Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts

Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A₁, E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.     

Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts

Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.     

Mesenteric lymph from rats with trauma-hemorrhagic shock causes abnormal cardiac myocyte function and induces myocardial contractile dysfunction

Myocardial contractile dysfunction develops following trauma-hemorrhagic shock (T/HS). We have previously shown that, in a rat fixed pressure model of T/HS (mean arterial pressure of 30-35 mmHg for 90 min), mesenteric lymph duct ligation before T/HS prevented T/HS-induced myocardial contractile depression. To determine whether T/HS lymph directly alters myocardial contractility, we examined the functional effects of physiologically relevant concentrations of mesenteric lymph collected from rats undergoing trauma-sham shock (T/SS) or T/HS on both isolated cardiac myocytes and Langendorff-perfused whole hearts. Acute application of T/HS lymph (0.1-2%), but not T/SS lymph, induced dual inotropic effects on myocytes with an immediate increase in the amplitude of cell shortening (1.4 ± 0.1-fold) followed by a complete block of contraction. Similarly, T/HS lymph caused dual, positive and negative effects on cellular Ca2? transients. These effects were associated with changes in the electrophysiological properties of cardiac myocytes; T/HS lymph initially prolonged the action potential duration (action potential duration at 90% repolarization, 3.3 ± 0.4-fold), and this was followed by a decrease in the plateau potential and membrane depolarization. Furthermore, intravenous infusion of T/HS lymph, but not T/SS lymph, caused myocardial contractile dysfunction at 24 h after injection, which mimicked actual T/HS-induced changes; left ventricular developed pressure (LVDP) and the maximal rate of LVDP rise and fall (±dP/dt(max)) were decreased and inotropic response to Ca2? was blunted. However, the contractile responsiveness to β-adrenergic receptor stimulation in the T/HS lymph-infused hearts remained unchanged. These results suggest that T/HS lymph directly causes negative inotropic effects on the myocardium and that T/HS lymph-induced changes in myocyte function are likely to contribute to the development of T/HS-induced myocardial dysfunction.     

Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1 mg/kg, i.p., 48 h). Echocardiographic, cardiomyocyte contractile and intracellular Ca^2 + properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca^2 + properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.     

Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via KCa1.1 upregulation

K_Ca1.1 regulates smooth muscle contractility by modulating membrane potential, and age‐associated changes in K_Ca1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K_Ca1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2‐null mice. The levels of C16‐ and C18‐ceramides, sphinganine, sphingosine, and sphingosine 1‐phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild‐type and young CerS2‐null mice. The altered SL composition upregulated K_Ca1.1 and increased K_Ca1.1 currents, while no change was observed in K_Ca1.1 channel activity. The upregulation of K_Ca1.1 impaired intracellular Ca²⁺ mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3‐kinase, protein kinase C_ζ, c‐Jun N‐terminal kinases, and nuclear factor kappa‐B were found to be involved in K_Ca1.1 upregulation. Our findings suggest that age‐associated changes in SL composition or CerS2 ablation upregulate K_Ca1.1 via the phosphoinositide 3‐kinase/protein kinase C_ζ/c‐Jun N‐terminal kinases/nuclear factor kappa‐B‐mediated pathway and impair Ca²⁺ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2‐null mice exhibited similar effects to aged wild‐type mice; therefore, CerS2‐null mouse models may be utilized for investigating the pathogenesis of aging‐associated motility disorders.     

Entering the lysosome through a transient gate by chaperone-mediated autophagy

A subset of cytosolic proteins can be selectively degraded in lysosomes through chaperone-mediated autophagy. The lysosomal-membrane protein type 2A (LAMP-2A) acts as the receptor for the substrates of chaperone-mediated autophagy (CMA), which should undergo unfolding before crossing the lysosomal membrane and reaching the lumen for degradation. Translocation of substrates is assisted by chaperones on both sides of the membrane, but the actual steps involved in this process and the characteristics of the translocation complex were, for the most part, unknown. We have now found that rather than a stable translocon at the lysosomal membrane, CMA substrates bind to monomers of LAMP-2A driving the organization of this protein into a high molecular weight multimeric complex that mediates translocation. Assembly and disassembly of LAMP-2A into and from this complex is dynamic and it is regulated by hsc70 and hsp90, the two lysosomal chaperones related to CMA. This work thus unveils a unique mechanism of protein translocation across the lysosomal membrane, which involves only transient discontinuity of the membrane. The possible advantages of this transitory lysosomal translocon are discussed in light of the unique properties of the lysosomal compartment.     

Entering the lysosome through a transient gate by chaperone-mediated autophagy

A subset of cytosolic proteins can be selectively degraded in lysosomes through chaperone-mediated autophagy. The lysosomal-membrane protein type 2A (LAMP-2A) acts as the receptor for the substrates of chaperone-mediated autophagy (CMA), which should undergo unfolding before crossing the lysosomal membrane and reaching the lumen for degradation. Translocation of substrates is assisted by chaperones on both sides of the membrane, but the actual steps involved in this process and the characteristics of the translocation complex were, for the most part, unknown. We have now found that rather than a stable translocon at the lysosomal membrane, CMA substrates bind to monomers of LAMP-2A driving the organization of this protein into a high molecular weight multimeric complex that mediates translocation. Assembly and disassembly of LAMP-2A into and from this complex is dynamic and it is regulated by hsc70 and hsp90, the two lysosomal chaperones related to CMA. This work thus unveils a unique mechanism of protein translocation across the lysosomal membrane, which involves only transient discontinuity of the membrane. The possible advantages of this transitory lysosomal translocon are discussed in light of the unique properties of the lysosomal compartment.

Addendum to: Bandyopadhyay U, Kaushik S, Vartikovski L, Cuervo AM. Dynamic organization of the receptor for chaperone-mediated autophagy at the lysosomal membrane. Mol Cell Biol 2008; 28:5747-63; DOI: 10.1128/MCB.02070-07.     

Hypoxia mimetic induces lipid accumulation through mitochondrial dysfunction and stimulates autophagy in murine preadipocyte cell line

BackgroundHypoxia occurs within adipose tissue of obese human and mice. However, its role in adipose tissue regulation is still controversial.MethodsWe used murine preadipocyte 3T3-L1 cells and hypoxia was induced by using hypoxia mimetic agents, as CoCl₂. To study adipocyte differentiation, we evaluated the adipocyte markers (PPARγ, C/EBPα and aP2), and a preadipocyte marker (pref-1) by qPCR, western blotting and immunofluorescence. Lipid accumulation was evaluated by Oil red-O assay and perilipin levels by western blotting and immunofluorescence. The effect of CoCl₂ in microRNA, miR-27a and miR-27b, levels was evaluated by qPCR. We also assessed the mitochondrial membrane potential and reactive oxygen species (ROS), superoxide and ATP production. The effect of hypoxia mimetic in autophagy was determined by LC3B and p62 level evaluation by western blotting.ResultsOur results show that the hypoxia mimetic cobalt chloride increases lipid accumulation with no expression of PPARγ2. Furthermore, using qPCR we observed that the hypoxia mimetic increases microRNAs miR-27a and miR-27b, which are known to block PPARγ2 expression. In contrast, cobalt chloride induces mitochondrial dysfunction, and increases ROS production and autophagy. Moreover, an antioxidant agent, glutathione, prevents lipid accumulation induced by hypoxia mimetic indicating that ROS are responsible for hypoxia-induced lipid accumulation.ConclusionsAll these results taken together suggest that hypoxia mimetic blocks differentiation and induces autophagy. Hypoxia mimetic also induces lipid accumulation through mitochondrial dysfunction and ROS accumulation.General significanceThis study highlights the importance of adipocyte response to hypoxia, which might impair adipocyte metabolism and compromise adipose tissue function.     

Intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart

Sleep apnea syndrome (SAS) is considered to be associated with heart failure (HF). It is known that autophagy is induced in various heart diseases thereby promotes survival, but its excess may be maladaptive. Intermittent hypoxia (IH) plays pivotal role in the pathogenesis of SAS. We aimed to clarify the relationships among IH, autophagy, and HF. Rats underwent IH at a rate of 20 cycles/h (nadir of 4% O2 to peak of 21% O2 with 0% CO2) or normal air breathing (control) for 8 h/d for 3 weeks. IH increased the cardiac LC3II/LC3I ratio. The IH induced upregulation of LC3II was attenuated by the administration of an inhibitor of autophagosome formation 3-methyladenine (3-MA), but enhanced by an inhibitor of autophagosome–lysosome fusion chloroquine (CQ), showing enhanced autophagic flux in IH hearts. Electron microscopy confirmed an increase in autophagosomes and lysosomes in IH. With 3-MA or CQ, IH induced progressive deterioration of fractional shortening (FS) on echocardiography over 3 weeks, although IH, 3-MA, or CQ alone had no effects. With CQ, IH for 4 weeks increased serum troponin T levels, reflecting necrosis. Western blotting analyses showed dephosphorylation of Akt and mammalian target of rapamycin (mTOR) at Akt (Ser2448, 2481) sites, suggesting the activation of autophagy via Akt inactivation. Conclusions. IH-mediated autophagy maintains contractile function, whereas when autophagy is inhibited, IH induces systolic dysfunction due to myocyte necrosis. General significance. This study highlighted the potential implications of autophagy in cardio-protection in early SAS patients without comorbidity, reproduced in normal rats by 3 ~ 4 weeks of IH.     

Mechanism of cytosol phospholipase C and sphingomyelinase-induced lysosome destabilization

Lysosomal disintegration may cause apoptosis, necrosis and some diseases. However, mechanisms for these events are still unclear. In this study, we measured lysosomal beta-hexosaminidase free activity, membrane potential and intralysosomal pH. The results revealed that the cytosolic extracts of rat hepatocytes could increase the lysosomal permeability to both potassium ions and protons, and osmotically destabilize lysosomes via K(+)/H(+) exchange. The effects of cytosol on lysosomes could be completely abolished by D609, which inhibited both phospholipase C and sphingomyelinase, and partly prevented by sphingomyelinase inhibitor Ara-AMP, but not by the inhibitors of PLA(2). Moreover, purified phospholipase C could destabilize the lysosomes while phospholipase A(2) and phospholipase D did not produce such effects. The cytosolic phospholipases hydrolyzed lysosomal membrane phospholipids by 50%, which could be prevented by D609. Disintegration of the cytosol-treated lysosomes biphasically depended on the cytosolic [Ca(2+)]. The cytosol did not disintegrate lysosomes below 100 nM or above 10 muM cytosolic [Ca(2+)], but markedly destabilized lysosomes at about 340 nM [Ca(2+)]. The results suggest that cytosolic phospholipase C and sphingomyelinase may be responsible for the alterations in lysosomal stability by increasing the ion permeability.     

Silver nanoparticles regulate autophagy through lysosome injury and cell hypoxia in prostate cancer cells

With the rapid development of nanotechnology, nanomaterials are now being used for cancer treatment. Although studies on the application of silver nanoparticles in cancer treatment are burgeoning, few studies have investigated the toxicology mechanisms of autophagy in cancer cells under exposure to sublethal silver nanoparticles. Here, we clarified the distinct mechanisms of silver nanoparticles for the regulation of autophagy in prostate cancer PC‐3 cells under sublethal exposure. Silver nanoparticle treatment caused lysosome injury, including the decline of lysosomal membrane integrity, decrease of lysosomal quantity, and attenuation of lysosomal protease activity, which resulted in blockage of autophagic flux. In addition, sublethal silver nanoparticle exposure activated AMP‐activated protein kinase/mammalian target of rapamycin‐dependent signaling pathway to modulate autophagy, which resulted from silver nanoparticles‐induced cell hypoxia and energy deficiency. Taken together, the results show that silver nanoparticles could regulate autophagy via lysosome injury and cell hypoxia in PC‐3 cells under sublethal dose exposure. This study will provide an experimental basis for the cancer therapy of nanomaterials.     

Peroxynitrite induces contractile dysfunction and lipid peroxidation in the diaphragm.

Peroxynitrite may be generated in and around muscles in several pathophysiological conditions (e.g., sepsis) and may induce muscle dysfunction in these disease states. The effect of peroxynitrite on muscle force generation has not been directly assessed. The purpose of the present study was to assess the effects of peroxynitrite administration on diaphragmatic force-generating capacity in 1) intact diaphragm muscle fiber bundles (to model the effects produced by exposure of muscles to extracellular peroxynitrite) and 2) single skinned diaphragm muscle fibers (to model the effects of intracellular peroxynitrite on contractile protein function) by examining the effects of both peroxynitrite and a peroxynitrite-generating solution, 3-morpholinosydnonimine, on force vs. pCa characteristics. In intact diaphragm preparations, peroxynitrite reduced diaphragm force generation and increased muscle levels of 4-hydroxynonenal (an index of lipid peroxidation). In skinned fibers, both peroxynitrite and 3-morpholinosydnonimine reduced maximum calcium-activated force. These data indicate that peroxynitrite is capable of producing significant diaphragmatic contractile dysfunction. We speculate that peroxynitrite-mediated alterations may be responsible for much of the muscle dysfunction seen in pathophysiological conditions such as sepsis.     

Arctigenin induces necroptosis through mitochondrial dysfunction with CCN1 upregulation in prostate cancer cells under lactic acidosis

Molecular and Cellular Biochemistry - Adipose tissue inflammation is closely associated with the development of obesity and insulin resistance. Free fatty acids (FFAs) are a major inducer of...     

IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes

Diabetic cardiomyopathy is characterized by impaired ventricular contraction and altered function of insulin-like growth factor I (IGF-I), a key factor for cardiac growth and function. Endogenous IGF-I has been shown to alleviate diabetic cardiomyopathy. This study was designed to evaluate exogenous IGF-I treatment on the development of diabetic cardiomyopathy. Adult rats were divided into four groups: control, control + IGF-I, diabetic, and diabetic + IGF-I. Streptozotocin (STZ; 55 mg/kg) was used to induce experimental diabetes immediately followed by a 7-wk IGF-I (3 mg. kg(-1). day(-1) ip) treatment. Mechanical properties were assessed in ventricular myocytes including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were evaluated as Ca(2+)-induced Ca(2+) release and Ca(2+) clearing constant. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and glucose transporter (GLUT4) were assessed by Western blot. STZ caused significant weight loss and elevated blood glucose, demonstrating the diabetic status. The diabetic state is associated with reduced serum IGF-I levels, which were restored by IGF-I treatment. Diabetic myocytes showed reduced PS and +/-dL/dt as well as prolonged TPS, TR(90), and intracellular Ca(2+) clearing compared with control. IGF-I treatment prevented the diabetes-induced abnormalities in PS, +/-dL/dt, TR(90), and Ca(2+) clearing but not TPS. The levels of SERCA and GLUT4, but not PLB, were significantly reduced in diabetic hearts compared with controls. IGF-I treatment restored the diabetes-induced decline in SERCA, whereas it had no effect on GLUT4 and PLB levels. These results suggest that exogenous IGF-I treatment may ameliorate contractile disturbances in cardiomyocytes from diabetic animals and could provide therapeutic potential in the treatment of diabetic cardiomyopathy.     

TMEPAI increases lysosome stability and promotes autophagy

Autophagy is emerging as a critical response of normal and cancer cells to environmental changes and plays an important role in cell metabolism and maintenance of damaged organelles. Transmembrane prostate androgen-induced protein (TMEPAI) is a pro-tumorigenic factor with high expression in tumor cells. In this study, we showed that depletion of TMEPAI leads to lysosomal labilization and inhibits autophagy. Further study showed that the inhibition of autophagy induced by the depletion of TMEPAI is involved in regulation of Beclin-1. Depletion of TMEPAI increases the sensitivity of cancer cells to chemotherapeutic drugs. Our study reveals the role of TMEPAI in promoting lysosome stability and autophagy, which might be used as a target for cancer chemotherapeutic treatment.     

Endocannabinoids acting at CB1 receptors mediate the cardiac contractile dysfunction in vivo in cirrhotic rats

Advanced liver cirrhosis is associated with hyperdynamic circulation consisting of systemic hypotension, decreased peripheral resistance, and cardiac dysfunction, termed cirrhotic cardiomyopathy. Previous studies have revealed the role of endocannabinoids and vascular CB(1) receptors in the development of generalized hypotension and mesenteric vasodilation in animal models of liver cirrhosis, and CB(1) receptors have also been implicated in the decreased beta-adrenergic responsiveness of isolated heart tissue from cirrhotic rats. Here we document the cardiac contractile dysfunction in vivo in liver cirrhosis and explore the role of the endocannabinoid system in its development. Rats with CCl(4)-induced cirrhosis developed decreased cardiac contractility, as documented through the use of the Millar pressure-volume microcatheter system, low blood pressure, and tachycardia. Bolus intravenous injection of the CB(1) antagonist AM251 (3 mg/kg) acutely increased mean blood pressure, as well as both load-dependent and -independent indexes of systolic function, whereas no such changes were elicited by AM251 in control rats. Furthermore, tissue levels of the endocannabinoid anandamide increased 2.7-fold in the heart of cirrhotic compared with control rats, without any change in 2-arachidonoylglycerol levels, whereas, in the cirrhotic liver, both 2-arachidonoylglycerol (6-fold) and anandamide (3.5-fold) were markedly increased. CB(1)-receptor expression in the heart was unaffected by cirrhosis, as verified by Western blotting. Activation of cardiac CB(1) receptors by endogenous anandamide contributes to the reduced cardiac contractility in liver cirrhosis, and CB(1)-receptor antagonists may be used to improve contractile function in cirrhotic cardiomyopathy and, possibly, in other forms of heart failure.