Gravitation is an important factor in maintaining cardiac contractility. Our study investigated whether simulated microgravity increases myocardial susceptibility to ischemia-reperfusion (IR) injury. Using the Langendorff-perfused heart model with 300 beats/min pacing, 4-week tail suspension (SUS) and control (CON) male Sprague-Dawley rats (n = 10 rats/group) were subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 120 min of reperfusion. Left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), creatine kinase (CK) and lactate dehydrogenase (LDH) activity, and infarct size were assessed. Data demonstrated that there were significantly increased LVEDP, CK, LDH, and infarct size in SUS compared with CON (P < 0.05), accompanied by decreased LVESP (P < 0.05). Furthermore, TUNEL-positive cardiomyocytes were higher in SUS than that in CON (P < 0.01), and AMP-activated protein kinase (AMPK) phosphorylation and Bcl-2/Bax in SUS were less compared with CON (P < 0.05). Similarly, isolated hearts pre-treated with A-769662 exhibited better recovery of cardiac function, increased AMPK phosphorylation, and reduced necrosis and apoptosis. Furthermore, AMPKα protein showed a significant suppression in 4-week hindlimb unweighting rats. These results suggest that AMPK deficiency increases myocardial susceptibility to IR injury in rats subjected to simulated microgravity.

Although immuno‐inflammatory response contributes to pathogenesis of AF, molecular and cellular mechanism in this process remains poorly understood. Recently, increasing evidence suggests that Programmed death‐1 (PD‐1)/PD‐1 ligand (PD‐L) pathway may be a potential pathway participating in AF pathogenesis. In this study, we detected the PD‐1 and PD‐L1, 2 expression on peripheral blood function cells by flow cytometry in 91 atrial fibrillation (AF) patients and 35 healthy volunteers. The expression of PD‐1 on CD4+ T cells and PD‐L1 on myeloid dendritic cells (mDCs) in AF patients is significantly down‐regulated compared with healthy volunteers. In addition, the extent of PD‐1/PD‐L1 down‐regulation is closely related with AF burden. More importantly, Allogeneic mixed leukocyte reactions (MLR) shows that the mDCs PD‐L1 down‐regulation is associated with increased T cell (CD4+ and CD8+) proliferation, increased type 1 effector cytokines (IL‐2 and IFN‐γ) secretion, and decreased type 2 effector cytokine (IL‐10) secretion. Then, PD‐L1 up‐regulation by the stimulation of IFN‐α can significantly convert this representation. Collectively, our report suggest that T(CD4+)/mDCs‐associated PD‐1/PD‐L1 pathway plays a key role in AF immune regulation. PD‐1/PD‐L1 down‐regulation in AF patients promotes T cells function and may contribute to AF pathogenesis.

Atrial fibrillation (AF) is a common disease with a poorly understood pathophysiological mechanism. Increasing evidence indicates that AF may be associated with immunologic inflammation responses, but it remains unclear whether activation of peripheral blood CD3(+) T-lymphocytes plays a role in the pathogenesis of AF. The aim of this study was to evaluate this phenomenon. Fifty paroxysmal AF patients and 56 persistent AF patients who underwent successful electrical cardioversion were enrolled. The percentages of CD69 and human leukocyte antigen DR (HLA-DR) positive peripheral blood CD3(+) T-lymphocytes, which indicate T-lymphocyte activation, were examined by flow cytometric analysis in the patients and 51 healthy controls. The patient groups had higher levels of CD69 and HLA-DR than the healthy controls. During the 3-month follow-up, 37 patients had recurrence of AF (recurrence group) and 50 patients remained in sinus (sinus group). The CD69 and HLA-DR levels in the sinus group were all significantly down-regulated at follow-up compared with before cardioversion. However, there were no statistically significant differences between the CD69 and HLA-DR levels in the recurrence group at follow-up and before cardioversion. Our findings suggest that activation of peripheral blood CD3(+) T-lymphocytes was associated with AF, and might be a diagnostic or therapeutic marker.

OBJECTIVE:Atherosclerosis is a progressive disease characterized by a series of inflammatory responses in the large and medium arteries. Th17 cells, a distinct T cell lineage which has recently been identified, have a proinflammatory role and are implicated in many inflammatory conditions in humans and mice. The present study was designed to assess whether Th17 cells are associated with human coronary atherosclerosis.DESIGN:Flow cytometry was used to examine Th17 cell frequencies in patients with coronary atherosclerosis and in healthy individuals. ELISA and real-time RT-PCR were performed to investigate circulating interleukin (IL)-17 (the signature cytokine of Th17 cells) and IL-8 (the cytokine induced by IL-17) protein and mRNA levels.RESULTS:Significantly increased Th17 cell frequencies are observed in patients with coronary artery disease compared to healthy controls. The protein and mRNA levels of IL-17 and IL-8 are also significantly elevated in patients with atherosclerosis compared to healthy volunteers. Furthermore, mRNA levels of IL-17 and IL-8 are correlated with each other and with peripheral neutrophil counts.CONCLUSIONS:Our findings indicate that Th17 cells and their signature cytokine are involved in the process of atherogenesis. These data suggest that Th17 cells link T cell activity with neutrophilic inflammation in atherosclerosis.

Objective Ascorbic acid has been reported to promote the differentiation of embryonic stem cells (ESCs) into cardiomyocytes (CMs). However, appropriate culture protocols are needed to improve the differentiation efficiency and produce adequate numbers of CMs for therapeutic cell transplantation. This study investigated the effect of co-culturing with native CMs on ascorbic acid-induced cardiomyogenic differentiation in embryonic stem cells, to develop a novel protocol for generating functional CMs from ESCs. Methods Native CMs were isolated from the hearts of 1-day-old Sprague-Dawley rats. Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with or without 0.1 mM of ascorbic acid (Sigma) for cardiomyogenic differentiation. They were divided into four groups: ascorbic acid & co-culture group, co-culture group, ascorbic acid group, and control group. In the co-culture system, EBs were co-cultured with native CMs by the hanging cell culture inserts (PET 1 μm) (Millicell; Millipore, Bedford, MA, USA). The native CMs were purposely placed on culture plate inserts to prevent direct contact with subnatant EBs. Both the ESCs and native CMs grew in the same medium but they were easy to separate. The structural and functional properties of ESC-derived CMs (ESCM) were evaluated by microscopic observation, immunocytochemistry, RT-PCR, and transmission electron microscopy. Results The average percentages of EBs exhibited rhythmic contractions in co-culture and ascorbic acid group, co-culture group, ascorbic acid group, and control group were 86.6±9.52%, 65.60±10.77%, 29.6±6.03%, and 17.76±5.99%, respectively. The percentage of beating EBs in co-culture & ascorbic acid group was much higher and the homogeneity of EBs were significantly improved over that seen in other groups (p<0.01), simultaneously, the automaticity of beating also maintained for more time. The majority (>90%) of cells in EBs were ESCM that acquired almost the same structural and functional properties as typical CMs. Conclusions The present study demonstrates the cardiomyogenic differentiation of ESCs can be efficiently controlled by co-cultured with native CMs, and this may lead to a practicable cocktail approach to generate ESC-derived CMs for stem cell-based regenerative medicine.

Although inflammatory cells contribute to immunopathogenesis of atherosclerosis, underlying molecular mechanisms remain largely undefined. Recently, it has been demonstrated in mouse model that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway plays a critical role in proatherogenic immune responses. Here we examined the expression of PD-1 and PD-L1 on peripheral blood mononuclear cells by flow cytometry in 76 patients with coronary artery disease (CAD), and 25 healthy volunteers. The expression of PD-1 and PD-L1 is significantly down-regulated on T cells and myeloid dendritic cells (mDCs) in CAD patients than in healthy individuals, respectively. More importantly, we found that decreased PD-L1 expression on mDCs is related with the increased T cell immune responses in CAD patients. In addition, stimulation of PD-L1 expression in vitro could attenuate the stimulatory ability on allogeneic T cell proliferation and its cytokine production, including IFN-gamma and IL-2, and also influence the production of IL-10 and IL-12 by mDCs. Taken together, we can draw a conclusion that PD-1/PD-L1 pathway plays a key role in the regulation of proatherogenic T cell immunity by intervening antigen presenting cell (APC)-dependent T cell activation, which associates with pro-inflammatory or anti-inflammatory cytokine production, and further studies need gain insight into that this pathway represents a strategy of immunotherapy for atherosclerosis.