Human adipose tissue‐derived stem cells protect impaired cardiomyocytes from hypoxia/reoxygenation injury through hypoxia‐induced paracrine mechanism

Growing studies have emerged on adipose‐derived stem cells (ADSCs), which hold the potential for cell‐based therapy in diseased injured hearts. Apart from their differentiation pluripotency, such benefits also result from the ability of paracrine. The results of this study showed that after a 24‐h hypoxia culture, ADSCs secreted amplified quantities of hepatocyte growth factor, interleukin‐1, vascular endothelial growth factor‐A, fibroblast growth factor‐2, and transforming growth factor‐β, all of which increased statistically compared with normoxia cultures. Resultantly, conditioned media (CM) from hypoxia‐treated ADSCs can promptly improve cardiac function in in vivo infarction model as well as ameliorate apoptosis of cardiomyocytes subjected to hypoxia/reoxygenation conditions, accompanied by changes of JNK signal activation. While SP600125, a specific JNK pathway inhibitor, partly decreased cardiac cytoprotection assessed by incremental caspase‐3 activation and subsequent TUNEL index, which led to no significantly different outcome between CM from ADSCs in normoxia culture and those in hypoxia culture. These data suggested that, in response to hypoxia, ADSCs could amplify expression of several protective soluble factors, which mediate direct cytoprotection. Furthermore, the improvement for impaired cardiomyocytes treated by hypoxia‐induced ADSCs‐CM was significant in part because of the involvement of the JNK signal pathway. Copyright © 2012 John Wiley & Sons, Ltd.     

Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.     

Activation of the prolyl-hydroxylase oxygen-sensing signal cascade leads to AMPK activation in cardiomyocytes

The proline hydroxylase domain-containing enzymes (PHD) act as cellular oxygen sensors and initiate a hypoxic signal cascade to induce a range of cellular responses to hypoxia especially in the aspect of energy and metabolic homeostasis regulation. AMP-activated protein kinase (AMPK) is recognized as a major energetic sensor and regulator of cardiac metabolism. However, the effect of PHD signal on AMPK has never been studied before. A PHD inhibitor (PHI), dimethyloxalylglycine and PHD2-specific RNA interference (RNAi) have been used to activate PHD signalling in neonatal rat cardiomyocytes. Both PHI and PHD2-RNAi activated AMPK pathway in cardiomyocytes effectively. In addition, the increased glucose uptake during normoxia and enhanced myocyte viability during hypoxia induced by PHI pretreatment were abrogated substantially upon AMPK inhibition with an adenoviral vector expressing a dominant negative mutant of AMPK-α1. Furthermore, chelation of intracellular Ca2+ by BAPTA, inhibition of calmodulin-dependent kinase kinase (CaMKK) with STO-609, or RNAi-mediated down-regulation of CaMKK α inhibited PHI-induced AMPK activation significantly. In contrast, down-regulation of LKB1 with adenoviruses expressing the dominant negative form did not affect PHI-induced AMPK activation. We establish for the first time that activation of PHD signal cascade can activate AMPK pathway mainly through a Ca(2+)/CaMKK-dependent mechanism in cardiomyocytes. Furthermore, activation of AMPK plays an essential role in hypoxic protective responses induced by PHI.     

Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Background

Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective

We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods

Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results

At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion

Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.     

Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis

Previous studies have shown that alpha-adrenergic activation reduces myocardial damages caused by ischemia/reperfusion. However, the molecular mechanisms of how alpha-adrenergic activation protects the myocardium are not completely understood. The objective of this study was to test the hypothesis that alpha-adrenergic activation protects the myocardium by, at least in part, inhibiting apoptosis in cardiomyocytes. The current data has shown that apoptosis in neonatal rat cardiomyocytes, induced by 24 h treatment with hypoxia (95% N2 and 5% CO2) and serum deprivation, was inhibited by co-treatment with phenylephrine. Pre-treatment with phenylephrine for 24 h also protected cardiomyocytes against subsequent 24 h treatment with hypoxia and serum deprivation. Exposure of cardiomyocytes to phenylephrine for up to 9 days under normoxic conditions did not cause apoptosis. The phenylephrine-mediated cytoprotection was blocked by an alpha-adrenergic antagonist, phentolamine. beta-adrenergic activation with isoproterenol did not protect cardiomyocytes against hypoxia and serum deprivation-induced apoptosis. Under hypoxic conditions, phenylephrine prevented the down-regulation of Bcl-2 and Bcl-X mRNA/protein and induced hypertrophic growth. Phenylephrine-mediated protection was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin and was mimicked by the caspase-9 peptidic inhibitor LEHD-fmk. These results suggest that alpha-adrenergic activation protects cardiomyocytes against hypoxia and serum deprivation-induced apoptosis through regulating the expression of mitochondrion-associated apoptosis regulatory genes, preventing activation of mitochondrial damage-induced apoptosis pathway (cytochrome C-caspase-9), and activating hypertrophic growth.     

Paracrine potential of adipose stromal vascular fraction cells to recover hypoxia-induced loss of cardiomyocyte function

Cell-based therapies show promising results in cardiac function recovery mostly through paracrine-mediated processes (as angiogenesis) in chronic ischemia. In this study, we aim to develop a 2D (two-dimensional) in vitro cardiac hypoxia model mimicking severe cardiac ischemia to specifically investigate the prosurvival paracrine effects of adipose tissue-derived stromal vascular fraction (SVF) cell secretome released upon three-dimensional (3D) culture. For the 2D-cardiac hypoxia model, neonatal rat cardiomyocytes (CM) were cultured for 5 days at < 1% (approaching anoxia) oxygen (O₂) tension. Typical cardiac differentiation hallmarks and contractile ability were used to assess both the cardiomyocyte loss of functionality upon anoxia exposure and its possible recovery following the 5-day-treatment with SVF-conditioned media (collected following 6-day-perfusion-based culture on collagen scaffolds in either normoxia or approaching anoxia). The culture at < 1% O ₂ for 5 days mimicked the reversible condition of hibernating myocardium with still living and poorly contractile CM (reversible state). Only SVF-medium conditioned in normoxia expressing a high level of the prosurvival hepatocyte-growth factor (HGF) and insulin-like growth factor (IGF) allowed the partial recovery of the functionality of damaged CM. The secretome generated by SVF-engineered tissues showed a high paracrine potential to rescue the nonfunctional CM, therefore resulting in a promising patch-based treatment of specific low-perfused areas after myocardial infarction.     

PO(2)-dependent differential regulation of multidrug resistance 1 gene expression by the c-Jun NH2-terminal kinase pathway

Hypoxia-induced multidrug resistance 1 (MDR1) gene expression is known to be mediated by c-Jun NH(2)-terminal kinase (JNK) activation. However, the molecular mechanisms underlying this action of JNK remain elusive. On the contrary, there has been increasing evidence for a negative correlation of JNK activity with MDR1 expression under normoxic conditions. Here, we present evidence that the JNK pathway represses MDR1 expression in normoxia and activates MDR1 expression in hypoxia. Our data show that JNK pathway-induced MDR1 repression in normoxia is mediated by increased c-Jun binding to activator protein 1 site, located in the MDR1 promoter, and requires the activity of histone deacetylase 5. In contrast, JNK pathway-induced MDR1 activation in hypoxia is independent of the activator protein 1 site. Rather, this action is dependent on increased hypoxia-inducible factor 1 (HIF1) binding to the hypoxia response element in the MDR1 promoter, which is promoted by the interaction of HIF1alpha with c-Jun in the nucleus and requires the activity of the p300/CBP (CREB-binding protein) coactivator.     

mIGF-1/JNK1/SirT1 signaling confers protection against oxidative stress in the heart

Oxidative stress contributes to the pathogenesis of aging-associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD(+) -dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin-like growth factor-1 (IGF-1) propeptide (mIGF-1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF-1/SirT1 signaling in vivo, we generated cardiac-specific mIGF-1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen-inducible and conditional fashion. Analysis of these mice confirmed that mIGF-1-induced SirT1 activity is necessary to protect the heart from paraquat (PQ)-induced oxidative stress and lethality. In cultured CM, mIGF-1 increases SirT1 expression through a c-Jun NH(2)-terminal protein kinase 1 (JNK1)-dependent signaling mechanism. Thus, mIGF-1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.     

High glucose protects single beating adult cardiomyocytes against hypoxia

In the heart, the opening of sarcolemmal ATP-sensitive K(+) (K(ATP)) channels seems to be crucial for the cardiac protection against hypoxia/ischaemia. In the present study, we have exposed cardiomyocytes under hypoxia to high extracellular glucose (30 mM). Under these conditions, intracellular concentration of 1,3-bisphosphoglycerate has increased confirming stimulation of glycolysis. Perforated patch-clamp electrophysiology revealed that hypoxia induces whole-cell K(+) current in cardiomyocytes more efficiently in the presence than in the absence of high glucose. Glucose significantly promoted survival of cardiomyocytes exposed to hypoxia. HMR 1098, an antagonist of sarcolemmal K(ATP) channels, inhibited glucose-induced activation of whole-cell K(+) current during hypoxia as well as glucose-mediated cytoprotection. An inhibitor of glyceraldehyde 3-phosphate dehydrogenase, iodoacetate, inhibited glycolysis in hypoxia and blocked the activation of sarcolemmal K(ATP) channels. Based on the obtained results, we conclude that the activation of sarcolemmal K(ATP) channels is involved in glucose-mediated cardioprotection.     

Conditioned medium from hypoxic cells protects cardiomyocytes against ischemia

The hypothesis of the present study is that cardiomyocytes subjected to prolonged ischemia, may release survival factors that will protect new cardiac cells from ischemic stress. We exposed neonatal rat cardiomyocyte primary cultures to hypoxia, collected the supernatant, treated intact cardiac cells by this posthypoxic supernatant, and exposed them to hypoxia. The results show cardioprotection of the treated cells compared with the untreated ones. We named the collected posthypoxic supernatant "conditioned medium" (CM), which acts in a dose-dependent manner to protect new cardiac cells from hypoxia: 100 or 75% of CM diluted in phosphate-buffered saline (PBS) protected cells as if they were not exposed to hypoxia (P < 0.001). When CM was removed from the cells before hypoxia, protection was not observed. CM also protected skeletal muscle cultures from hypoxia, but not cardiac cells against H(2)O(2)-induced cell damage. Finally, CM treatment protected the isolated heart in Langendorff set-up against ischemia. Smaller infarct size (9.9 ± 4.4% vs. 28.3 ± 8.5%, P < 0.05), better Rate Pressure Product (67 ± 11% vs. 48.6 ± 13.4%, P < 0.05) and better rate of contraction and relaxation were observed following ischemia and reperfusion (1341 ± 399 mmHg/s vs. 951 ± 349 mmHg/s, P < 0.05 and 1053 ± 347 mmHg/s vs. 736 ± 314 mmHg/s, P < 0.05). To conclude, there are factors that are released from the heart cells subjected to ischemia/hypoxia that protects cardiomyocytes from ischemic stress.     

Hypoxia-induced bFGF gene expression is mediated through the JNK signal transduction pathway

Although the synthesis of angiogenic factors in hypoxic regions of solid tumors is recognized as one of the critical steps in tumor growth and metastasis, the signal transduction pathway involved in hypoxic induction of basic fibroblast growth factor (bFGF) gene expression is still obscure. In the study described here, we investigated the intracellular responses to hypoxia and the mechanisms triggering the initiation of angiogenic activity in drug-resistant human breast carcinoma MCF-7/ADR cells. Northern blots showed an increase in the level of c-jun, c-fos, and bFGF mRNA during hypoxia. Gel mobility-shift analysis of nuclear extracts from hypoxia-exposed cells showed an increase in AP-1 binding activity. In addition, hypoxic treatment strongly activated c-Jun N-terminal kinase 1 (JNK1), leading to phosphorylation and activation of c-Jun. Expression of a dominant negative mutant of JNK1 suppressed hypoxia-induced JNK1 activation as well as bFGF gene expression. Taken together, hypoxia-induced bFGF gene expression is mediated through the stress-activated protein kinase (SAPK) signal transduction pathway.     

The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice

Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.     

USP49 inhibits ischemia–reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1–JNK1/2 signaling

Dual-specificity protein phosphatases (DUSP) also known as mitogen-activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. DUSP1-mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia–reperfusion (I/R) injury. However, the regulation of DUSP1–JNK pathway remains unclear. In the current study, ubiquitin-specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real-time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl-2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK-8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time-dependently increased in AC16 cardiomyocytes following I/R. I/R-induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown-induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1–JNK1/2 signaling pathway with a protective role in cardiac I/R injury.     

Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells

Hypoxia/reoxygenation causes cellular injury and death associated with a number of pathophysiological conditions, including myocardial ischemia/reperfusion injury and stroke. The cell death pathways induced by hypoxia/reoxygenation and their underlying regulatory mechanisms remain poorly understood. Recent studies have shown that hypoxia/reoxygenation can induce Bax translocation and cytochrome c release. Using murine lung endothelial cells as a model, we found that the induction of apoptosis by hypoxia/reoxygenation involved the activation of both Bax-dependent and death receptor-mediated pathways. We demonstrated the activation of the death-inducing signal complex and Bid pathway after hypoxia/reoxygenation. Hepatocyte growth factor markedly inhibited hypoxia/reoxygenation-induced endothelial cell apoptosis. The cytoprotection afforded by hepatocyte growth factor was mediated in part by the stimulation of FLICE-like inhibiting protein expression, the attenuation of death-inducing signal complex formation, and the inhibition of Bid and Bax activation. Hepatocyte growth factor also prevented cell injury and death by increasing the expression of the antiapoptotic Bcl-XL protein. The inhibition of Bid/Bax-induced cell death by hepatocyte growth factor primarily involved p38 MAPK and in part Akt-dependent pathways but not ERK1/ERK2.     

MCP-1/CCL2 protects cardiac myocytes from hypoxia-induced apoptosis by a G(alphai)-independent pathway

Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of G(alphai) signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms.     

ADSCs differentiated into cardiomyocytes in cardiac microenvironment

The microenvironment plays a critical role in directing the progression of stem cells into differentiated cells. So we investigated the role that cardiac microenvironment plays in directing this differentiation process. Adipose tissue-derived stem cells (ADSCs) were cultured with cardiomyocytes directly (“co-culture directly”) or by cell culture insert (“co-culture indirectly”). For co-culture indirectly, differentiated ADSCs were collected and identified. For co-culture directly, ADSCs were labeled with carboxyfluorescein succinimidyl ester (CFSE), Fluorescence-activated cell sorting was used to extract and examine the differentiated ADSCs. The ultrastructure and the expression of cardiac specific proteins and genes were analyzed by SEM, TEM, western blotting, and RT-PCR, respectively. Differentiated ADSCs experienced the co-culture presented cardiac ultrastructure and expressed cardiac specific genes and proteins, and the fractions of ADSCs expressing these markers by co-culture directly were higher than those of co-culture indirectly. These data indicate that in addition to soluble signaling molecules, direct cell-to-cell contact is obligatory in relaying the external cues of the microenvironment controlling the differentiation of ADSCs to cardiomyocytes.     

EGF Enhances ADSCs Secretion via ERK and JNK Pathways

The objective of this work was to study the effect of epidermal growth factor (EGF) induced secretions of angiogenesis factors in adipose-derived stem cells (ADSCs) and the involvement of mitogen-activated protein kinases (MAPK). ADSCs were cultured and ELISA assays were performed to quantify the vascular endothelial growth factor, the hepatocyte growth factor, and the stromal derived factor-1 in ADSC-conditioned medium before and after EGF treatments and after pharmacological inhibition of MAPKs with PD98059, SB203580, and SP600125. The tube formation assay was used to test the effects of EGF treated and inhibitor treated ADSCs on the human umbilical vein endothelial cells (HUVECs) tube formation. Liposuction was applied and ADSCs were cultured successfully. The ADSCs released a variety of angiogenic factors, with the EGF treatments enhancing secretions and promoting the HUVEC tube formation. The MAPK inhibitors PD98059 and SP600125 increased the paracrine to promote tubular formation, while the SB203580 played an opposite role. In conclusion, (1) the in vitro cultured ADSCs secrete various angiogenic factors and the EGF amplifies the secretion and can enhance the ADSCs on the HUVEC tube formation. (2) ERK1/2 and JNK pathway may be involved in the enhanced secretion capacity of ADSCs while the p38 pathway may exert an opposite effect.     

低氧与心肌细胞凋亡

细胞凋亡是心肌细胞低氧损伤的主要死亡形式之一。低氧引起心肌细胞凋亡可以通过外部的死亡受体通路以及内部的线粒体通路,两条通路之间又存在复杂的交互作用,其中,线粒体通路在低氧诱导的心肌细胞凋亡中起重要作用。另外,心肌细胞本身也具有多种内源性的凋亡抑制因子。因此,低氧时心肌细胞凋亡的产生是多种因素综合作用的结果,Bcl-2家族蛋白、线粒体通透性改变、细胞色素c的释放以及caspases的活化等参与了低氧引起的心肌细胞凋亡的调控。对低氧时心肌细胞凋亡的认识和深入研究,为人类在缺血性心脏病的防治中提供了一个新的治疗措施。     

MKP1 overexpression reduces TNF-α-induced cardiac injury via suppressing mitochondrial fragmentation and inhibiting the JNK–MIEF1 pathways

Mitochondrial stress has been acknowledged as the pathogenesis for tumor necrosis factor-α (TNF-α)-induced septic cardiomyopathy. Recently, MAP kinase phosphatase 1 (MKP1) downregulation and mitochondrial fragmentation modulate the mitochondrial stress via multiple molecular mechanisms. Thereby, the goal of our current work is to figure out the functional role of mitochondrial fragmentation in TNF-α-induced septic cardiomyopathy. Our results exhibited that MKP1 expression was significantly repressed in hearts treated by TNF-α. Overexpression of MKP1 sustained cardiac function and attenuated cardiomyocytes death in TNF-α-treated hearts. At the molecular levels, decreased MKP1 induced mitochondrial stress, as indicated by mitochondrial calcium overloading, mitochondrial oxidative stress, mitochondrial antioxidant downregulation, mitochondrial membrane potential reduction, mitochondrial bioenergetics suppression, mitochondrial proapoptotic factors liberation, and caspase-9 apoptotic pathway activation. To the end, we illustrated that MKP1-modulated mitochondrial stress via mitochondrial fragmentation; reactivation of mitochondrial fragmentation abolished the protective effect of MKP1 overexpression on mitochondrial function. Further, MKP1 affected mitochondrial division in a mechanism through the JNK–MIEF1 axis. Blockade of JNK pathway abolished the regulatory actions of MKP1 on mitochondrial division. Altogether, our results identify MKP1 as a novel cardioprotective factor in TNF-α-related septic cardiomyopathy via affecting mitochondrial division by the way of JNK–MIEF1 signaling pathway. Therefore, MKP1 expression, mitochondrial fragmentation modification, and JNK–MIEF1 pathway modulation may be considered as potential therapeutic targets for the treatment of cardiac injury induced by sepsis.     

Combined hypoxia and sodium nitrite pretreatment for cardiomyocyte protection in vitro

Methods that increase cardiomyocyte survival upon exposure to ischemia, hypoxia and reoxygenation injuries are required to improve the efficacy of cardiac cell therapy and enhance the viability and function of engineered tissues. We investigated the effect of combined hypoxia/NaNO₂ pretreatment on rat neonatal cardiomyocyte (CM), cardiac fibroblast, and human embryonic stem cell‐derived CM (hESC‐CM) survival upon exposure to hypoxia/reoxygenation (H/R) injury in vitro. Cells were pretreated with and without hypoxia and/or various concentrations of NaNO₂ for 20 min, then incubated for 2 h under hypoxic conditions, followed by 2 h in normoxia. The control cells were maintained under normoxia for 4 h. Pretreatment with either hypoxia or NaNO₂ significantly increased CM viability but had no effect on cardiac fibroblast viability. Combined hypoxia/NaNO₂ pretreatment significantly increased CM viability but significantly decreased cardiac fibroblast viability. In rat neonatal CMs, cell death, as determined by lactate dehydrogenase (LDH) activity, was significantly reduced with hypoxia/NaNO₂ pretreatment; and in hESC‐CMs, hypoxia/NaNO₂ pretreatment increased the BCL‐2/BAX gene expression ratio, suggesting that hypoxia/NaNO₂ pretreatment promotes cell viability by downregulating apoptosis. Additionally, we found a correlation between the prosurvival effect of hypoxia/NaNO₂ pretreatment and the myoglobin content of the cells by comparing neonatal rat ventricular and atrial CMs, which express high and low myoglobin respectively. Functionally, hypoxia/NaNO₂ pretreatment significantly improved the excitation threshold upon H/R injury to the level observed for uninjured cells, whereas pretreatment did not affect the maximum capture rate. Hence, hypoxia/NaNO₂ pretreatment may serve as a strategy to increase CM survival in cardiac regenerative therapy applications and tissue engineering. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:482–492, 2015