-knockout in CKO mice accelerated peripartum cardiac fibrosis, inflammation, heart failure, and mortality. CONCLUSION: In PPCM patients, circulating and cardiac PAI-1 expression are up-regulated. While circulating PAI-1 may add 16 kDa-PRL to induce vascular impairment via the uPAR/NF-κB/miR-146a pathway, experimental data suggest that cardiac PAI-1 expression seems to protect the PPCM heart from fibrosis. Thus, measuring circulating PAI-1 and miR-146a, together with an uPAR/NF-κB-activity assay could be developed into a specific diagnostic marker assay for PPCM, but unrestricted reduction of PAI-1 for therapy may not be advised.

Selenium and outcome in heart failure.

AIMS: Severe deficiency of the essential trace element selenium can cause myocardial dysfunction although the mechanism at cellular level is uncertain. Whether, in clinical practice, moderate selenium deficiency is associated with worse symptoms and outcome in patients with heart failure is unknown. METHODS AND RESULTS: BIOSTAT-CHF is a multinational, prospective, observational cohort study that enrolled patients with worsening heart failure. Serum concentrations of selenium were measured by inductively coupled plasma mass spectrometry. Primary endpoint was a composite of all-cause mortality and hospitalization for heart failure; secondary endpoint was all-cause mortality. To investigate potential mechanisms by which selenium deficiency might affect prognosis, human cardiomyocytes were cultured in absence of selenium, and mitochondrial function and oxidative stress were assessed. Serum selenium concentration (deficiency) was <70 µg/L in 485 (20.4%) patients, who were older, more often women, had worse New York Heart Association class, more severe signs and symptoms of heart failure and poorer exercise capacity (6-min walking test) and quality of life (Kansas City Cardiomyopathy Questionnaire). Selenium deficiency was associated with higher rates of the primary endpoint [hazard ratio (HR) 1.23; 95% confidence interval (CI) 1.06-1.42] and all-cause mortality (HR 1.52; 95% CI 1.26-1.86). In cultured human cardiomyocytes, selenium deprivation impaired mitochondrial function and oxidative phosphorylation, and increased intracellular reactive oxygen species levels. CONCLUSIONS: Selenium deficiency in heart failure patients is independently associated with impaired exercise tolerance and a 50% higher mortality rate, and impaired mitochondrial function in vitro, in human cardiomyocytes. Clinical trials are needed to investigate the effect of selenium supplements in patients with heart failure, especially if they have low plasma concentrations of selenium.

The role of cathepsin D in the pathophysiology of heart failure and its potentially beneficial properties: a translational approach.

AIMS: Cathepsin D is a ubiquitous lysosomal protease that is primarily secreted due to oxidative stress. The role of circulating cathepsin D in heart failure (HF) is unknown. The aim of this study is to determine the association between circulating cathepsin D levels and clinical outcomes in patients with HF and to investigate the biological settings that induce the release of cathepsin D in HF. METHODS AND RESULTS: Cathepsin D levels were studied in 2174 patients with HF from the BIOSTAT-CHF index study. Results were validated in 1700 HF patients from the BIOSTAT-CHF validation cohort. The primary combined outcome was all-cause mortality and/or HF hospitalizations. Human pluripotent stem cell-derived cardiomyocytes were subjected to hypoxic, pro-inflammatory signalling and stretch conditions. Additionally, cathepsin D expression was inhibited by targeted short hairpin RNAs (shRNA). Higher levels of cathepsin D were independently associated with diabetes mellitus, renal failure and higher levels of interleukin-6 and N-terminal pro-B-type natriuretic peptide (P < 0.001 for all). Cathepsin D levels were independently associated with the primary combined outcome [hazard ratio (HR) per standard deviation (SD): 1.12; 95% confidence interval (CI) 1.02-1.23], which was validated in an independent cohort (HR per SD: 1.23, 95% CI 1.09-1.40). In vitro experiments demonstrated that human stem cell-derived cardiomyocytes released cathepsin D and troponin T in response to mechanical stretch. ShRNA-mediated silencing of cathepsin D resulted in increased necrosis, abrogated autophagy, increased stress-induced metabolism, and increased release of troponin T from human stem cell-derived cardiomyocytes under stress. CONCLUSIONS: Circulating cathepsin D levels are associated with HF severity and poorer outcome, and reduced levels of cathepsin D may have detrimental effects with therapeutic potential in HF.

Concise Review: The Current State of Human In Vitro Cardiac Disease Modeling: A Focus on Gene Editing and Tissue Engineering.

Until recently, in vivo and ex vivo experiments were the only means to determine factors and pathways involved in disease pathophysiology. After the generation of characterized human embryonic stem cell lines, human diseases could readily be studied in an extensively controllable setting. The introduction of human-induced pluripotent stem cells, a decade ago, allowed the investigation of hereditary diseases in vitro. In the field of cardiology, diseases linked to known genes have successfully been studied, revealing novel disease mechanisms. The direct effects of various mutations leading to hypertrophic cardiomyopathy, dilated cardiomyopathy, arrythmogenic cardiomyopathy, or left ventricular noncompaction cardiomyopathy are discovered as a result of in vitro disease modeling. Researchers are currently applying more advanced techniques to unravel more complex phenotypes, resulting in state-of-the-art models that better mimic in vivo physiology. The continued improvement of tissue engineering techniques and new insights into epigenetics resulted in more reliable and feasible platforms for disease modeling and the development of novel therapeutic strategies. The introduction of CRISPR-Cas9 gene editing granted the ability to model diseases in vitro independent of induced pluripotent stem cells. In addition to highlighting recent developments in the field of human in vitro cardiomyopathy modeling, this review also aims to emphasize limitations that remain to be addressed; including residual somatic epigenetic signatures induced pluripotent stem cells, and modeling diseases with unknown genetic causes. Stem Cells Translational Medicine 2019;8:66-74.

Iron deficiency impairs contractility of human cardiomyocytes through decreased mitochondrial function.

AIMS: Iron deficiency is common in patients with heart failure and associated with a poor cardiac function and higher mortality. How iron deficiency impairs cardiac function on a cellular level in the human setting is unknown. This study aims to determine the direct effects of iron deficiency and iron repletion on human cardiomyocytes. METHODS AND RESULTS: Human embryonic stem cell-derived cardiomyocytes were depleted of iron by incubation with the iron chelator deferoxamine (DFO). Mitochondrial respiration was determined by Seahorse Mito Stress test, and contractility was directly quantified using video analyses according to the BASiC method. The activity of the mitochondrial respiratory chain complexes was examined using spectrophotometric enzyme assays. Four days of iron depletion resulted in an 84% decrease in ferritin (P < 0.0001) and significantly increased gene expression of transferrin receptor 1 and divalent metal transporter 1 (both P < 0.001). Mitochondrial function was reduced in iron-deficient cardiomyocytes, in particular ATP-linked respiration and respiratory reserve were impaired (both P < 0.0001). Iron depletion affected mitochondrial function through reduced activity of the iron-sulfur cluster containing complexes I, II and III, but not complexes IV and V. Iron deficiency reduced cellular ATP levels by 74% (P < 0.0001) and reduced contractile force by 43% (P < 0.05). The maximum velocities during both systole and diastole were reduced by 64% and 85%, respectively (both P < 0.001). Supplementation of transferrin-bound iron recovered functional and morphological abnormalities within 3 days. CONCLUSION: Iron deficiency directly affects human cardiomyocyte function, impairing mitochondrial respiration, and reducing contractility and relaxation. Restoration of intracellular iron levels can reverse these effects.

EGR1 controls divergent cellular responses of distinctive nucleus pulposus cell types.

BACKGROUND: Immediate early genes (IEGs) encode transcription factors which serve as first line response modules to altered conditions and mediate appropriate cell responses. The immediate early response gene EGR1 is involved in physiological adaptation of numerous different cell types. We have previously shown a role for EGR1 in controlling processes supporting chondrogenic differentiation. We recently established a unique set of phenotypically distinct cell lines from the human nucleus pulposus (NP). Extensive characterization showed that these NP cellular subtypes represented progenitor-like cell types and more functionally mature cells. METHODS: To further understanding of cellular heterogeneity in the NP, we analyzed the response of these cell subtypes to anabolic and catabolic factors. Here, we test the hypothesis that physiological responses of distinct NP cell types are mediated by EGR1 and reflect specification of cell function using an RNA interference-based experimental approach. RESULTS: We show that distinct NP cell types rapidly induce EGR1 exposure to either growth factors or inflammatory cytokines. In addition, we show that mRNA profiles induced in response to anabolic or catabolic conditions are cell type specific: the more mature NP cell type produced a strong and more specialized transcriptional response to IL-1ß than the NP progenitor cells and aspects of this response were controlled by EGR1. CONCLUSIONS: Our current findings provide important substantiation of differential functionality among NP cellular subtypes. Additionally, the data shows that early transcriptional programming initiated by EGR1 is essentially restrained by the cells' epigenome as it was determined during development and differentiation. These studies begin to define functional distinctions among cells of the NP and will ultimately contribute to defining functional phenotypes within the adult intervertebral disc.