Inhibition of Rac1 signaling by lovastatin protects against anthracycline-induced cardiac toxicity

Normal tissue damage limits the efficacy of anticancer therapy. For anthracyclines, the clinically most relevant adverse effect is cardiotoxicity. The mechanisms involved are poorly understood and putative cardioprotectants are controversially discussed. Here, we show that the lipid-lowering drug lovastatin protects rat H9c2 cardiomyoblasts from doxorubicin in vitro. Protection by lovastatin is related to inhibition of the Ras-homologous GTPase Rac1. It rests on a reduced formation of DNA double-strand breaks, resulting from the inhibition of topoisomerase II by doxorubicin. Doxorubicin transport and reactive oxygen species are not involved. Protection by lovastatin was confirmed in vivo. In mice, lovastatin mitigated acute doxorubicin-induced heart and liver damage as indicated by reduced mRNA levels of the pro-fibrotic cytokine connective tissue growth factor (CTGF) and pro-inflammatory cytokines, respectively. Lovastatin also protected from doxorubicin-provoked subacute cardiac damage as shown by lowered mRNA levels of CTGF and atrial natriuretic peptide. Increase in the serum concentration of troponin I and cardiac fibrosis following doxorubicin treatment were also reduced by lovastatin. Whereas protecting the heart from harmful doxorubicin effects, lovastatin augmented its anticancer efficacy in a mouse xenograft model with human sarcoma cells. These data show that statins lower the incidence of cardiac tissue injury after anthracycline treatment in a Rac1-dependent manner, without impairing the therapeutic efficacy.     

Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface

Agonist-induced dimerization of TLR4 Toll/IL-1R (TIR) domains initiates intracellular signaling. Therefore, identification of the TLR4-TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating decoy peptides, each of which represents a nonfragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides--4R1, 4R3, 4BB, 4R9, and 4αE--potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by F?rster resonance energy transfer using time-resolved fluorescence spectroscopy, Bodipy-TMR-X-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean expressed in HeLa or HEK293T cells, whereas 4R3 was partially active, and 4R9 was least active. These findings suggest that the area between the BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the decoy peptide approach, in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function, and then their specific targets are identified by F?rster resonance energy transfer to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions.     

Cellular FLICE-like inhibitory protein protects against cardiac hypertrophy by blocking ASK1/p38 signaling in mice

Cellular FLICE-like inhibitory protein (Flip) is a negative regulator of nuclear factor κB signaling which has been shown previously to complicate with cardiac hypertrophy. In the present study, we tested the hypothesis that the knockout of Flip would increase cardiac hypertrophy in vivo and in vitro. The effects of Flip knockout on cardiac hypertrophy were investigated using in vitro and in vivo models. Flip was downregulated in transverse aortic constriction (TAC)-induced animal hearts and cardiomyocytes that had been treated with angiotensin II or phenylephrine for 1 h. An in vivo, heart hypertrophy model, was performed by TAC in Flip knockdown and sham mice. The extent of hypertrophy of heart was quantitated by echocardiography, and further confirmed by pathological and molecular examination of heart tissue samples. Conditional knockout of Flip in the murine heart increases the hypertrophic response induced by TAC, whereas cardiac function was preserved with reduced Flip levels in response to hypertrophic stimuli. Western blot experiments further showed Flip knockout activated markedly ASK1/P38 signaling cascades in vivo and in vitro. In conclusion, Flip preserves cardiac functions and inhibits cardiac hypertrophy partially by blocking ASK1/P38 signaling.     

d-Propranolol protects against oxidative stress and progressive cardiac dysfunction in iron overloaded rats

d-Propranolol (d-Pro: 2-8?mg·(kg body mass)(-1)·day(-1)) protected against cardiac dysfunction and oxidative stress during 3-5?weeks of iron overload (2?mg Fe-dextran·(g body mass)(-1)·week(-1)) in Sprague-Dawley rats. At 3?weeks, hearts were perfused in working mode to obtain baseline function; red blood cell glutathione, plasma 8-isoprostane, neutrophil basal superoxide production, lysosomal-derived plasma N-acetyl-β-galactosaminidase (NAGA) activity, ventricular iron content, and cardiac iron deposition were assessed. Hearts from the Fe-treated group of rats exhibited lower cardiac work (26%) and output (CO, 24%); end-diastolic pressure rose 1.8-fold. Further, glutathione levels increased 2-fold, isoprostane levels increased 2.5-fold, neutrophil superoxide increased 3-fold, NAGA increased 4-fold, ventricular Fe increased 4.9-fold; and substantial atrial and ventricular Fe-deposition occurred. d-Pro (8?mg) restored heart function to the control levels, protected against oxidative stress, and decreased cardiac Fe levels. After 5?weeks of Fe treatment, echocardiography revealed that the following were depressed: percent fractional shortening (%FS, 31% lower); left ventricular (LV) ejection fraction (LVEF, 17%), CO (25%); and aortic pressure maximum (P(max), 24%). Mitral valve E/A declined by 18%, indicating diastolic dysfunction. Cardiac CD11b+ infiltrates were elevated. Low d-Pro (2?mg) provided modest protection, whereas 4-8?mg greatly improved LVEF (54%-75%), %FS (51%-81%), CO (43%-78%), P(max) (56%-100%), and E/A?>100%; 8?mg decreased cardiac inflammation. Since d-Pro is an antioxidant and reduces cardiac Fe uptake as well as inflammation, these properties may preserve cardiac function during Fe overload.     

Low-dose Tadenan protects the rabbit bladder from bilateral ischemia/reperfusion-induced contractile dysfunction

Recent studies indicate that focal ischemia/reperfusion (I/R) can cause the contractile dysfunctions induced in animal models of partial bladder outlet obstruction. Tadenan (Pygeum africanum) pretreatment can prevent the rabbit bladder from developing the contractile and biochemical dysfunctions induced by partial outlet obstruction, possibly by protecting the bladder from ischemic injury. The current study was designed to determine whether pre-treating rabbits with a clinically relevant dose of Tadenan could prevent the bladder from developing the contractile dysfunctions that are induced by bilateral ischemia followed by reperfusion. New Zealand White rabbits were separated into two groups. One group was pre-treated by oral gavage for 3 weeks with Tadenan (3.0 mg/kg body wt./day). The second group was treated with vehicle (peanut oil). Five rabbits from each group were subjected to either bilateral ischemia for 1 or 3 h and than reperfused for either 1 h or 1 week. Five rabbits from each group were subjected to sham surgery and run with each of the experimental groups. The results of the current study show that Tadenan pre-treatment at the clinically relevant dose of 3.0 mg/kg body wt./day protected the bladder from the contractile dysfunctions induced by bilateral ischemia followed by reperfusion. These data are consistent with the assertion that Tadenan therapy in both rabbits and humans acts by protecting the bladder smooth muscle against cellular damage caused by ischemia and reperfusion.     

Antioxidant treatment protects diabetic rats from cardiac dysfunction by preserving contractile protein targets of oxidative stress

BackgoundAnimal studies suggest that reactive oxygen species (ROS) play an important role in the development of diabetic cardiomyopathy.HypothesisMatrix metalloproteinase-2 (MMP-2) is activated by ROS and contributes to the acute loss of myocardial contractile function by targeting and cleaving susceptible proteins including troponin I (TnI) and α-actinin.MethodsUsing the streptozotocin-induced diabetic rat model, we evaluated the effect of daily in vivo administration of sodium selenate (0.3 mg/kg; DMS group), or a pure omega-3 fish oil with antioxidant vitamin E (omega-3E; 50 mg/kg; DMFA group), which has antioxidant-like effects, for 4 weeks on heart function and on several biochemical parameters related to oxidant stress and MMP-2.ResultsAlthough both treatments prevented the diabetes-induced depression in left ventricular developed pressure (LVDP) as well as the rates of changes in developed pressure (±dP/dt) (P<.001), the improvement in LVDP of the DMS group was greater compared to that of the DMFA group (P<.001). Moreover, these treatments reduced the diabetes-induced increase in myocardial oxidized protein sulfhydryl and nitrite concentrations (P<.001). Gelatin zymography and Western blot data indicated that the diabetes-induced changes in myocardial levels of MMP-2 and tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) and the reduction in TnI and α-actinin protein levels were improved in both the DMS and DMFA groups (P<.001).ConclusionsThese results suggest that diabetes-induced alterations in MMP-2 and TIMP-4 contribute to myocardial contractile dysfunction by targeting TnI and α-actinin and that sodium selenate or omega-3E could have therapeutic benefits in diabetic cardiomyopathy.     

Damping excessive inflammation and tissue damage in Mycobacterium tuberculosis infection by Toll IL-1 receptor 8/single Ig IL-1-related receptor, a negative regulator of IL-1/TLR signaling

Toll IL-1R 8/single Ig IL-1-related receptor (TIR8/SIGIRR) is a member of the IL-1R family, expressed by epithelial tissues and immature dendritic cells, and is regarded as a negative regulator of TLR/IL-1R signaling. Tir8-deficient mice were rapidly killed by intranasal administration of low doses of Mycobacterium tuberculosis, despite controlling efficiently the number of viable bacilli in different organs. Tir8(-/-)-infected mice showed an increased number of neutrophils and macrophages in the lungs; however, mycobacteria-specific CD4 and CD8 T cells were similar in Tir8(-/-) and Tir8(+/+) mice. Exaggerated mortality of Tir8(-/-) mice was due to massive liver necrosis and was accompanied by increased levels of IL-1beta and TNF-alpha in lung mononuclear cells and serum, as well as by increased production of IL-1beta and TNF-alpha by M. tuberculosis-infected dendritic cells in vitro. Accordingly, blocking IL-1beta and TNF-alpha with a mix of anti-cytokine Abs, significantly prolonged survival of Tir8(-/-) mice. Thus, TIR8/SIGIRR plays a key role in damping inflammation and tissue damage in M. tuberculosis infection.     

Allicin protects against cardiac hypertrophy and fibrosis via attenuating reactive oxygen species-dependent signaling pathways

Increased oxidative stress has been associated with the pathogenesis of chronic cardiac hypertrophy and heart failure. Since allicin suppresses oxidative stress in vitro and in vivo, we hypothesized that allicin would inhibit cardiac hypertrophy through blocking oxidative stress-dependent signaling. We examined this hypothesis using primary cultured cardiac myocytes and fibroblasts and one well-established animal model of cardiac hypertrophy. Our results showed that allicin markedly inhibited hypertrophic responses induced by Ang II or pressure overload. The increased reactive oxygen species (ROS) generation and NADPH oxidase activity were significantly suppressed by allicin. Our further investigation revealed this inhibitory effect on cardiac hypertrophy was mediated by blocking the activation of ROS-dependent ERK1/2, JNK1/2 and AKT signaling pathways. Additional experiments demonstrated allicin abrogated inflammation and fibrosis by blocking the activation of nuclear factor-κB and Smad 2/3 signaling, respectively. The combination of these effects resulted in preserved cardiac function in response to cardiac stimuli. Consequently, these findings indicated that allicin protected cardiac function and prevented the development of cardiac hypertrophy through ROS-dependent mechanism involving multiple intracellular signaling.     

Muscle-specific Pten deletion protects against insulin resistance and diabetes

Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.     

BRD4 inhibition by JQ1 protects against LPS-induced cardiac dysfunction by inhibiting activation of NLRP3 inflammasomes

Molecular Biology Reports - JQ1, a BRD4 inhibitor, first identified its therapeutic role in cancer, has gradually demonstrated a protective effect on the heart in recent years; however, it is...     

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson''s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.     

Impairment of insulin-stimulated Akt/GLUT4 signaling is associated with cardiac contractile dysfunction and aggravates I/R injury in STZ-diabetic Rats

In this study, we established systemic in-vivo evidence from molecular to organism level to explain how diabetes can aggravate myocardial ischemia-reperfusion (I/R) injury and revealed the role of insulin signaling (with specific focus on Akt/GLUT4 signaling molecules). The myocardial I/R injury was induced by the left main coronary artery occlusion for 1 hr and then 3 hr reperfusion in control, streptozotocin (STZ)-induced insulinopenic diabetes, and insulin-treated diabetic rats. The diabetic rats showed a significant decrease in heart rate, and a prolonged isovolumic relaxation (tau) which lead to decrease in cardiac output (CO) without changing total peripheral resistance (TPR). The phosphorylated Akt and glucose transporter 4 (GLUT 4) protein levels were dramatically reduced in both I/R and non-I/R diabetic rat hearts. Insulin treatment in diabetes showed improvement of contractile function as well as partially increased Akt phosphorylation and GLUT 4 protein levels. In the animals subjected to I/R, the mortality rates were 25%, 65%, and 33% in the control, diabetic, and insulin-treated diabetic group respectively. The I/R-induced arrhythmias and myocardial infarction did not differ significantly between the control and the diabetic groups. Consistent with its anti-hyperglycemic effects, insulin significantly reduced I/R-induced arrhythmias but had no effect on I/R-induced infarctions. Diabetic rat with I/R exhibited the worse hemodynamic outcome, which included systolic and diastolic dysfunctions. Insulin treatment only partially improved diastolic functions and elevated P-Akt and GLUT 4 protein levels. Our results indicate that cardiac contractile dysfunction caused by a defect in insulin-stimulated Akt/GLUT4 may be a major reason for the high mortality rate in I/R injured diabetic rats.     

SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling

Accumulating evidence suggests that neurodegeneration induced by pathogenic proteins depends on contributions from surrounding glia. Here we demonstrate that NF-kappaB signaling in microglia is critically involved in neuronal death induced by amyloid-beta (Abeta) peptides, which are widely presumed to cause Alzheimer disease. Constitutive inhibition of NF-kappaB signaling in microglia by expression of the nondegradable IkappaBalpha superrepressor blocked neurotoxicity, indicating a pivotal role for microglial NF-kappaB signaling in mediating Abeta toxicity. Stimulation of microglia with Abeta increased acetylation of RelA/p65 at lysine 310, which regulates the NF-kappaB pathway. Overexpression of SIRT1 deacetylase and the addition of the SIRT1 agonist resveratrol markedly reduced NF-kappaB signaling stimulated by Abeta and had strong neuroprotective effects. Our results support a glial loop hypothesis by demonstrating a critical role for microglial NF-kappaB signaling in Abeta-dependent neurodegeneration. They also implicate SIRT1 in this pathway and highlight the therapeutic potential of resveratrol and other sirtuin-activating compounds in Alzheimer disease.     

A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling

The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is a member of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor (TIR) family signaling cascade. We have identified a novel splice variant, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. IRAK1c expression was confirmed by both RNA and protein detection. Although both IRAK1 and IRAK1c are expressed in most tissues tested, IRAK1c is the predominant form of IRAK1 expressed in the brain. Unlike IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated by IRAK4. However, IRAK1c retains the ability to strongly interact with IRAK2, MyD88, Tollip, and TRAF6. Overexpression of IRAK1c suppressed NF-kappaB activation and blocked IL-1beta-induced IL-6 as well as lipopolysaccharide- and CpG-induced tumor necrosis factor alpha production in multiple cellular systems. Mechanistically, we provide evidence that IRAK1c functions as a dominant negative by failing to be phosphorylated by IRAK4, thus remaining associated with Tollip and blocking NF-kappaB activation. The presence of a regulated, alternative splice variant of IRAK1 that functions as a kinase-dead, dominant-negative protein adds further complexity to the variety of mechanisms that regulate TIR signaling and the subsequent inflammatory response.     

Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction

Mitochondrial quality control plays a vital role in the maintenance of optimal mitochondrial function. However, its roles and regulation remain ill-defined in cardiac pathophysiology. Here, we tested the hypothesis that PARK2/Parkin, an E3-ligase recently described as being involved in the regulation of cardiac mitophagy, is important for (1) the maintenance of normal cardiac mitochondrial function; and (2) adequate recovery from sepsis, a condition known to induce reversible mitochondrial injury through poorly understood mechanisms. Investigations of mitochondrial and cardiac function were thus performed in wild-type and Park2-deficient mice at baseline and at 2 different times following administration of a sublethal dose of E. coli lipopolysaccharide (LPS). LPS injection induced cardiac and mitochondrial dysfunctions that were followed by complete recovery in wild-type mice. Recovery was associated with morphological and biochemical evidence of mitophagy, suggesting that this process is implicated in cardiac recovery from sepsis. Under baseline conditions, multiple cardiac mitochondrial dysfunctions were observed in Park2-deficient mice. These mild dysfunctions did not result in a visibly distinct cardiac phenotype. Importantly, Park2-deficient mice exhibited impaired recovery of cardiac contractility and constant degradation of mitochondrial metabolic functions. Interestingly, autophagic clearance of damaged mitochondria was still possible in the absence of PARK2 likely through compensatory mechanisms implicating PARK2-independent mitophagy and upregulation of macroautophagy. Together, these results thus provide evidence that in vivo, mitochondrial autophagy is activated during sepsis, and that compensation for a lack of PARK2 is only partial and/or that PARK2 exerts additional protective roles in mitochondria.     

Activation of hypoxia-inducible factor-1 protects airway epithelium against oxidant-induced barrier dysfunction

The respiratory epithelium forms an important barrier against inhaled pollutants and microorganisms, and its barrier function is often compromised during inflammatory airway diseases. Epithelial activation of hypoxia-inducible factor-1 (HIF-1) represents one feature of airway inflammation, but the functional importance of HIF-1 within the respiratory epithelium is largely unknown. Using primary mouse tracheal epithelial (MTE) cells or immortalized human bronchial epithelial cells (16HBE14o-), we evaluated the impact of HIF-1 activation on loss of epithelial barrier function during oxidative stress. Exposure of either 16HBE14o- or MTE cells to H(2)O(2) resulted in significant loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-dextran (4 kDa), and this was attenuated significantly after prior activation of HIF-1 by preexposure to hypoxia (2% O(2); 6 h) or the hypoxia mimics CoCl(2) or dimethyloxalylglycine (DMOG). Oxidative barrier loss was associated with reduced levels of the tight junction protein occludin and with hyperoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO(2)H), events that were also attenuated by prior activation of HIF-1. Involvement of HIF-1 in these protective effects was confirmed using the pharmacological inhibitor YC-1 and by short-hairpin RNA knockdown of HIF-1α. The protective effects of HIF-1 were associated with induction of sestrin-2, a hypoxia-inducible enzyme known to reduce oxidative stress and minimize Prx hyperoxidation. Together, our results suggest that loss of epithelial barrier integrity by oxidative stress is minimized by activation of HIF-1, in part by induction of sestrin-2.     

Zinc supplementation protects against diabetic endothelial dysfunction via GTP cyclohydrolase 1 restoration

This study explored whether zinc supplementation alleviates diabetic endothelial dysfunction and the possible mechanisms underlying. We found that high glucose exposure significantly increased reactive oxygen species (ROS) and decreased guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) and tetrahydrobiopterin (BH4) levels in bovine aortic endothelial cells (BAECs) in a time-dependent manner. High glucose increased zinc release from GTPCH1 in a similar trend. Zinc supplementation restored GTPCH1 and BH4 levels and blocked ROS accumulation in both BACEs and wild type GTPCH1 transfected HEK293 cells, but not in the zinc-free C141R mutant of GTPCH1 transfected ones. In vivo experiments showed that exogenous supplementation of zinc to streptozotocin (STZ)-induced diabetic mice partially improved the impaired maximal endothelium-dependent vasorelaxation, reversed the aberrant reduction of GTPCH1 and BH4, and suppressed the elevation of ROS in the aortas. In conclusion, our study demonstrated a novel mechanism that via GTPCH1 restoration zinc supplementation exerts a protective benefit on diabetic endothelial dysfunction.     

Impairment of cardiac insulin signaling and myocardial contractile performance in high-cholesterol/fructose-fed rats

Although insulin resistance is recognized as a potent and prevalent risk factor for coronary heart disease, less is known as to whether insulin resistance causes an altered cardiac phenotype independent of coronary atherosclerosis. In this study, we investigated the relationship between insulin resistance and cardiac contractile dysfunctions by generating a new insulin resistance animal model with rats on high cholesterol-fructose diet. Male Sprague-Dawley rats were given high cholesterol-fructose (HCF) diet for 15 wk; the rats developed insulin resistance syndrome characterized by elevated blood pressure, hyperlipidemia, hyperinsulinemia, impaired glucose tolerance, and insulin resistance. The results show that HCF induced insulin resistance not only in metabolic-response tissues (i.e., liver and muscle) but also in the heart as well. Insulin-stimulated cardiac glucose uptake was significantly reduced after 15 wk of HCF feeding, and cardiac insulin resistance was associated with blunted Akt-mediated insulin signaling along with glucose transporter GLUT4 translocation. Basal fatty acid transporter FATP1 levels were increased in HCF rat hearts. The cardiac performance of the HCF rats exhibited a marked reduction in cardiac output, ejection fraction, stroke volume, and end-diastolic volume. It also showed decreases in left ventricular end-systolic elasticity, whereas the effective arterial elasticity was increased. In addition, the relaxation time constant of left ventricular pressure was prolonged in the HCF group. Overall, these results indicate that insulin resistance reduction of cardiac glucose uptake is associated with defects in insulin signaling. The cardiac metabolic alterations that impair contractile functions may lead to the development of cardiomyopathy.     

Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.