Buforin IIb induces endoplasmic reticulum stress-mediated apoptosis in HeLa cells

Buforin IIb, a novel cell-penetrating anticancer peptide derived from histone H2A, has been reported to induce mitochondria-dependent apoptosis in tumor cells. However, increasing evidence suggests that endoplasmic reticulum and mitochondria cooperate to signal cell death. In this study, we investigated the mechanism of buforin IIb-induced apoptosis in human cervical carcinoma HeLa cells by focusing on ER stress-mediated mitochondrial membrane permeabilization. Two-dimensional PAGE coupled with MALDI-TOF and western blot analysis showed that buforin IIb treatment of HeLa cells resulted in upregulation of ER stress proteins. PBA (ER stress inhibitor) and BAPTA/AM (Ca²⁺ chelator) pretreatment rescued viability of buforin IIb-treated cells through abolishing phosphorylation of SAPK/JNK and p38 MAPK. SP600125 (SAPK/JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated down-regulation of Bcl-xL/Bcl-2, mitochondrial translocation of Bax, and cytochrome c release from mitochondria. Taken together, our data suggest that the ER stress pathway has an important role in the buforin IIb-induced apoptosis in HeLa cells.     

Candidate mechanical stimuli for hypertrophy during volume overload.

A myocyte system that senses and responds to mechanical inputs might be activated by any number of features of the time-varying length or force signals experienced by the myocytes. We therefore characterized left ventricular volume and wall stress signals during early volume overload with high spatial and temporal resolution. Left ventricular pressure and volume were measured in open-chest isoflurane-anesthetized male Sprague-Dawley rats 4 and 7 days after surgical creation of an infrarenal arteriovenous fistula or sham operation. Mean wall stresses were calculated by using a simple thick-walled ellipsoidal model. Consistent with previous reports, this surgical model produced a 66% increase in cardiac output and a 10% increase in left ventricular mass by day 7. A number of features of the time-varying volume signal (maximum, mean, amplitude, rates of rise and fall) were significantly altered during early volume overload, whereas many other proposed hypertrophic stimuli, including peak systolic wall stress and diastolic strain, were not. Treating hemodynamic variables more generally as time-varying signals allowed us to identify a wider range of candidate mechanical stimuli for hypertrophy (including some not previously proposed in the literature) than focusing on standard time points in the cardiac cycle. We conclude that features of the time-varying ventricular volume signal and related local deformations may drive hypertrophy during volume overload and propose that those features of the volume signal that also change during pressure overload might be the most interesting candidates for further exploration.     

Cryptotanshinone induces ER stress-mediated apoptosis in HepG2 and MCF7 cells

The endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that functions in protein synthesis and maturation, and also functions as a calcium storage organelle. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress can activate apoptotic pathways in damaged cells. For this reason, pharmacological interventions that effectively enhance tumor death through ER stress have been the subject of a great deal of attention for anti-cancer therapy. Cryptotanshinone, the major active constituent isolated from the root of Salvia miltiorrhiza Bunge, has been recently evaluated for its anti-cancer activity, but the molecular mechanisms underlying these activities remain poorly understood. In particular, it remains completely unknown as to whether or not cryptotanshinone can induce ER stress. Herein, we identify cryptotanshinone as a potent stimulator of ER stress, leading to apoptosis in many cancer cell lines, including HepG2 hepatoma and MCF7 breast carcinoma, and also demonstrate that mitogen-activated protein kinases function as mediators in this process. Reactive oxygen species generated by cryptotanshinone have been shown to play a critical role in ER stress-induced apoptosis. Cryptotanshinone also evidenced sensitizing effects to a broad range of anti-cancer agents including Fas/Apo-1, TNF-α, cisplatin, etoposide or 5-FU through inducing ER stress, highlighting the therapeutic potential in the treatment of human hepatoma and breast cancer.     

Constriction ability of coronary artery in volume overload cardiac hypertrophy

The constrictor response of the rabbit conduit coronary artery from hypertrophied heart (volume-overload stabilized hypertrophy) was studied to vasoactive substances. The heart/body weight ratio was 2.67 +/- 0.95 in the experimental group and 1.90 +/- 0.09 in the controls. The responses to acetylcholine, serotonin and potassium chloride was dose-dependent in the controls: the maximum amounted to 9.07 +/- 2.03 mN, 6.00 +/- 1.79 and 10.94 +/- 1.64 mN, respectively. Remarkably lower responses were detected in coronary arteries from hypertrophied hearts in the whole range of concentrations applied; the maximum was only 22.34 +/- 8.32% of the control response to acetylcholine, 17.83 +/- 11.37% to serotonin, and 21.74 +/- 5.50% to potassium chloride. A disbalance between stabilized cardiac hypertrophy and the remarkably low constrictor ability of the conduit coronary artery has been described.     

Normal myocardial function in severe right ventricular volume overload hypertrophy

Severe left ventricular volume overloading causes myocardial and cellular contractile dysfunction. Whether this is also true for severe right ventricular volume overloading was unknown. We therefore created severe tricuspid regurgitation percutaneously in seven dogs and then observed them for 3.5-4.0 yr. All five surviving operated dogs had severe tricuspid regurgitation and right heart failure, including massive ascites, but they did not have left heart failure. Right ventricular cardiocytes were isolated from these and from normal dogs, and sarcomere mechanics were assessed via laser diffraction. Right ventricular cardiocytes from the tricuspid regurgitation dogs were 20% longer than control cells, but neither the extent (0.171 +/- 0.005 microm) nor the velocity (2.92 +/- 0.12 microm/s) of sarcomere shortening differed from controls (0.179 +/- 0.005 microm and 3.09 +/- 0.11 microm/s, respectively). Thus, despite massive tricuspid regurgitation causing overt right heart failure, intrinsic right ventricular contractile function was normal. This finding for the severely volume-overloaded right ventricle stands in distinct contrast to our finding for the left ventricle severely volume overloaded by mitral regurgitation, wherein intrinsic contractile function is depressed.     

Mitofusin-2 is a major determinant of oxidative stress-mediated heart muscle cell apoptosis

An inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure. Here, we have provided several lines of evidence to demonstrate that mitofusin-2 (Mfn-2; also called hyperplasia suppressor gene), a member of the mitofusin family, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. First, oxidative stress with H(2)O(2) led to concurrent increases in Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes. Second, overexpression of Mfn-2 to a level similar to that induced by H(2)O(2) was sufficient to trigger myocyte apoptosis, which is associated with profound inhibition of Akt activation without altering ERK1/2 signaling. Third, Mfn-2 silencing inhibited oxidative stress-induced apoptosis in H9C2 cells, a cardiac muscle cell line. Furthermore, Mfn-2-induced myocyte apoptosis was abrogated by inhibition of caspase-9 (but not caspase-8) and by overexpression of Bcl-x(L) or enhanced activation of phosphatidylinositol 3-kinase-Akt, suggesting that inhibition of Akt signaling and activation of the mitochondrial death pathway are essentially involved in Mfn-2-induced heart muscle cell apoptosis. These results indicate that increased cardiac Mfn-2 expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that Mfn-2 deregulation may be a crucial pathogenic element and a potential therapeutic target for heart failure.     

Regulation of CD2-associated protein influences podocyte endoplasmic reticulum stress-mediated apoptosis induced by albumin overload

The adaptation of microorganisms to pesticide biodegradation relies on the recruitment of catabolic genes by horizontal gene transfer and homologous recombination mediated by insertion sequences (IS). This environment-friendly function is maintained in the degrading population but it has a cost which could diminish its fitness. The loss of genes in the course of evolution being a major mechanism of ecological specialization, we mimicked evolution in vitro by sub-culturing the atrazine-degrading Pseudomonas sp. ADP in a liquid medium containing cyanuric acid as the sole source of nitrogen. After 120 generations, a new population evolved, which replaced the original one. This new population grew faster on cyanuric acid but showed a similar cyanuric acid degrading ability. Plasmid profiles and Southern blot analyses revealed the deletion of a 47 kb region from pADP1 containing the atzABC genes coding for the enzymes that turn atrazine into cyanuric acid. Long PCR and sequencing analyses revealed that this deletion resulted from a homologous recombination between two direct repeats of a 110-bp, identical to ISPps1 of Pseudomonas huttiensis, flanking the deleted 47 kb region. The loss of a region containing three functional genes constitutively expressed thereby constituting a genetic burden under cyanuric acid selection pressure was responsible for the gain in fitness of the new population. It highlights the IS-mediated plasticity of the pesticide-degrading potential and shows that IS not only favours the expansion of the degrading genetic potential thanks to dispersion and duplication events but also contribute to its reduction thanks to deletion events.     

A high-fructose diet worsens eccentric left ventricular hypertrophy in experimental volume overload

The development of left ventricular (LV) hypertrophy (LVH) can be affected by diet manipulation. Concentric LVH resulting from pressure overload can be worsened by feeding rats with a high-fructose diet. Eccentric LVH is a different type of hypertrophy and is associated with volume overload (VO) diseases. The impact of an abnormal diet on the development of eccentric LVH and on ventricular function in chronic VO is unknown. This study therefore examined the effects of a fructose-rich diet on LV eccentric hypertrophy, ventricular function, and myocardial metabolic enzymes in rats with chronic VO caused by severe aortic valve regurgitation (AR). Wistar rats were divided in four groups: sham-operated on control diet (SC; n = 13) or fructose-rich diet (SF; n = 13) and severe aortic regurgitation fed with the same diets [aortic regurgitation on control diet (ARC), n = 16, and aortic regurgitation on fructose-rich diet (ARF), n = 13]. Fructose-rich diet was started 1 wk before surgery, and the animals were euthanized 9 wk later. SF and ARF had high circulating triglycerides. ARC and ARF developed significant LV eccentric hypertrophy after 8 wk as expected. However, ARF developed more LVH than ARC. LV ejection fraction was slightly lower in the ARF compared with ARC. The increased LVH and decreased ejection fraction could not be explained by differences in hemodynamic load. SF, ARC, and ARF had lower phosphorylation levels of the AMP kinase compared with SC. A fructose-rich diet worsened LV eccentric hypertrophy and decreased LV function in a model of chronic VO caused by AR in rats. Normal animals fed the same diet did not develop these abnormalities. Hypertriglyceridemia may play a central role in this phenomenon as well as AMP kinase activity.     

Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 +/- 0.013 vs. 0.31 +/- 0.008), HW/TL by 47.2% (42.7 +/- 1.30 vs. 29.0 +/- 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 +/- 0.006 vs. 0.44 +/- 0.013) and HW/TL by 22.3% (33.2 +/- 0.95 vs. 42.7 +/- 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-kappaB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.     

Cardiac myocyte hypertrophy and proliferating cell nuclear antigen expression in Wistar rats infected with Trypanosoma cruzi

Chagasic cardiomyopathy is a major life-threatening complication of Trypanosoma cruzi infection in human beings. This study focuses on the hypertrophic and hyperplastic mechanisms underlying the structural changes of the heart during experimental infection. Proliferating cell nuclear antigen (PCNA) expression, transversal diameter, nuclear area, and number of nuclei per unit volume were determined in the ventricular myocytes of T. cruzi-infected Wistar rats. PCNA expression was enhanced throughout the inflamed myocardium and in the spared areas of the left ventricular wall and the septum. Myocyte width increased from 26 to 75% at the inflammation-free myocardium (P < 0.0001), whereas it decreased 25% at the inflamed left ventricular wall areas (P < 0.001). Nuclear size increased in the inflammation-free myocardium of the left ventricle and the septum (> 10-36%, P < 0.01 and >0.2-32%, P < 0.03, respectively) and decreased at the inflamed areas of the left ventricular wall (10-22%. P < 0.02) with respect to the controls. The number of nuclei per unit volume decreased at the inflamed myocardium regardless of topographical location (36-65%) with respect to the controls (P < 0.0001) and in the inflammation-free myocardium of the right ventricle and the septum (<21-37%, P < 0.002 and <8-39%, P < 0.002, respectively). These results show that the heart responds to T. cruzi infection with DNA repair and cell multiplication in the inflamed sites and with hypertrophy of the unaffected myocardium.     

Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stress-mediated apoptosis

Aims

Proliferation is a ‘multiplier’ for extracellular matrix production and contraction of activated hepatic stellate cells (HSC) in fibrotic liver. Transient receptor potential melastatin-like 7 channels (TRPM7) are implicated in the survival and proliferation of several kinds of cells. This study was aimed to investigate the effect of TRPM7 blocker 2-APB on survival and proliferation of HSC and the underlying mechanisms.

Main methods

Rat HSC were stimulated by 2-APB for 24 h and then collected for further use. Cell viability was detected by MTT, and apoptosis was determined by AnnexinV/PI staining and TUNEL assay. Gene expressions of TRPM7, α-SMA, bcl-2, bax, and endoplasmic reticulum (ER) stress key members CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin were evaluated with quantitative RT-PCR. Quantifications of α-SMA, TRPM7, CHOP and GRP78 proteins were carried out by Western blot. Transmission electron microscopy and Xbp1 mRNA splicing analysis were also used for detection of ER stress.

Key findings

2-APB decreased TRPM7 and α-SMA expressions in primary HSC, and inhibited proliferation of activated HSC in a dose-dependent manner. 2-APB also decreased total count of activated HSC and increased the number of apoptotic cells. 2-APB increased expressions of bax and ER stress key factors CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin. Meanwhile, ultra-structural ER changes and spliced Xbp1 mRNA were also observed in 2-APB treated HSC.

Significance

Blockage of TRPM7 could inhibit activation and proliferation of primary HSC and induce apoptotic death of activated cells, in which ER stress was identified as one of possible underlying molecular bases.     

Characterization of cardiac hypertrophy and heart failure due to volume overload in the rat.

Alterations in general characteristics and morphology of the heart, as well as changes in hemodynamics, myosin heavy chain isoforms, and beta-adrenoceptor responsiveness, were determined in Sprague-Dawley rats at 1, 2, 4, 8, and 16 wk after aortocaval fistula (shunt) was induced by the needle technique. Three stages of cardiac hypertrophy due to volume overload were recognized during the 16-wk period. Developing hypertrophy occurred within the first 2 wk after aortocaval shunt was induced and was characterized by a rapid increase of cardiac mass in both left and right ventricles. Compensated hypertrophy occurred between 2 and 8 wk after aortocaval shunt where normal or mild depression in hemodynamic function was observed. Decompensated hypertrophy or heart failure occurred between 8 and 16 wk after aortocaval shunt and was characterized by circulatory congestion, decreased in vivo and in vitro cardiac function, and a shift in myosin heavy chain isozyme expression. However, the positive inotropic effect of isoproterenol was augmented at all times during the 16-wk period. Characterization of beta-adrenoceptor binding in failing hearts at 16 wk revealed a significant increase in beta(1)-receptor density, whereas beta(2)-receptor density was unchanged. Consistent with this, basal adenylyl cyclase activity was significantly increased, and both isoproterenol- and forskolin-stimulated adenylyl cyclase activities were also increased. These results indicate that upregulation of beta-adrenoceptor signal transduction is a unique feature of cardiac hypertrophy and failure induced by volume overload.