Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

• Keywords
• c-kit, cardiovascular progenitors, dilated cardiomyopathy, duchenne muscular dystrophy, genomic instability, mdx mouse,
• MeSH
• Cardiomyopathy, Dilated genetics   metabolism   pathology   MeSH
• Muscular Dystrophy, Duchenne genetics   metabolism   pathology   MeSH
• Dystrophin deficiency   genetics   MeSH
• Myocytes, Cardiac metabolism   pathology   MeSH
• Cardiovascular System metabolism   pathology   MeSH
• Stem Cells metabolism   pathology   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Myocardium metabolism   pathology   MeSH
• Mice, Inbred mdx genetics   MeSH
• Mice MeSH
• DNA Damage genetics   MeSH
• Proto-Oncogene Proteins c-kit genetics   MeSH
• Gene Expression Regulation genetics   MeSH
• Aging genetics   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Dystrophin MeSH
• Proto-Oncogene Proteins c-kit MeSH

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 < 50% (n = 8), B/ patients with LGE and LVEF ≥ 50% (n = 13), and C/ patients without LGE and LVEF ≥ 50% (n = 30). MAPSE and global LV strains of the 3 DMD groups were compared to controls (n = 18). Groups A and B had significantly reduced values of MAPSE, global longitudinal strain (GLS), global circumferential strain (GCS), and global radial strain (GRS) in comparison to controls (p < 0.05). The values of MAPSE (11.6 ± 1.9 v 13.7 ± 2.7 mm) and GCS (- 26.2 ± 4.2 v - 30.0 ± 5.1%) were significantly reduced in group C compared to the controls (p < 0.05). CONCLUSION: DMD patients had decreased LV systolic function measured by MAPSE and global LV strain even in the case of normal LV EF and the absence of LGE. FT and MAPSE measurement provide sensitive assessment of early cardiac involvement in DMD patients.

• Keywords
• Cardiac magnetic resonance, Duchenne muscular dystrophy, Feature tracking, Strain analysis,
• MeSH
• Muscular Dystrophy, Duchenne * diagnostic imaging   MeSH
• Ventricular Dysfunction, Left * diagnostic imaging   MeSH
• Ventricular Function, Left MeSH
• Cardiomyopathies * MeSH
• Humans MeSH
• Myocardium MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• Keywords
• IPSC, aminophylline, arrhythmogenic effects, atomic force microscopy, cardiomyocytes, drug cardiotoxicity, hESC, methylxanthines,
• Publication type
• Journal Article MeSH

To assess subclinical cardiac function impairment in Duchenne dystrophy (DMD) female carriers. Forty-four female subjects proved as DMD carriers underwent echocardiographic examination including tissue Doppler imaging (TDI) of mitral and tricuspid annulus. Seventeen age-matched healthy female subjects served as controls. A significant differences in peak systolic annular velocity (Sa) between carriers and controls were found for lateral and septal part of the mitral annulus and for tricuspid annulus (0.09 vs. 0.11 m/s, p < 0.001, 0.08 vs. 0.09 m/s, p < 0.01 and 0.13 vs. 0.14 m/s, p = 0.02 respectively). There was also difference in early diastolic velocity (Ea) of the septal part of the mitral annulus (0.11 vs. 0.13 m/s, p = 0.03). The subclinical deterioration of systolic function is presented even in asymptomatic DMD female carriers.

• MeSH
• Echocardiography, Doppler MeSH
• Adult MeSH
• Muscular Dystrophy, Duchenne diagnostic imaging   genetics   physiopathology   MeSH
• Ventricular Dysfunction, Left diagnostic imaging   physiopathology   MeSH
• Heterozygote * MeSH
• Middle Aged MeSH
• Humans MeSH
• Mitral Valve diagnostic imaging   MeSH
• Blood Flow Velocity MeSH
• Case-Control Studies MeSH
• Systole MeSH
• Tricuspid Valve diagnostic imaging   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• Keywords
• DMD, adrenergic response, cardiomyocyte death, cardiomyocytes, duchenne muscular dystrophy, excitation-contraction coupling, human pluripotent stem cells, intracellular calcium,
• Publication type
• Journal Article MeSH

UNLABELLED: 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.

• Keywords
• Becker muscular dystrophy, C-kit, Cardiomyopathy, Cardiovascular progenitor cells, Dystrophin, Heart failure,
• MeSH
• Cardiomyopathy, Dilated * MeSH
• Muscular Dystrophy, Duchenne * MeSH
• Dystrophin MeSH
• Stem Cells MeSH
• Humans MeSH
• Myocardium MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dystrophin MeSH

Mild hypoxia (5% O2) as well as FGFR1-induced activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and MAPK signaling pathways markedly support pluripotency in human pluripotent stem cells (hPSCs). This study demonstrates that the pluripotency-promoting PI3K/AKT signaling pathway is surprisingly attenuated in mild hypoxia compared to the 21% O2 environment. Hypoxia is known to be associated with lower levels of reactive oxygen species (ROS), which are recognized as intracellular second messengers capable of upregulating the PI3K/AKT signaling pathway. Our data denote that ROS downregulation results in pluripotency upregulation and PI3K/AKT attenuation in a hypoxia-inducible factor 1 (HIF-1)-dependent manner in hPSCs. Using specific MAPK inhibitors, we show that the MAPK pathway also downregulates ROS and therefore attenuates the PI3K/AKT signaling-this represents a novel interaction between these signaling pathways. This inhibition of ROS initiated by MEK1/2-ERK1/2 may serve as a negative feedback loop from the MAPK pathway toward FGFR1 and PI3K/AKT activation. We further describe the molecular mechanism resulting in PI3K/AKT upregulation in hPSCs-ROS inhibit the PI3K's primary antagonist PTEN and upregulate FGFR1 phosphorylation. These novel regulatory circuits utilizing ROS as second messengers may contribute to the development of enhanced cultivation and differentiation protocols for hPSCs. Since the PI3K/AKT pathway often undergoes an oncogenic transformation, our data could also provide new insights into the regulation of cancer stem cell signaling.

• Keywords
• HIF-1, MAPK, PI3K/AKT, hPSCs, hypoxia, reactive oxygen species,
• Publication type
• Journal Article MeSH