Reprogramming of human keratinocytes into functional cardiomyocytes

Purpose. Induced pluripotent stem cells (iPS) can be generated by patients intro- ducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. However, the various iPS cell lines are characterized by different properties such as differentiation efficiency and potential safety hazards. Among several readily available primary human somatic cell types, keratinocytes can be isolated easily from human skin or hair follicle, and therefore represent an attractive cell source for reprogramming. The aims of the present study were: 1. To assess the cardiomyogenic potential of human keratynocytes-derived iPS. 2. To increase the differentiation efficiency in order to obtain a homogeneous population of beating cardiomyocytes, also overcoming the limitation of embryonic body formation.Methods. Established iPS cell line obtained from human keratinocytes was cultured in monolayer and exposed sequentially to Ascorbic Acid, 5-Azacytidine, BMP4, ActivinA, VEGF up to 20 days. Differentiation was evaluated monitoring the expression of Nkx2.5, Gata4, sarcomeric α-actinin, α cardiac myosin heavy chain, cardiac T-troponin and Connexin43 and β-adrenoceptors as cardiac markers, by Western Blot, immunofluorescent and cytometric analyses. An ImageStream analysis for a simulta- neous quantitative and morphological evaluation was also performed.Results. During the differentiation induction, iPS became positive to all the analyzed cardiac markers. In particular, we observed a rapid and significant increase of Nkx2.5 and Gata4 expression (14.7±0.7% vs 43.2±2.1% Nkx2.5+ and 21.7±1.2 vs 65.9±3.6% Gata4+ in iPS and 15 days after the induction respectively, p<.005, analy- sis by ImageStream), with a significant nuclear translocation (nuclear+ cells:7.2±0.8% vs 18.8±2.1% of the Nkx2.5+ cells in iPS and 15 days after the induction respectively, p<.005, analysis by ImageStream). The differentiation process was accompanied by a modulation of β1- and β2-adrenoceptors expression. Foci of spontaneous contraction were observed after only 5 days from the induction and after 15 days the beating cells represented about the 60-90% of the entire population. Moreover, differentiated cardio- myocytes responded to adrenergic stimulation by increasing the contraction rate. Conclusion. iPS obtained from hKeratinocytes efficiently differentiate into cardi- omyocytes giving rise to a homogenous population with cardiac-specific molecular and functional properties

Effect of short-term aerobic exercise on cardiovascular remodelling in post-menopause women

It is well known that menopause is associated with an increase of cardiovascular (CV) risk factors, including changes on metabolic profile, body composition, haemdynamic loads and cardiovascular remodelling. The latter starts with an increase of arterial elastance and early concentric left ventricular remodeling, manifested by an increment of the relative wall thickness. We investigated morphological and functional effects of short-term aerobic exercise at moderate intensity without diet restriction on cardiovascular apparatus in post-menopause women. Seventy-six post-menopausal healthy sedentary postwomen (56± 4 yrs) underwent clinical history, physician and anthropometric exam, 12-lead electrocardiography. Echocardiography was used to assess LV geometry and systolic and diastolic functions. Relative diastolic wall thickness (RWT), midwall fractional shortening (MFS) and arterial elastance (AE) were calculated to evaluate the LV concentric remodeling, the intrinsic systolic function and the vascular load, respectively. Data were collected before and after 14 weeks of moderate aerobic exercise training (four time per week). After the intervention program, systolic and diastolic arterial pressure, heart rate and hip circumference were significantly decreased (P = 0,01; P = 0,00; P = 0,00; P = 0,01, respectively). Left ventricular mass/height 2.7, ejection fraction and MFS were higher (p< 0,00; P= 0,00; P =0,00), while relative wall thickness (RWT) and AE were lower (P<0,03;P=0,00). Mitral E wave and E/A were higher (P = 0,001; P<0,0001). Left ventricular mass/height 2.7 was correlated to BMI (P = 0,04), WC (P = 0,034) and waist/hip ratio (P = 0,007). In post-menopause women aerobic exercise improves CV function (concentric remodelling, diastolic and systolic function) acting both on haemodynamic factors and body composition. Our data underline the role of non pharmacological interventions in the CV disease prevention

hAFSC expressing a specific panel of stem cell markers give rise to fully differentiate cardiomyocytes

Human amniotic fluid-derived stem cells (hAFSC) represent a novel class of multipotent stem cells sharing characteristics of both embryonic and adult stem cells. In fact, hAFSC proliferate rapidly, are able to differentiate into cells of all the three embryonic germ layers, but do not form teratoma. It has been already reported that hAFSC have a cardiac potential, but a high variability between hAFSC donors in differentiation efficiency has been described. Aim of this study was to phenotypically identify the hAFSC able to differentiate into mature cardiomyocytes. hAFSCs from 10 different donors were characterized for the immunophenotypic expression of stemness markers and then cultured in differentiatve conditions. hAFSC differed for both stemness markers expression and for differentiation efficiency. Only the hAFSC expressing specific stem cell antigens were able to differentiate into a homogeneous population of cells that highly express cardiac cytoskeletal proteins and the structural and functional sarcoplasmatic reticulum proteins. Our results demonstrate that only hAFSC showing a specific stem cell pattern phenotype can fully differentiate into myocytes giving rise to a homogenous population characterized by cardiac-specific molecular, structural, and functional properties

Combining Core Training and Sensory Refinement: effects on physical performance

The concept of “the Core” describes the complex of anatomical components of the trunk, pelvis and shoulder girdle that are responsible for maintaining the stability of the spine and pelvis and are critical for the transfer of energy from larger torso to the smaller extremities, during many sport and daily-living activities (1). This concept rooted in sport science and rehabilitation and recently Core Training (CT) became very popular as a method to prevent injuries and improve sport performance and physical fitness. It consists in the progressive training of the musculature of the Core with special emphasis in posture and lumbar spine stability. This aspect requires a fine coordination and body awareness that often are poor developed or regressed after an injury. Then, it is important to include exercises of Sensorial Refinement (SR) that may stimulate the refinement of perceptually neglected areas (2). The aim of the study was to evaluate the effect of combined CT and SR on physical performance and to compare these effects with traditional core training. Furthermore, the effect on retention after 4 weeks of detraining was evaluated. Two groups of participants were recruited (age >30 < 50) and assigned to experimental (CT and SR: EXP, n = 9) or control (CT: CON, n = 9) group. Both groups trained ten weeks, with a frequency of two sessions per week. Training consisted in 10’ of warm up, 40’ of workout and 10’ of cool down. Workout of EXP group consisted in 20’ of SR and 20’ of CT whereas CON group performs 40’ of CT. Participants where tested by: Star Excursion Balance Test (SEBT) for the dynamic balance of lower body, Upper Quarter Y Balance Test (YBT-UQ) to assess upper extremities function in a closed-chain position and McGill test to evaluate muscular endurance of the core. Both groups improved core endurance after training and worsen it at follow up; moreover, both groups ameliorate upper an lower body control (SEBT and YBT-UQ) after training but only EXP group improved or maintained it at follow up. Since the information about the movements of the body are elaborated in the somatomotor cortex for fine coordination, the combination of CT and SR should better promote the consolidation of motor memory and long-term body control

The phosphoinositide 3-kinase role in IL-6 induction to differentiation of rat embryonic cardiomyocytes

Purpose. Cardiomyocytes express IL-6 and its signal transducer, 130-kDa glycoprotein (gp130) that influence cell growth, apoptosis, differentiation and survival by an autocrine pathway. High levels of circulating IL-6 have been reported in patients with congestive heart failure (HF) and after myocardial infarction and IL-6-gp130-signaling participates in HF prevention and compensatory hypertrophy, influencing remodeling processes and inducing Protein kinase C (PKC)-dependent apoptosis. Recent studies demonstrated the presence of precursor cells able to differentiate into cardiomyocytes in the adult heart through signaling pathways similar to those in the embryonic heart thus participating to the physiologic repair of the injured heart. In this line, the study of the embryonic signaling may represent an important cue to understand the molecular bases of the regenerative capacity myocardial progenitors. Aim of this study was to investigate the signal transduction pathways evoked by IL-6 treatment on cardiomyoblasts and its possible differentiating effects. Methods. H9c2 cells cultured in medium supplemented with 1% FBS (differentiation-promoting medium) in presence of IL-6 (10 ng/ml) up to three days. Results. Western Blot and Immunofluorence analysis demonstrated that Il-6 after 3 days of treatment induced a marked increase of the expression of α-myosin heavy chain (α-MHC; terminal cardiac differentiation marker) together with a microfilament reorganization and morphological modifications, that included mono-nucleated cells elongation and fusion into multinucleated tubes. This process was accompanied by an evident nuclear translocation of Nkx2.5 (early myocardial development transcription factor), and by a sub-cellular redistribution of gp130. Results suggest that the biological effects evoked by IL6 were at least partially mediated by PI3K through Akt and PKCzeta pathways modulation, as confirmed by the varied phosphorylated PI3K level observed after 30 min of IL-6 treatment and PI3K induction of downstream PKCzeta phosphorilation, but not PKCalpha and PKCdelta, whereas Akt de-phosphorylation was detected. Conclusions. Our observations evidenced that PI3K is a key regulator of IL-6- induction of embryonic cardiomyocytes differentiation by PKCzeta and Akt-signalling pathways. These data suggest a possible role of IL-6 dependent pathways in regenerative capacity of myocardial cells following injuries

Angiogenetic effects of physical exercise in menopausal women

Introduction: With menopause women face many changes that may lead to the loss of health related fitness, especially if sedentary. In particular, the estrogens deficiency affects the endothelial function thus increasing the incidence of the cardiovascular diseases. The cardio-protective effects of physical exercise is at least partially due to its ability to improve the health of arterial walls by influencing the endothelial function. Nonetheless, a direct angiogenetic of the physical exercise cannot be ruled out. VEGF is an important modulator of vascular growth but there are contrasting results about its response to physical exercise. Aim of our study was to compare the effects of two aerobic training on the VEGF levels and on the angiogenesis in postmenopausal women. Material and Methods. 34 Postmenopausal women underwent a 13 weeks training. In order to analyse the angiogentic effects, plasmatic VEGF levels were analysed before (T0) and after the training (T1). Moreover, the ability of the T0 and T1 serum to chemoattract endothelial cells and to induce them to form tubes was analyzed. Results: All post-menopausal women increase VEGF (P=0,014) after training. In vitro tests evidenced that when cultured in presence of T1 serum, HUVEC cells improved their ability to form tube (P&lt;0,001) as well as their calibre (P&lt;0,001). Moreover, migration assays evidenced that after training the serum chemoattractive capacity increased significantly (P&lt;0,001) capacity. Conclusion: Our data evidence that aerobic training influence the arterial wall physiology also inducing a angiogenetic affect

Human Cardiopoietic Amniotic Fluid cell population: characterization and terminal differentiation

Rationale. Human amniotic fluid-derived (hAF) stem cells are considered a novel class of multipotent stem cells, sharing characteristics of both embryonic and adult stem cells. In fact, they proliferate rapidly, are able to differentiate into cells of all the embryonic germ layers, but do not form teratoma. It has been already reported that the embryoid bodies (EBs) obtained from hAFs have a cardiac potential, but it has not been described a functional terminal differentiation in cardiomyocytes (CMs) yet. Objective. Aim of this study was to foster the cardiomyogenic potential of hAFs in order to obtain a cellular population with morphological and functional features of CMs. Methods and Results. AFCs were exposed sequentially to inducing factors (Ascorbic Acid, 5-Azacytidine, BMP4, ActivinA, VEGF) up to 15 days and differentiation was monitored. Only the hAF samples expressing the multipotency markers SSEA4, OCT4 and CD90 (CardiopoieticAF) responded to the differentiation process loosing their stemness and increasing the cardiac nuclear factors NKX2.5 and GATA4. After the differentiation cells expressed high levels of the sarcomeric proteins (cTnT, αMHC and αSA), the gap junction marker Connexin43 and both atrial and ventricular markers; moreover, up to 90% of the cells was positive for CACNA1C and SERCA2a, cardiac calcium pumps involved in the excitation/contraction coupling, and about 30% of the CardiopoieticAF-derived cells presented spontaneous intracellular Ca2+ waves and Ca2+ fluctuation in response to caffeine or adrenergic stimulation. Some spontaneous beating foci were also observed. Conclusion. Our results demonstrate that CardiopoietichAFs can fully differentiate into a homogenous population of CM-like cells, characterized by cardiac-specific molecular, structural, and functional properties. Thus, CardiopoietichAFs can hold great promise for the development of in vitro models of cardiac genetic disorders, for drug discovery and testing, and for the emerging field of cardiovascular regenerative medicine

VEGF response to aerobic training in postmenopause: walking training vs nordic walking

Menopausal transition leads to increased risk of non-communicable chronic diseases, which are characterized by endothelial disruption and dysfunction. Aerobic physical exercise determines an increase of vascular endothelial growth factor (VEGF), a signal protein that stimulates angiogenesis, and recent studies suggest a direct correlation between exercise intensity and VEGF production (1). However, high intensity training is often not recommended for postmenopausal women as a result of cardiovascular and orthopaedic contraindications. The aim of the study was to determine if exercising at the same intensity (i.e. moderate intensity) but involving a more extended muscle mass might induce higher circulating levels of VEGF. Nordic Walking (NW), a form of physical activity where the active use of a pair of dedicated poles is added to regular walking, was compared to walking training (WT). Thirty postmenopausal women were enrolled in the study (57.93±3.55 years old) and randomly assigned to WT (n=15) and NW (n=15). Serum VEGF levels were determined by ELISA before and after exercise training, consisting in three workouts of 40-50 minutes for 13 weeks at intensity between 11 and 13 of a 15-category scale of the ratings of the perceived exertion. The only difference was the use of poles with appropriated technique in NW group. RM-ANOVA with repeated measure for the factor time revealed an effect for time (P=.01) and an interaction effect time x group (P=.041). Post-hoc analysis, consisting in a paired sample t-test for each group, showed that NW increased VEGF whereas WT showed only a tendency (WT: T0=39.68±2.90 T1=40.22±2.56, P=.012; NW: T0=38.22±1.60 T1=42.52±5.97, P=.57). In conclusion, independently from exercise intensity, NW seems to increase VEGF more efficiently than WT, probably as a result of a larger capillary bed actively involved by exercising muscles. This outcome should be taken in consideration when programming exercise training for postmenopausal women

Measuring the perception of quality physical education in Latin American professionals

Um plano instável para o desenvolvimento de currículos e questões de apoio na educac¸ão física (PE) criou confusão entre os profissionais. O objetivo desta pesquisa é investigar os fatores percebidos como importantes no desenvolvimento da educac¸ão física de qualidade (QPE) por profissionais de países da América Latina (AL). Um questionário composto por 24 itens com base no QPE foi respondido por 468 profissionais coletados em 6 cidades da América Latina. Uma análise fatorial exploratória dos 24 itens usando extrac¸ão ML e rotac¸ão obliminar direta foram aplicados, e os 17 itens retidos foram agrupados em uma soluc¸ão de três fatores denominada Elementos de Desenvolvimento e Suporte para QPE na Escola (DSFQPE) ( = 0,935), Valor essencial do QPE (CVPE) ( = 0,890) e Arranjo Curricular das Atividades Físicas (CAPA) ( = 0,850). Os itens retidos indicaram propriedades excelentes e o referencial básico percebido pelos profissionais de EF em países da América Latina como importante na investigac¸ão do PEQ. © 2018 Colegio ´ Brasileiro de Ciencias ˆ do Esporte. Publicado por Elsevier Editora Ltda. Este e´ um artigo Open Access sob uma licenc¸a CC BY-NC-N