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Abstract The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential.

Abstract Background Achondroplasia (ACH) is one of the most prevalent genetic forms of short-limbed skeletal dysplasia, caused by gain-of-function mutations in the receptor tyrosine kinase FGFR3. In August 2021, the C-type natriuretic peptide (CNP) analog vosoritide was approved for the treatment of ACH. A total of six other inhibitors of FGFR3 signaling are currently undergoing clinical evaluation for ACH. This progress creates an opportunity for children with ACH, who may gain early access to the treatment by entering clinical trials before the closure of their epiphyseal growth plates and cessation of growth. Pathophysiology associated with the ACH, however, demands a long observational period before admission to the interventional trial. Public patient registries can facilitate the process by identification of patients suitable for treatment and collecting the data necessary for the trial entry. Results In 2015, we established the prospective ACH registry in the Czechia and the Slovak Republic ( http://www.achondroplasia-registry.cz ). Patient data is collected through pediatric practitioners and other relevant specialists. After informed consent is given, the data is entered to the online TrialDB system and stored in the Oracle 9i database. The initial cohort included 51 ACH children (average age 8.5 years, range 3 months to 14 years). The frequency of selected neurological, orthopedic, or ORL diagnoses is also recorded. In 2015–2021, a total of 89 measurements of heights, weights, and other parameters were collected. The individual average growth rate was calculated and showed values without exception in the lower decile for the appropriate age. Evidence of paternal age effect was found, with 58.7% of ACH fathers older than the general average paternal age and 43.5% of fathers older by two or more years. One ACH patient had orthopedic limb extension and one patient received growth hormone therapy. Low blood pressure or renal impairment were not found in any patient. Conclusion The registry collected the clinical information of 51 pediatric ACH patients during its 6 years of existence, corresponding to ~ 60% of ACH patients living in the Czechia and Slovak Republic. The registry continues to collect ACH patient data with annual frequency to monitor the growth and other parameters in preparation for future therapy.

Aims: This study aims to compare procedural parameters and clinical efficacy of remote magnetic navigation (RMN) vs. manual navigation (MAN) approach for radiofrequency ablation (RFA) in patients with atrial fibrillation (AF). Methods: 146 patients with AF were enrolled in the study. In the RMN group (n=57), patients were treated with the CARTO® 3 in combination with the Niobe ES system. In the MAN group (n=89), ablation was performed with the EnSite Velocity and TactiCath™ Quartz catheter with direct contact force measurement. Procedural time, ablation time, fluoroscopy time, radiation dose and ablation counts were measured and compared between the groups. Recurrence of AF was evaluated after 6 months of follow-up. Results: Mean procedure times (236.87±64.31 vs. 147.22±45.19 min, P

Abstract We describe the association of Becker muscular dystrophy (BMD) derived heart failure with the impairment of tissue homeostasis and remodeling capabilities of the affected heart tissue. We report that BMD heart failure is associated with a significantly decreased number of cardiovascular progenitor cells, reduced cardiac fibroblast migration, and ex vivo survival. Background Becker muscular dystrophy belongs to a class of genetically inherited dystrophin deficiencies. It affects male patients and results in progressive skeletal muscle degeneration and dilated cardiomyopathy leading to heart failure. It is a relatively mild form of dystrophin deficiency, which allows patients to be on a heart transplant list. In this unique situation, the explanted heart is a rare opportunity to study the degenerative process of dystrophin-deficient cardiac tissue. Heart tissue was excised, dissociated, and analyzed. The fractional content of c-kit+/CD45− cardiovascular progenitor cells (CVPCs) and cardiac fibroblast migration were compared to control samples of atrial tissue. Control tissue was obtained from the hearts of healthy organ donor’s during heart transplantation procedures. Results We report significantly decreased CVPCs (c-kit+/CD45−) throughout the heart tissue of a BMD patient, and reduced numbers of phase-bright cells presenting c-kit positivity in the dystrophin-deficient cultured explants. In addition, ex vivo CVPCs survival and cardiac fibroblasts migration were significantly reduced, suggesting reduced homeostatic support and irreversible tissue remodeling. Conclusions Our findings associate genetically derived heart failure in a dystrophin-deficient patient with decreased c-kit+/CD45− CVPCs and their resilience, possibly hinting at a lack of cardioprotective capability and/or reduced homeostatic support. This also correlates with reduced plasticity of the explanted cardiac tissue, related to the process of irreversible remodeling in the BMD patient’s heart.

Abstract Background Gigantic left atrium is defined in the current literature as an excessive dilatation of the left atrium above 65mm. Chronic mitral valve disease is associated with the development of thrombus in the left atrium in up to 19% of all cases of mitral insufficiency and appropriate treatment must be initiated to prevent thromboembolic events. In order to diagnose thrombi in the left atrium or left atrial appendage, various imaging methods may be used, including cardiac magnetic resonance. Case presentation The case report describes a 73-year-old male who developed recurrent sessile thrombus on the posterior wall of the gigantic left atrium. A large thrombus was first detected following mitral valve surgery despite effective vitamin K antagonist anticoagulation therapy. Echocardiography and cardiac magnetic resonance were used within the diagnostic procedure and to monitor the treatment outcomes. Cardiac magnetic resonance was shown to be beneficial as it provided a more precise description of the intra-atrial masses located on the posterior left atrial wall, and in such situations, is of greater benefit than standard echocardiography. This led to the surgical removal of the intra-atrial mass; nevertheless, it was quickly followed by the recurrence of the thrombus. The anticoagulant therapy was adjusted and fortified by the introduction of acetylsalicylic acid and sequentially clopidogrel, but this also did not resolve the thrombus formation. Finally, employing a combination of rivaroxaban and clopidogrel resulted in partial thrombus regression. Therefore, various pathophysiological aspects of thrombus formation and used anticoagulation strategies are discussed. Conclusions We describe a unique case of a recurrent thrombus located on the posterior wall of the gigantic left atrium. Cardiac magnetic resonance was shown to be beneficial in providing a more precise description of the intra-atrial masses located on the posterior left atrial wall as compared to standard echocardiographic examination. Development of a thrombus after mitral valve surgery despite effective anticoagulant therapy and its final resolution by introducing a combination of rivaroxaban and clopidogrel highlights the complex etiopathogenesis of thrombus formation. This supports the potential use of this combination in tailoring an individual personalized therapy for patients with recurrent atrial thrombi.

Cardiac side effects of some pulmonary drugs are observed in clinical practice. Aminophylline, a methylxanthine bronchodilator with documented proarrhythmic action, may serve as an example. Data on the action of aminophylline on cardiac cell electrophysiology and contractility are not available. Hence, this study was focused on the analysis of changes in the beat rate and contraction force of human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) and HL-1 cardiomyocytes in the presence of increasing concentrations of aminophylline (10 µM–10 mM in hPSC-CM and 8–512 µM in HL-1 cardiomyocytes). Basic biomedical parameters, namely, the beat rate (BR) and contraction force, were assessed in hPSC-CMs using an atomic force microscope (AFM). The beat rate changes under aminophylline were also examined on the HL-1 cardiac muscle cell line via a multielectrode array (MEA). Additionally, calcium imaging was used to evaluate the effect of aminophylline on intracellular Ca2+ dynamics in HL-1 cardiomyocytes. The BR was significantly increased after the application of aminophylline both in hPSC-CMs (with 10 mM aminophylline) and in HL-1 cardiomyocytes (with 256 and 512 µM aminophylline) in comparison with controls. A significant increase in the contraction force was also observed in hPSC-CMs with 10 µM aminophylline (a similar trend was visible at higher concentrations as well). We demonstrated that all aminophylline concentrations significantly increased the frequency of rhythm irregularities (extreme interbeat intervals) both in hPSC-CMs and HL-1 cells. The occurrence of the calcium sparks in HL-1 cardiomyocytes was significantly increased with the presence of 512 µM aminophylline. We conclude that the observed aberrant cardiomyocyte response to aminophylline suggests an arrhythmogenic potential of the drug. The acquired data represent a missing link between the arrhythmic events related to the aminophylline/theophylline treatment in clinical practice and describe cellular mechanisms of methylxanthine arrhythmogenesis. An AFM combined with hPSC-CMs may serve as a robust platform for direct drug effect screening.

IntroductionPulsed field ablation (PFA) exploits the delivery of short high-voltage shocks to induce cells death via irreversible electroporation. The therapy offers a potential paradigm shift for catheter ablation of cardiac arrhythmia. We designed an AC-burst generator and therapeutic strategy, based on the existing knowledge between efficacy and safety among different pulses. We performed a proof-of-concept chronic animal trial to test the feasibility and safety of our method and technology.MethodsWe employed 6 female swine – weight 53.75 ± 4.77 kg – in this study. With fluoroscopic and electroanatomical mapping assistance, we performed ECG-gated AC-PFA in the following settings: in the left atrium with a decapolar loop catheter with electrodes connected in bipolar fashion; across the interventricular septum applying energy between the distal electrodes of two tip catheters. After procedure and 4-week follow-up, the animals were euthanized, and the hearts were inspected for tissue changes and characterized. We perform finite element method simulation of our AC-PFA scenarios to corroborate our method and better interpret our findings.ResultsWe applied square, 50% duty cycle, AC bursts of 100 μs duration, 100 kHz internal frequency, 900 V for 60 pulses in the atrium and 1500 V for 120 pulses in the septum. The inter-burst interval was determined by the native heart rhythm – 69 ± 9 bpm. Acute changes in the atrial and ventricular electrograms were immediately visible at the sites of AC-PFA – signals were elongated and reduced in amplitude (p < 0.0001) and tissue impedance dropped (p = 0.011). No adverse event (e.g., esophageal temperature rises or gas bubble streams) was observed – while twitching was avoided by addition of electrosurgical return electrodes. The implemented numerical simulations confirmed the non-thermal nature of our AC-PFA and provided specific information on the estimated treated area and need of pulse trains. The postmortem chest inspection showed no peripheral damage, but epicardial and endocardial discolorations at sites of ablation. T1-weighted scans revealed specific tissue changes in atria and ventricles, confirmed to be fibrotic scars via trichrome staining. We found isolated, transmural and continuous scars. A surviving cardiomyocyte core was visible in basal ventricular lesions.ConclusionWe proved that our method and technology of AC-PFA is feasible and safe for atrial and ventricular myocardial ablation, supporting their systematic investigation into effectiveness evaluation for the treatment of cardiac arrhythmia. Further optimization, with energy titration or longer follow-up, is required for a robust atrial and ventricular AC-PFA.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by the lack of functional dystrophin. DMD is associated with progressive dilated cardiomyopathy, eventually leading to heart failure as the main cause of death in DMD patients. Although several molecular mechanisms leading to the DMD cardiomyocyte (DMD-CM) death were described, mostly in mouse model, no suitable human CM model was until recently available together with proper clarification of the DMD-CM phenotype and delay in cardiac symptoms manifestation. We obtained several independent dystrophin-deficient human pluripotent stem cell (hPSC) lines from DMD patients and CRISPR/Cas9-generated DMD gene mutation. We differentiated DMD-hPSC into cardiac cells (CC) creating a human DMD-CC disease model. We observed that mutation-carrying cells were less prone to differentiate into CCs. DMD-CCs demonstrated an enhanced cell death rate in time. Furthermore, ion channel expression was altered in terms of potassium (Kir2.1 overexpression) and calcium handling (dihydropyridine receptor overexpression). DMD-CCs exhibited increased time of calcium transient rising compared to aged-matched control, suggesting mishandling of calcium release. We observed mechanical impairment (hypocontractility), bradycardia, increased heart rate variability, and blunted β-adrenergic response connected with remodeling of β-adrenergic receptors expression in DMD-CCs. Overall, these results indicated that our DMD-CC models are functionally affected by dystrophin-deficiency associated and recapitulate functional defects and cardiac wasting observed in the disease. It offers an accurate tool to study human cardiomyopathy progression and test therapies in vitro.

(1) Background: Computer tomography (CT) is an imaging modality used in the pre-planning of radiofrequency catheter ablation (RFA) procedure in patients with cardiac arrhythmias. However, it is associated with a considerable ionizing radiation dose for patients. This study aims to develop and validate low-dose CT scanning protocols of the left atrium (LA) for RFA guidance. (2) Methods: 68 patients scheduled for RFA of atrial fibrillation were sequentially assigned to four groups of ECG-gated scanning protocols, based on the set tube current (TC): Group A (n = 20, TC = 33 mAs), Group B (n = 18, TC = 67 mAs), Group C (n = 10, TC = 135 mAs), and control Group D (n = 20, TC = 600 mAs). We used a 256-row multidetector CT with body weight-dependent tube voltage of 80 kVp (90 kg). We evaluated scanning parameters including radiation dose, total scanning procedure time and signal-to-noise ratio (SNR). (3) Results: The average effective radiation dose (ED) was lower in Group A in comparison to Group B, C and D (0.83 (0.76–1.10), 1.55 (1.36–1.67), 2.91 (2.32–2.96) and 9.35 (8.00–10.04) mSv, p < 0.05). The total amount of contrast media was not significantly different between groups. The mean SNR was 6.5 (5.8–7.3), 7.1 (5.7–8.2), 10.8 (10.1–11.3), and 12.2 (9.9–15.7) for Group A, B, C and D, respectively. The comparisons of SNR in group A vs. B and C vs. D were without significant differences. (4) Conclusions: Optimized pre-ablation CT scanning protocols of the LA can reduce an average ED by 88.7%. Three dimensional (3D) models created with the lowest radiation protocol are useful for the integration of electro-anatomic-guided RFA procedures.

Duchenne muscular dystrophy (DMD) affects 1:3500–5000 newborn boys and manifests with progressive skeletal muscle wasting, respiratory failure and eventual heart failure. Symptoms show different onset from patients' childhood to the second decade of age. We reprogrammed fibroblasts from two independent DMD patients with a complete loss of dystrophin expression, carrying deletions of exons 45–50 and 48–50. The resulting hiPSCs show expression of pluripotency markers (NANOG, OCT4, SSEA4), differentiation capacity into all three germ layers, normal karyotype, genetic identity to the originating parental fibroblasts and the patient-specific dystrophin mutation.

The cell biomechanical properties play a key role in the determination of the changes during the essential cellular functions, such as contraction, growth, and migration. Recent advances in nano-technologies have enabled the development of new experimental and modeling approaches to study cell biomechanics, with a level of insights and reliability that were not possible in the past. The use of atomic force microscopy (AFM) for force spectroscopy allows nanoscale mapping of the cell topography and mechanical properties under, nearly physiological conditions. A proper evaluation process of such data is an essential factor to obtain accurate values of the cell elastic properties (primarily Young's modulus). Several numerical models were published in the literature, describing the depth sensing indentation as interaction process between the elastic surface and indenting probe. However, many studies are still relying on the nowadays outdated Hertzian model from the nineteenth century, or its modification by Sneddon. The lack of comparison between the Hertz/Sneddon model with their modern modifications blocks the development of advanced analysis software and further progress of AFM promising technology into biological sciences. In this work, we applied a rationalized use of mechanical models for advanced postprocessing and interpretation of AFM data. We investigated the effect of the mechanical model choice on the final evaluation of cellular elasticity. We then selected samples subjected to different physicochemical modulators, to show how a critical use of AFM data handling can provide more information than simple elastic modulus estimation. Our contribution is intended as a methodological discussion of the limitations and benefits of AFM-based advanced mechanical analysis, to refine the quantification of cellular elastic properties and its correlation to undergoing cellular processes in vitro.

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor’s involvement in the disease pathology often leading to the DMD patient’s death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.

Abstract Background Female carriers of dystrophin gene mutations (DMD-FC) were previously considered non-manifesting, but in recent decades, cardiomyopathy associated with muscular dystrophy and myocardial fibrosis has been described. Our study aimed to assess prospectively myocardial fibrosis in asymptomatic DMD-FC compared to a sex-matched control group (CG) with similar age distribution using native T1 mapping and extracellular volume (ECV) quantification by cardiovascular magnetic resonance (CMR) imaging. Materials and methods 38 DMD-FC with verified genetic mutation and 22 healthy volunteers were included. Using CMR, native T1 relaxation time and ECV quantification were determined in each group. Late gadolinium enhancement (LGE) was assessed in all cases. Results There were 38 DMD-FC (mean age 39.1 ± 8.8 years) and 22 healthy volunteers (mean age 39.9 ± 12.6 years) imagined by CMR. The mean global native T1 relaxation time was similar for DMD-FC and CG (1005.1 ± 26.3 ms vs. 1003.5 ± 25.0 ms; p-value = 0.81). Likewise, the mean global ECV value was also similar between the groups (27.92 ± 2.02% vs. 27.10 ± 2.89%; p-value = 0.20). The segmental analysis of mean ECV values according to the American Heart Association classification did not show any differences between DMD-FC and CG. There was a non-significant trend towards higher mean ECV values of DMD-FC in the inferior and inferolateral segments of the myocardium (p-value = 0.075 and 0.070 respectively). Conclusion There were no statistically significant differences in the mean global and segmental native T1 relaxation times and the mean global or segmental ECV values. There was a trend towards higher segmental mean ECV values of DMD-FC in the inferior and inferolateral walls of the myocardium.

In this prospective study involving 37 Duchenne muscular dystrophy (DMD) patients aged 8–18 years and older, we examined the impact of neurological and cardiac factors on quality of life (QoL). Our findings revealed a negative correlation between upper limb movement and overall mobility, self-service, and usual activities. Ambulatory and non-ambulatory DMD patients showed significant differences in mobility-related parameters. Cardiac evaluations demonstrated associations between mitral annular plane systolic excursion (MAPSE) and mobility-related aspects. The PEDSQL 3.0 neuromuscular model questionnaire further highlighted age-related and movement-related correlations with QoL. The loss of ambulatory status and reduced upper limb movement were negatively associated with QoL, while upper limb movement positively correlated with septal MAPSE. However, no significant associations were found between MAPSE and anxiety/depression. These findings underscore the multifaceted impact of DMD on QoL and emphasize the importance of considering both neurological and cardiac factors in comprehensive patient care.

Abstract Atrial fibrillation (AF) is an abnormal and irregular heartbeat caused by uncoordinated electrical impulses in the left atrium (LA), which could induce lasting changes in the heart tissue or could be a consequence of underlying cardiac disease. This study aimed to assess the left atrial phasic function and deformation in paroxysmal AF (PAF) patients—who had not received radiofrequency ablation and had no signs of permanent AF—using the cardiovascular magnetic resonance (CMR) feature-tracking (FT) technique. Fifty subjects (27 PAF patients and 23 controls) were included and examined with CMR. Their LA volume, LA function, LA longitudinal strain (LS) and LA strain rate were assessed in the LA reservoir, conduit, and contractile phases. PAF patients exhibited higher LA volumes than controls, while their LA emptying fraction and LA LS was significantly lower in all three phases. In contrast, the corresponding emptying volumes (total, passive and active) were similar in both groups. The LA volumetric rates from CMR-derived volume curves differed significantly in PAF patients vs controls in the reservoir and contractile phases. In contrast, the equivalent LV volumetric rates were similar. This study suggests that assessing the LA phasic function could offer insight into early LA impairments for PAF patients.

Aims: Gross pathology inspection (patho) is the gold standard for the morphological evaluation of focal myocardial pathology. Examination with 9.4 T magnetic resonance imaging (MRI) is a new method for very accurate display of myocardial pathology. The aim of this study was to demonstrate that lesions can be measured on high-resolution MRI images with the same accuracy as on pathological sections and compare these two methods for the evaluation of radiofrequency (RF) ablation lesion dimensions in swine heart tissue during animal experiment.Methods: Ten pigs underwent radiofrequency ablations in the left ventricle during animal experiment. After animal euthanasia, hearts were explanted, flushed with ice-cold cardioplegic solution to relax the whole myocardium, fixed in 10% formaldehyde and scanned with a 9.4 T magnetic resonance system. Anatomical images were processed using ImageJ software. Subsequently, the hearts were sliced, slices were photographed and measured in ImageJ software. Different dimensions and volumes were compared.Results: The results of both methods were statistically compared. Depth by MRI was 8.771 ± 2.595 mm and by patho 9.008 ± 2.823 mm; p = 0.198. Width was 10.802 ± 2.724 mm by MRI and 11.125 ± 2.801 mm by patho; p = 0.049. Estuary was 2.006 ± 0.867 mm by MRI and 2.001 ± 0.872 mm by patho; p = 0.953. The depth at the maximum diameter was 4.734 ± 1.532 mm on MRI and 4.783 ± 1.648 mm from the patho; p = 0.858. The volumes of the lesions calculated using a formula were 315.973 ± 257.673 mm3 for MRI and 355.726 ± 255.860 mm3 for patho; p = 0.104. Volume directly measured from MRI with the “point-by-point” method was 671.702 ± 362.299 mm3.Conclusion: Measurements obtained from gross pathology inspection and MRI are fully comparable. The advantage of MRI is that it is a non-destructive method enabling repeated measurements in all possible anatomical projections.

Abstract Background Duchenne muscular dystrophy (DMD) manifests in males mainly by skeletal muscle impairment, but also by cardiac dysfunction. The assessment of the early phases of cardiac involvement using echocardiography is often very difficult to perform in these patients. The aim of the study was to use cardiac magnetic resonance (CMR) strain analysis and mitral annular plane systolic excursion (MAPSE) in the detection of early left ventricular (LV) dysfunction in DMD patients. Methods and results In total, 51 male DMD patients and 18 matched controls were examined by CMR. MAPSE measurement and functional analysis using feature tracking (FT) were performed. Three groups of patients were evaluated: A/ patients with LGE and LV EF

Abstract Background The progressive cardiomyopathy that develops in boys with Duchenne and Becker muscular dystrophy (DMD/BMD) is presumed to be a secondary consequence of the fibrosis within the myocardium. There are only limited data on using parametric imaging in these patients. The purpose of this study was to assess native T1 and extracellular volume (ECV) values in DMD patients. Methods The Czech population of males with DMD/BMD was screened. All eligible patients fulfilling the inclusion criteria were included. Forty nine males underwent cardiac magnetic resonance (MR) examination including T1 native and post-contrast mapping measurements. One DMD patient and all BMD patients were excluded from statistical analysis. Three groups were compared – Group D1 - DMD patients without late gadolinium enhancement (LGE) (n = 23), Group D2 - DMD patients with LGE (n = 20), and Group C – gender matched controls (n = 13). Results Compared to controls, both DMD groups had prolonged T1 native relaxation time. These results are concordant in all 6 segments as well as in global values (1041 ± 31 ms and 1043 ± 37 ms vs. 983 ± 15 ms, both p

The transcriptional co-activator YAP is known to operate downstream of mechanical signals arising from the cell niche. Here the authors demonstrate that YAP controls cell mechanics, force development and adhesion strength by promoting the transcription of genes related to focal adhesions.

Cryopreservation of cells (mouse embryonic fibroblasts) is a fundamental task for wide range of applications. In practice, cells are protected against damage during freezing by applications of specific cryoprotectants and freezing/melting protocols. In this study by using AFM and fluorescence microscopy we showed how selected cryoprotectants (dimethyl sulfoxide and polyethylene glycol) affected the cryopreserved cells mechanical properties (stiffness) and how these parameters are correlated with cytoskeleton damage and reconstruction. We showed how cryopreserved (frozen and thawed) cells' stiffness change according to type of applied cryoprotectant and its functionality in extracellular or intracellular space. We showed that AFM can be used as technique for investigation of cryopreserved cells surfaces state and development ex vivo. Our results offer a new perspective on the monitoring and characterization of frozen cells recovery by measuring changes in elastic properties by nanoindentation technique. This may lead to a new and detailed way of investigating the post-thaw development of cryopreserved cells which allows to distinguish between different cell parts.

Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.