α,β‐Unsaturated aldehyde pollutant acrolein suppresses cardiomyocyte contractile function: Role of TRPV1 and oxidative stress

Air pollution is associated with an increased prevalence of heart disease and is known to trigger a proinflammatory response via stimulation of transient receptor potential vanilloid cation channels (TRPV1, also known as the capsaicin receptor). This study was designed to examine the effect of acrolein, an essential α,β‐unsaturated aldehyde pollutant, on myocardial contractile function and the underlying mechanism involved with a focus on TRPV1 and oxidative stress. Cardiomyocyte mechanical and intracellular Ca²⁺ properties were evaluated using an IonOptix MyoCam® system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time‐to‐PS (TPS), time‐to‐90% relengthening (TR₉₀), fura‐2 fluorescence intensity (FFI) and intracellular Ca²⁺ decay. Changes in apoptosis and TRPV1 were evaluated using Western blot analysis. The degree of oxidative stress was assessed using the ratio between reduced and oxidized glutathione. Results obtained revealed that exposure of cardiomyocytes to acrolein acutely compromised contractile and intracellular Ca²⁺ properties including depressed PS, ± dL/dt and ΔFFI, as well as prolonged TR₉₀ and intracellular Ca²⁺ decay. In addition, acrolein exposure upregulated TRPV1 associated with an increase in both apoptosis and oxidative stress. However, the acrolein‐induced cardiomyocyte contractile and intracellular Ca²⁺ anomalies, as well as apoptosis (as evidenced by Bcl‐2, Bax, FasL, Caspase‐3 and ?8), were negated by the reactive oxygen species (ROS) scavenger glutathione or the TRPV1 antagonist capsazepine. Collectively these data suggest that the α,β‐unsaturated aldehyde pollutant acrolein may play a role in the pathogenesis and sequelae of air pollution‐induced heart disease via a TRPV1‐ and oxidative stress‐dependent mechanism. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 638–647, 2015.     

Cisplatin compromises myocardial contractile function and mitochondrial ultrastructure: Role of endoplasmic reticulum stress

1. Cisplatin is a potent chemotherapeutic agent with broad‐spectrum antineoplastic activity against various types of tumours. However, a major factor limiting treatment with cisplatin is its acute and cumulative cardiotoxicity. The aim of the present study was to explore the effect of cisplatin on myocardial contractile function and the possible underlying cellular mechanisms. 2. C57 mice were treated with cisplatin (10 mg/kg per day, i.v.) or vehicle (0.9% NaCl) for 1 week and myocardial function was assessed using the Langendorff and cardiomyocyte edge‐detection systems. Transmission electron microscopy, mitochondrial membrane potential, indices of endoplasmic reticulum (ER) stress and caspase 3 activity were evaluated. 3. Cisplatin‐treated mice developed myocardial contractile dysfunction, as evidenced by a reduction in left ventricular developed pressure (LVDP) and the first derivative of LVDP (+/?dP/dt). Cisplatin treatment significantly prolonged time to 90% relengthening, depressed peak shortening, maximal velocity of shortening/relengthening (+/?dL/dt) and augmented the frequency‐elicited depression in peak shortening. The JC‐1 fluorescent assay demonstrated that cispatin‐induced cardiac dysfunction was associated with mitochondrial membrane depolarization. Transmission electron microscopy revealed that cisplatin induces ultrastructural abnormalities of the mitochondria. Following cisplatin treatment, cardiomyocytes show activation of the ER stress response, increased caspase 3 activity and increased terminal deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labelling (TUNEL) staining. 4. The data indicate that cisplatin is cardiotoxic and may lead to left ventricular dysfunction and depressed cardiomyocyte contraction associated with mitochondrial abnormalities, enhanced ER stress and apoptosis. This work should shed some light on the management of cisplatin‐induced cardiac injury.     

Akt2 knockout mitigates chronic iNOS inhibition-induced cardiomyocyte atrophy and contractile dysfunction despite persistent insulin resistance

Increased levels of inducible nitric oxide synthase (iNOS) during cardiac stress such as ischemia-reperfusion, sepsis and hypertension may display both beneficial and detrimental roles in cardiac contractile performance. However, the precise role of iNOS in the maintenance of cardiac contractile function remains elusive. This study was designed to determine the impact of chronic iNOS inhibition on cardiac contractile function and the underlying mechanism involved with a special focus on the NO downstream signaling molecule Akt. Male C57 or Akt2 knockout [Akt2(−/−)] mice were injected with the specific iNOS inhibitor 1400W (2 mg/kg/d) or saline for 7 days. Both 1400W and Akt2 knockout dampened glucose and insulin tolerance without additive effects. Treatment of 1400W decreased heart and liver weights as well as cardiomyocyte cross-sectional area in C57 but not Akt2 knockout mice. 1400W but not Akt2 knockout compromised cardiomyocyte mechanical properties including decreased peak shortening and maximal velocity of shortening/relengthening, prolonged relengthening duration, reduced intracellular Ca²⁺ release and decay rate, the effects of which were ablated or attenuated by Akt2 knockout. Akt2 knockout but not 1400W increased the levels of intracellular Ca²⁺ regulatory proteins including SERCA2a and phospholamban phosphorylation. 1400W reduced the level of anti-apoptotic protein Bcl-2, the effect of which was unaffected by Akt2 knockout. Neither 1400W nor Akt2 knockout significantly affected ER stress, autophagy, the post-insulin receptor signaling Akt, GSK3β and AMPK, as well as the stress signaling IκB, JNK, ERK and p38 with the exception of elevated IκB phosphorylation with jointed effect of 1400W and Akt2 knockout. Taken together, these data indicated that an essential role of iNOS in the maintenance of cardiac morphology and function possibly through an Akt2-dependent mechanism.     

AQP4‐knockout aggravation of isoprenaline‐induced myocardial injury is mediated by p66Shc and endoplasmic reticulum stress

Aquaporin 4 (AQP4) is a type of water channel protein that maintains the water balance of cardiomyocytes. However, the physiological role of AQP4 in cardiovascular disease is poorly understood. We wanted to explore whether p66Shc and endoplasmic reticulum stress participates in AQP4 knockout (KO)‐mediated cardiac injury. There were two types of mice: AQP4 knockout and wild‐type mice. Each type was randomly divided into three groups: Control group, isoprenaline stimulation group (ISO, 1 mg/kg, s.c., 5 days), and apocynin treatment group (APO, 100 mg/kg, p.o., 3 days). H9c2 rat cardiomyocytes were cultured for RNA interference of AQP4. Results showed increased left ventricular weight index and more severe myocardial inflammation were induced in AQP4 knockout mice relative to wild‐type mice, accompanied by significantly increased levels of the oxidative stress biomarkers MDA and NOX4. In addition, the expressions of p66Shc, ER stress markers PERK, GRP78 and CHOP and proinflammatory factors such as ET_A, IL6 and TNFα were upregulated in the myocardium of AQP4 knockout mice or AQP4 siRNA treated cardiomyocytes, whereas CASQ2 was downregulated. ISO stimulation aggravated these abnormalities, which were significantly attenuated by apocynin. This study showed that AQP4 knockout mice were susceptible to cardiac injury induced by ISO. The mechanism was closely connected with p66Shc and proinflammatory factors. Endoplasmic reticulum stress was also involved in the pathological process.     

Toll-like receptor 4 knockout protects against anthrax lethal toxin-induced cardiac contractile dysfunction: role of autophagy

BACKGROUND AND PURPOSE

Anthrax lethal toxin (LeTx) is known to induce circulatory shock and death, although the underlying mechanisms have not been elucidated. This study was designed to evaluate the role of toll-like receptor 4 (TLR4) in anthrax lethal toxin-induced cardiac contractile dysfunction.

EXPERIMENTAL APPROACH

Wild-type (WT) and TLR4 knockout (TLR^−/−) mice were challenged with lethal toxin (2 µg·g⁻¹, i.p.), and cardiac function was assessed 18 h later using echocardiography and edge detection. Small interfering RNA (siRNA) was employed to knockdown TLR4 receptor or class III PI3K in H9C2 myoblasts. GFP–LC3 puncta was used to assess autophagosome formation. Western blot analysis was performed to evaluate autophagy (LC3, Becline-1, Agt5 and Agt7) and endoplasmic reticulum (ER) stress (BiP, eIF2α and calreticulin).

KEY RESULTS

In WT mice, lethal toxin exposure induced cardiac contractile dysfunction, as evidenced by reduced fractional shortening, peak shortening, maximal velocity of shortening/re-lengthening, prolonged re-lengthening duration and intracellular Ca²⁺ derangement. These effects were significantly attenuated or absent in the TLR4 knockout mice. In addition, lethal toxin elicited autophagy in the absence of change in ER stress. Knockdown of TLR4 or class III PI3 kinase using siRNA but not the autophagy inhibitor 3-methyladenine significantly attenuated or inhibited lethal toxin-induced autophagy in H9C2 cells.

CONCLUSION AND IMPLICATIONS

Our results suggest that TLR4 may be pivotal in mediating the lethal cardiac toxicity induced by anthrax possibly through induction of autophagy. These findings suggest that compounds that negatively modulate TLR4 signalling and autophagy could be used to treat anthrax infection-induced cardiovascular complications.     

Inhibition of DNA methylation attenuates low‐dose cadmium‐induced cardiac contractile and intracellular Ca2+ anomalies

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Osthole induced apoptosis in human normal liver cells by regulating cell proliferation and endoplasmic reticulum stress

Osthole (Ost) is often used in treatment for cancer, inflammation and rheumatism in clinic. However, Ost‐induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of Ost‐induced hepatotoxicity in human normal liver cells (L02). When cells were exposed to Ost, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, Ost altered apoptotic related proteins levels, including Bcl‐2, Bax, Cleaved‐Caspase‐9/‐8/‐3, and Pro‐Caspase‐3/‐8. In addition, Ost enhanced the levels of endoplasmic reticulum (ER) stress proteins (GRP78/Bip, CHOP, Caspase‐4, IRE1α, PERK, JNK, P‐JNK, and ATF4), decreased the cell proliferation and cycle‐associated protein (Phospho‐Histone H3, P‐Cdc25C, Cdc25C, P‐Cdc2, Cdc2, and Cyclin B1) level. The results show that Ost has toxic effects on L02 cells. Furthermore, it induces apoptosis by inhibiting cell proliferation, arresting cell cycle at the G2/M phase and activating ER stress.     

Influence of gender on oxidative stress, lipid peroxidation, protein damage and apoptosis in hearts and brains from spontaneously hypertensive rats

1. Hypertension leads to oxidative stress, lipid and protein damage, apoptosis and impaired cardiac contractile function. However, impact of gender on these hypertension-associated abnormalities has not been elucidated. 2. The present study evaluated the oxidative stress, lipid/protein damage, apoptosis in heart and brain tissues as well as cardiomyocyte contractile function in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) of both genders. Oxidative stress, lipid peroxidation, protein damage and apoptosis were assessed by glutathione (GSH) : reduced glutathione (GSSG) ratio, malondialdehyde (MDA) levels, protein carbonyl levels and caspase-3 activity, respectively. Cardiomyocyte contractile function was examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/-dL/dt). The SHR cardiomyocytes displayed reduced PS and +/-dL/dt compared with gender-matched WKY counterparts. Male but not female SHR cardiomyocytes possessed longer resting cell length, normal TPS and prolonged TR90. All mechanical parameters were comparable between male and female WKY rats with the exception of a higher TR90 in females. Hypertension did not significantly affect the GSH : GSSG ratio in the heart and brain tissues of either gender. Brain from female WKY rats displayed a reduced GSH : GSSG ratio. The MDA levels were unchanged and elevated, respectively, in SHR heart and SHR brain tissues from both genders. Protein carbonyl formation and caspase-3 activity were elevated in male but not female SHR hearts. Nonetheless, brain protein carbonyl level and caspase-3 activity were unaffected by hypertension or gender. 3. In summary, these results suggest that gender affects hypertension-associated oxidative stress, lipid and protein damage, apoptosis in heart and brain tissues and cardiomyocyte contractile function.     

2,4‐dichlorophenol induces ER stress‐mediated apoptosis via eIF2α dephosphorylation in vitro

2,4‐Dichlorophenol (2,4‐DCP) has been widely used to produce herbicides and pharmaceutical intermediates, which exhibits various toxic effects including apoptosis. However, the mechanisms underlying 2,4‐DCP‐induced apoptosis, especially mediated by endoplasmic reticulum (ER) stress, are still unknown. In the present study, the mouse embryonic fibroblasts (MEFs) were used as an in vitro model system to figure out whether 2,4‐DCP could induce ER stress, and further to elucidate the role of ER stress in 2,4‐DCP‐induced apoptosis. The results showed that 2,4‐DCP dramatically caused the decrease of cell viability, the increase of apoptotic cells, the collapse of mitochondrial membrane potential (MMP) and the activation of caspase‐3, suggesting that 2,4‐DCP did induce apoptosis. Meanwhile, 2,4‐DCP acted similarly as ER stress agonist tunicamycin (Tu) to activate all three branches (IRE1α, ATF6 and eIF2α) of ER stress. Furthermore, repression of ER stress or inhibition of eIF2α dephosphorylation significantly alleviated 2,4‐DCP‐induced apoptosis. Taking these results together, the present study firstly showed that 2,4‐DCP induced ER stress‐mediated apoptosis via eIF2α dephosphorylation in mammalian cells. These findings will provide new insights into the mechanisms underlying apoptosis after chlorophenols exposure. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 245–255, 2016.     

Association of p38MAPK‐p53‐Fas aggregation in S‐allyl cysteine mediated regulation of hepatocarcinoma

Bioactive components of dietary phytochemicals have been reported to possess antitumor activities. Evidences suggested key role of stress responsive p38MAPK in the induction of nutraceuticals mediated apoptosis in hepatocellular carcinoma (HCC). Current study demonstrated detailed molecular bagatelle associated with p38 MAPK mediated effective suppression of cell growth both in HepG2 and chemically induced liver carcinoma after S‐allyl cysteine (SAC) treatment. SAC promoted p38MAPK activity responsible for p53 phosphorylation, its stabilization followed by nuclear translocation leading to induction in expression and oligomerization of Fas protein. Distinctive p38MAPK‐p53 axis dependent Fas‐FasL‐FADD mediated caspase activities along with perturbed cell cycling became normalized with continuation of SAC treatment for another month to diethylnitrosamine induced liver carcinoma. Co‐treatment with SB203580, the p38MAPK inhibitor, prevented pro‐apoptotic effect of SAC by altering p53 phosphorylation and death inducing signaling complex conformation in HepG2 and induced HCC. Collectively study suggested significant contribution of p38MAPK‐p53‐DISC‐Caspase pathway in the regulation of anti‐neoplastic activity of SAC against HCC.     

Epigenetic modification of H3K4 and oxidative stress are involved in MC‐LR‐induced apoptosis in testicular cells of SD rats

Microcystin‐leucine arginine (MC‐LR) is a cyclic heptapeptide, produced by aquatic cyanobacteria such as microcystis, with strong reproductive toxicity which poses greater threat to the reproductive abilities of humans and animals. By exploring the role of trimethylation of histone H3 at lysine 4 (H3K4me3) and the role of oxidative stress in MC‐LR‐induced apoptosis in testicular Sertoli cells in Sprague‐Dawley (SD) rats, this study indicated that MC‐LR increased the expression levels of apoptosis‐related genes by raising the levels of H3K4me3. 5′‐Deoxy‐5′‐methylthioadenosine (MTA), the inhibitor of H3K4me3, reduced apoptosis, indicating for the first time that epigenetic modification is closely related to the testicular reproductive toxicity induced by MC‐LR. MC‐LR also induced oxidative stress by stimulating the generation of reactive oxygen species (ROS), and subsequently triggering mitochondria‐mediated apoptotic pathway by decreasing mitochondrial membrane potential and increasing the levels of Bax, Bcl‐2, Caspase‐3, and so on. MC‐LR‐induced apoptosis of testicular cells could be decreased after pretreatment with oxidative stress inhibitor N‐acetyl‐cysteine (NAC). Furthermore, the pathological damage to mitochondria and testes were observed in SD rats. These results show that MC‐LR can induce apoptosis by raising the levels of H3K4me3, and pretreatment with MTA can ameliorate the MC‐LR‐induced apoptosis of cocultured cells by lowering the levels of H3K4me3. Furthermore, NAC has a protective effect on MC‐LR‐induced apoptosis of testicular cells in SD rats by inhibiting the oxidative stress.     

Heavy metal scavenger metallothionein attenuates ER stress-induced myocardial contractile anomalies: Role of autophagy

Endoplasmic reticulum (ER) stress increases the risk of cardiovascular morbidity and mortality although the underlying mechanism remains elusive. This study was designed to examine the impact of cardiac over-expression of metallothionein, a cysteine-rich heavy metal scavenger, on ER stress-induced changes in myocardial function and underlying mechanism involved with a focus on autophagy. Wild-type friendly virus B (FVB) and metallothionein transgenic mice were subjected to the ER stress inducer tunicamycin (1 mg/kg). Our results showed that ER stress led to compromised echocardiographic and cardiomyocyte contractile function, intracellular Ca²⁺ mishandling. Tunicamycin promoted ER stress and oxidative stress, increased left ventricular end systolic and diastolic diameter, as well as suppressed fractional shortening and whole heart contractility, the effects of which were significantly attenuated or ablated by metallothionein. Levels of the autophagy markers such as phosphorylated ULK1, Atg5, Atg7, LC3B and the autophagy adaptor p62 were significantly upregulated. These ER stress-induced changes in myocardial function, autophagy and autophagy signaling were distinctly mitigated or alleviated by metallothionein. Inhibition of autophagy using 3-methyladenine in vitro reversed ER stress-induced cardiomyocyte contractile defects. Meanwhile, ER stress-induced cardiomyocyte dysfunction was attenuated by the antioxidant N-acetylcysteine. Collectively, these findings suggested that metallothionein protects against ER stress-induced cardiac anomalies possibly through attenuation of cardiac autophagy.     

Proanthocyanidin protects against cisplatin‐induced oxidative liver damage through inhibition of inflammation and NF‐κβ/TLR‐4 pathway

Although cisplatin (CIS) is a highly effective anticancer drug, hepatotoxicity is one of the most common adverse effects associated with its use. Recently, reactive oxygen species (ROS) and inflammation are suggested to be key factors in the pathophysiology of CIS‐induced acute liver damage. The aim of this study is to investigate the possible protective effect of proanthocyanidin (PRO) against CIS‐induced acute hepatotoxicity. Rats were divided into four groups: 1, Control; 2, PRO; 3, CIS; and 4, PRO + CIS. Biochemical studies and histopathology were used to assess liver damage. ROS, inflammatory cytokines, nuclear factor kappa beta (NF‐κβ), inducible cyclooxygenase enzyme (COX‐2), inducible nitric oxide synthase (iNOS), toll‐like receptor‐4 (TLR‐4) gene expression, and apoptotic markers were also assessed. PRO pretreatment protected the liver against CIS‐induced toxicity as indicated by decreased plasma levels of liver function enzymes and the normal liver histopathology observed in the PRO + CIS group. PRO pretreatment also diminished indicators of oxidative stress in the liver, including nitric oxide (NO) and malondialdehyde (MDA). It also increased the antioxidants, reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) in the liver. Plasma interleukin‐1 beta (IL‐1β), IL‐6, and tumor necrosis factor‐alpha (TNF‐α) were all reduced. Liver gene expression of NF‐κβ, COX‐2, iNOS, and TLR‐4 were all downregulated. Furthermore, PRO administration downregulated the liver expression of the apoptotic marker, Bax, while upregulated the antiapoptotic marker, Bcl2. In conclusion, our results revealed that PRO may protect against CIS‐induced acute liver damage mainly through inhibition of ROS, inflammation, and apoptosis.     

Mono(2‐ethylhexyl) phthalate induces apoptosis in p53‐silenced L02 cells via activation of both mitochondrial and death receptor pathways

Mono(2‐ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2‐ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10‐fold more potent than its parent compound in toxicity in vitro. MEHP‐induced apoptosis is mediated by either p53‐dependent or ‐independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP‐induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 μM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 μM) induced apoptosis in p53‐silenced L02 cells, along with the up‐regulations of Fas and FasL proteins as well as increased the Bax/Bcl‐2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF‐016) and Caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐ fluoromethylketone (Z‐VAD‐FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP‐induced apoptosis in L02 cells involving a Caspases‐mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP‐induced L02 cell apoptosis. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1178–1191, 2015.     

Deep sea minerals prolong life span of streptozotocin‐induced diabetic rats by compensatory augmentation of the IGF‐I‐survival signaling and inhibition of apoptosis

Consumption of deep sea minerals (DSM), such as magnesium, calcium, and potassium, is known to reduce hypercholesterolemia‐induced myocardial hypertrophy and cardiac‐apoptosis and provide protection against cardiovascular diseases. Heart diseases develop as a lethal complication among diabetic patients usually due to hyperglycemia‐induced cardiac‐apoptosis that causes severe cardiac‐damages, heart failure, and reduced life expectancy. In this study, we investigated the potential of DSM and its related cardio‐protection to increase the life expectancy in diabetic rats. In this study, a heart failure rat model was developed by using streptozotocin (65 mg kg^?1) IP injection. Different doses of DSM‐1× (37 mg kg^?1 day^?1), 2× (74 mg kg^?1 day^?1) and 3× (111 mg kg^?1 day^?1), were administered to the rats through gavages for 4 weeks. The positive effects of DSM on the survival rate of diabetes rats were determined with respect to the corresponding effects of MgSO₄. Further, to understand the mechanism by which DSM enhances the survival of diabetic rats, their potential to regulate cardiac‐apoptosis and control cardiac‐dysfunction were examined. Echocardiogram, tissue staining, TUNEL assay, and Western blotting assay were used to investigate modulations in the myocardial contractile function and related signaling protein expression. The results showed that DSM regulate apoptosis and complement the cardiomyocyte proliferation by enhancing survival mechanisms. Moreover DSM significantly reduced the mortality rate and enhanced the survival rate of diabetic rats. Experimental results show that DSM administration can be an effective strategy to improve the life expectancy of diabetic subjects by improving cardiac‐cell proliferation and by controlling cardiac‐apoptosis and associated cardiac‐dysfunction. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 769–781, 2016.     

Creatine kinase inhibitor iodoacetamide antagonizes calcium-stimulated inotropy in cardiomyocytes

1. Inhibition of creatine kinase is known to suppress cardiac contractile reserve in intact hearts, although the underlying mechanism has not been elucidated. 2. The present study was designed to examine whether cardiac depression induced by creatine kinase inhibition was due to action at the level of the essential contractile element, namely cardiomyocytes. Adult rat cardiomyocytes were perfused with the creatine kinase inhibitor iodoacetamide (90 micromol/L) for 90 min. Mechanical and intracellular Ca(2+) properties were evaluated using edge-detection and fluorescence microscopy, respectively. Myocytes were superfused with normal (1.3 mmol/L) or high (3.3 mmol/L) extracellular Ca(2+) contractile buffer. Mechanical function was examined, including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time to 90% PS (TPS(90)), time to 90% relengthening (TR(90)) and integration of shortening/relengthening (normalized to PS). Intracellular Ca(2+) transients were evaluated using the following indices: resting and rise of fura-2 fluorescence intensity (Delta FFI) and intracellular Ca(2+) decay time constant. 3. The results indicate that elevated extracellular Ca(2+) stimulated cardiomyocyte positive inotrope, manifested as increased PS, +/-dL/dt, area of shortening, resting FFI and Delta FFI associated with a shortened TR(90) and intracellular Ca(2+) decay time constant. High extracellular Ca(2+) did not affect TPS(90) and area of relengthening. Iodoacetamide ablated high Ca(2+)-induced increases in PS, +/-dL/dt, area of shortening, resting FFI, Delta FFI and shortened TR(90) and intracellular Ca(2+) decay time constant. Iodoacetamide itself significantly enhanced the area of relengthening and TR(90) without affecting other indices. 4. Collectively, these data demonstrate that inhibition of creatine kinase blunts high extracellular Ca(2+)-induced increases in cardiomyocyte contractile response (i.e. cardiac contractile reserve).     

4-Phenylbutyric acid prevent cytotoxicity induced by thapsigargin in rat cardiac fibroblast

Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail.Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot.In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA.Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.     

Fisetin‐induced apoptosis of human oral cancer SCC‐4 cells through reactive oxygen species production,endoplasmic reticulum stress,caspase‐, and mitochondria‐dependent signaling pathways

Oral cancer is one of the cancer‐related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin‐induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin‐induced cell death and associated signal pathways on human oral cancer SCC‐4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca²⁺, mitochondria membrane potential (ΔΨ_m), and caspase‐8, ‐9, and ‐3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca²⁺ production, and decreased the level of ΔΨ_m and increased caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin‐induced cell apoptosis in SCC‐4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl‐2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC‐4 cells. We also used ATF‐6α, ATF‐6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria‐, and caspase‐dependent pathways.