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

Can off-training physical behaviors influence recovery in athletes? A sccoping review

Recently, the attention on recovery in sport increased enormously although there is lack of scientific evidence on the role of lifestyle in terms of movement [i.e., physical behaviors (PBs)], apart from sleep. Few studies assessed physical activity (PA) and sedentary behavior (SB) in athletes. The aims of this scoping review were to answer to the following scientific questions: (1) How active/inactive are competitive athletes out of training? (2) Do off-training PBs affect recovery, performance, and health? (3) What strategies can be implemented to improve recovery using off-training PBs, apart from sleep? From 1,116 potentially relevant articles, nine were eligible for inclusion in this review. The main issues identified were related to the heterogeneity concerning the types of sports, age category, gender, competitive level, sample size, and instruments/devices adopted, the paucity of studies investigating the effects of PBs while awake on recovery, and the lack of experimental designs manipulating PBs while awake to accelerate recovery. Furthermore, PA and SB domains were rarely investigated, while no research articles focused on the combined effect of 24-h PBs. Eight out of nine studies measured PA, seven SB, and two included sleep. Three studies included training practice into PA measurement by the means of accelerometry. Overall, almost the totality of the athletes achieved recommended PA levels although they sustained prolonged SB. In conclusion, more descriptive researches are needed in different athletic populations and settings. Furthermore, experimental designs aimed at investigating the effects of PBs manipulation on recovery and the putative mechanisms are encouraged

Nordic walking increases circulating VEGF more than traditional walking training in postmenopause.

OBJECTIVES Nordic walking (NW) is widely practiced by postmenopausal women. Its effects are peculiar owing to the involvement of more muscle groups than in traditional walking training (WT). Since mechanical load promotes secretion of vascular endothelial growth factor (VEGF) from both skeletal muscle and muscle endothelium, the aim of the study was to compare the effect of NW and WT on VEGF levels. METHOD Thirty postmenopausal women were randomly assigned to NW or WT. Both groups trained 40-50 min/day, three times per week, at a mean intensity of 12 on a 15-category scale of the ratings of perceived exertion. Since VEGF is also released from adipocytes, anthropometric parameters were assessed. RESULTS NW increased circulating VEGF more than WT (p = 0.041). Furthermore, both study groups exhibited an average decrease in weight (p = 0.023), body mass index (p = 0.024), hip circumference (p = 0.001), and arm fat index, although WT participants had higher values for this index at baseline (p < 0.001) and thus exhibited a greater net decrease compared with the NW participants (p < 0.011). CONCLUSIONS These data imply that NW increases the level of circulating VEGF more than does traditional walking when the intensity of training is equivalent

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

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

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&lt; 0,00; P= 0,00; P =0,00), while relative wall thickness (RWT) and AE were lower (P&lt;0,03;P=0,00). Mitral E wave and E/A were higher (P = 0,001; P&lt;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

Study of cardiomyogenic potential of human Amniotic Fluid Stem Cells

It has been shown that Amniotic fluid stem cells (AFSCs) have characteristics intermediate between pluripotent embryonic and lineage-restricted adult stem cells, and are non-tumorigenic and low immunogenic. Moreover, they are obtained without destroying human embryos, so that preventing most of the ethical and social controversy. Human AFSCs express some genes specific of both embryonic (OCT3/4, NANOG, c-MYC) and primordial germ stem cells (Fragilis, Stella, c-KIT). We have demonstrated that hAFSCs form in vitro embryoid bodies (EBs) and express markers of three germ layers. Studies reported the ability of hAFSCs to differentiate in vitro into adipocytes and osteocytes, but only few data are available on their cardiomyogenic potential. Aim of this study is to analyze hAFSCs differentiation through the cardiac pathway. Embryonic Bodies (EBs) were obtained from hAFSCs cultured in presence of ascorbic acid and 5-aza-2’-deoxycytidine. Cardiomyogenic potential of hAFSCs and EBs was explored by WB and immunoflorescent analyses of specific markers. Simultaneous quantitative detection and cellular localization analysis of cardiomyogenic markers were conducted with an ImageStream multispectral imaging flow cytometer (Amnis-Seattle, WA) equipped with IDEAS statistical software. We evidenced that both AFSCs and EBs at early stage express Nkx2.5, a transcription factor expressed by cardiomyocytes precursor cells. Moreover, ImageStream imaging cytometer analysis evidenced that EBs formation was accompanied by an up-regulation of Nkx2.5 expression (36.54±1.83% and 64.68±3.23% positive cells in hAFSC and EBs respectively, p&lt;.005) and by a significant nuclear translocation (12.98±0.64% and 37.98±1.9% nuclear positive cells in hAFSC and EBs respectively, p&lt;.005). Microscopical analysis evidenced inside the EBs cells positive for the presence of cardiac α-Myosin heavy chain protein structurally organized in oriented filaments. Moreover, we detected cells expressing Connexin43. These results evidenced that EBs obtained from hAFSCs cultured in permissive conditions terminally differentiate into cardiomyocytes and suggest them as possible model to study the cardiac differentiation

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