Moderate ethanol administration accentuates cardiomyocyte contractile dysfunction and mitochondrial injury in high fat diet-induced obesity.

Light to moderate drinking confers cardioprotection although it remains unclear with regards to the role of moderate drinking on cardiac function in obesity. This study was designed to examine the impact of moderate ethanol intake on myocardial function in high fat diet intake-induced obesity and the mechanism(s) involved with a focus on mitochondrial integrity. C57BL/6 mice were fed low or high fat diet for 16 weeks prior to ethanol challenge (1g/kg/d for 3 days). Cardiac contractile function, intracellular Ca(2+) homeostasis, myocardial histology, and mitochondrial integrity [aconitase activity and the mitochondrial proteins SOD1, UCP-2 and PPARγ coactivator 1α (PGC-1α)] were assessed 24h after the final ethanol challenge. Fat diet intake compromised cardiomyocyte contractile and intracellular Ca(2+) properties (depressed peak shortening and maximal velocities of shortening/relengthening, prolonged duration of relengthening, dampened intracellular Ca(2+) rise and clearance without affecting duration of shortening). Although moderate ethanol challenge failed to alter cardiomyocyte mechanical property under low fat diet intake, it accentuated high fat diet intake-induced changes in cardiomyocyte contractile function and intracellular Ca(2+) handling. Moderate ethanol challenge failed to affect fat diet intake-induced cardiac hypertrophy as evidenced by H&E staining. High fat diet intake reduced myocardial aconitase activity, downregulated levels of mitochondrial protein UCP-2, PGC-1α, SOD1 and interrupted intracellular Ca(2+) regulatory proteins, the effect of which was augmented by moderate ethanol challenge. Neither high fat diet intake nor moderate ethanol challenge affected protein or mRNA levels as well as phosphorylation of Akt and GSK3ß in mouse hearts. Taken together, our data revealed that moderate ethanol challenge accentuated high fat diet-induced cardiac contractile and intracellular Ca(2+) anomalies as well as mitochondrial injury.

α,ß-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,ß-unsaturated aldehyde pollutant, on myocardial contractile function and the underlying mechanism involved with a focus on TRPV1 and oxidative stress. Cardiomyocyte mechanical and intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) properties including depressed PS, ± dL/dt and ΔFFI, as well as prolonged TR90 and intracellular Ca(2+) 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 Ca(2+) 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,ß-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.

α,ß-Unsaturated aldehyde crotonaldehyde triggers cardiomyocyte contractile dysfunction: role of TRPV1 and mitochondrial function.

Recent evidence has suggested that cigarette smoking is associated with an increased prevalence of heart diseases. Given that cigarette smoking triggers proinflammatory response via stimulation of the capsaicin-sensitive transient receptor potential cation channel TRPV1, this study was designed to evaluate the effect of an essential α,ß-unsaturated aldehyde from cigarette smoke crotonaldehyde on myocardial function and the underlying mechanism with a focus on TRPV1 and mitochondria. Cardiomyocyte mechanical and intracellular Ca2+ properties were evaluated 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), intracellular Ca2+ decay and SERCA activity. Apoptosis and TRPV1 were evaluated using Western blot analysis. Production of reactive oxygen species (ROS) and DNA damage were measured using the intracellular fluoroprobe 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and 8-hydroxy-2'-deoxyguanosine (8-OHdG), respectively. Our data revealed that crotonaldehyde interrupted cardiomyocyte contractile and intracellular Ca2+ property including depressed PS, ±dL/dt, ΔFFI and SERCA activity, as well as prolonged TR90 and intracellular Ca2+ decay. Crotonaldehyde exposure increased TRPV1 and NADPH oxidase levels, promoted apoptosis, mitochondrial injury (decreased aconitase activity, PGC-1α and UCP-2) as well as production of ROS and 8-OHdG. Interestingly, crotonaldehyde-induced cardiac defect was obliterated by the ROS scavenger glutathione and the TRPV1 inhibitor capsazepine. Capsazepine (not glutathione) ablated crotonaldehyde-induced mitochondrial damage. Capsazepine, glutathione and the NADPH inhibitor apocynin negated crotonaldehyde-induced ROS accumulation. Our data suggest a role of crotonaldehyde compromises cardiomyocyte mechanical function possibly through a TRPV1- and mitochondria-dependent oxidative stress mechanism.

Inhibition of protein kinase C ßII isoform rescues glucose toxicity-induced cardiomyocyte contractile dysfunction: role of mitochondria.

AIMS: Hyperglycemia leads to cytotoxicity in the heart. Although theories were postulated for glucose toxicity-induced cardiomyocyte dysfunction including oxidative stress, the mechanism involved still remains unclear. Recent evidence has depicted a role of protein kinase C (PKC) in diabetic complications while high concentrations of glucose stimulate PKC. This study examined the role of PKCßII in glucose toxicity-induced cardiomyocyte contractile and intracellular Ca(2+) aberrations. MAIN METHODS: Adult rat cardiomyocytes were maintained in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) medium for 12 h. Contractile and intracellular Ca(2+) properties were measured using a video edge-detection system including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), rise in intracellular Ca(2+) Fura-2 fluorescence intensity and intracellular Ca(2+) decay. Production of ROS/O2(-) and mitochondrial integrity were examined using fluorescence imaging, aconitase activity and Western blotting. KEY FINDINGS: High glucose triggered abnormal contractile and intracellular Ca(2+) properties including reduced PS, ±dL/dt, prolonged TR90, decreased electrically-stimulated rise in intracellular Ca(2+) and delayed intracellular Ca(2+) clearance, the effects of which were ablated by the PKCßII inhibitor LY333531. Inhibition of PKCßII rescued glucose toxicity-induced generation of ROS and O2(-), apoptosis, cell death and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCßII inhibition-induced beneficial effects were mimicked by the NADPH oxidase inhibitor apocynin and were canceled off by mitochondrial uncoupling using FCCP. SIGNIFICANCE: These findings suggest the therapeutic potential of specific inhibition of PKCßII isoform in the management of hyperglycemia-induced cardiac complications.

Inhibition of CYP2E1 attenuates chronic alcohol intake-induced myocardial contractile dysfunction and apoptosis.

Alcohol intake is associated with myocardial contractile dysfunction and apoptosis although the precise mechanism is unclear. This study was designed to examine the effect of the cytochrome P450 enzyme CYP2E1 inhibition on ethanol-induced cardiac dysfunction. Adult male mice were fed a 4% ethanol liquid or pair-fed control diet for 6weeks. Following 2weeks of diet feeding, a cohort of mice started to receive the CYP2E1 inhibitor diallyl sulfide (100mg/kg/d, i.p.) for the remaining feeding duration. Cardiac function was assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate CYP2E1, heme oxygenase-1 (HO-1), iNOS, the intracellular Ca(2+) regulatory proteins sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)Ca(2+) exchanger and phospholamban, pro-apoptotic protein cleaved caspase-3, Bax, c-Jun-NH(2)-terminal kinase (JNK) and apoptosis signal-regulating kinase (ASK-1). Ethanol led to elevated levels of CYP2E1, iNOS and phospholamban, decreased levels of HO-1 and Na(+)Ca(2+) exchanger, cardiac contractile and intracellular Ca(2+) defects, cardiac fibrosis, overt O(2)(-) production, and apoptosis accompanied with increased phosphorylation of JNK and ASK-1, the effects were significantly attenuated or ablated by diallyl sulfide. Inhibitors of JNK and ASK-1 but not HO-1 inducer or iNOS inhibitor obliterated ethanol-induced cardiomyocyte contractile dysfunction, substantiating a role for JNK and ASK-1 signaling in ethanol-induced myocardial injury. Taken together, these findings suggest that ethanol metabolism through CYP2E1 may contribute to the pathogenesis of alcoholic cardiomyopathy including myocardial contractile dysfunction, oxidative stress and apoptosis, possibly through activation of JNK and ASK-1 signaling.

Involvement of AMPK in alcohol dehydrogenase accentuated myocardial dysfunction following acute ethanol challenge in mice.

OBJECTIVES: Binge alcohol drinking often triggers myocardial contractile dysfunction although the underlying mechanism is not fully clear. This study was designed to examine the impact of cardiac-specific overexpression of alcohol dehydrogenase (ADH) on ethanol-induced change in cardiac contractile function, intracellular Ca(2+) homeostasis, insulin and AMP-dependent kinase (AMPK) signaling. METHODS: ADH transgenic and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Oral glucose tolerance test, cardiac AMP/ATP levels, cardiac contractile function, intracellular Ca(2+) handling and AMPK signaling (including ACC and LKB1) were examined. RESULTS: Ethanol exposure led to glucose intolerance, elevated plasma insulin, compromised cardiac contractile and intracellular Ca(2+) properties, downregulated protein phosphatase PP2A subunit and PPAR-gamma, as well as phosphorylation of AMPK, ACC and LKB1, all of which except plasma insulin were overtly accentuated by ADH transgene. Interestingly, myocardium from ethanol-treated FVB mice displayed enhanced expression of PP2Calpha and PGC-1alpha, decreased insulin receptor expression as well as unchanged expression of Glut4, the response of which was unaffected by ADH. Cardiac AMP-to-ATP ratio was significantly enhanced by ethanol exposure with a more pronounced increase in ADH mice. In addition, the AMPK inhibitor compound C (10 microM) abrogated acute ethanol exposure-elicited cardiomyocyte mechanical dysfunction. CONCLUSIONS: In summary, these data suggest that the ADH transgene exacerbated acute ethanol toxicity-induced myocardial contractile dysfunction, intracellular Ca(2+) mishandling and glucose intolerance, indicating a role of ADH in acute ethanol toxicity-induced cardiac dysfunction possibly related to altered cellular fuel AMPK signaling cascade.

Cardiac-specific overexpression of insulin-like growth factor I (IGF-1) rescues lipopolysaccharide-induced cardiac dysfunction and activation of stress signaling in murine cardiomyocytes.

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, plays a key role in cardiac dysfunction in sepsis. Low circulating levels of insulin-like growth factor 1 (IGF-1) are found in sepsis, although the influence of IGF-1 on septic cardiac defect is unknown. This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca2+ dysfunction, activation of stress signal and endoplasmic reticulum (ER) stress. Mechanical and intracellular Ca2+ properties were examined in cardiomyocytes from Fast Violet B and cardiac-specific IGF-1 overexpression mice treated with or without LPS (4 mg kg(-1), 6 h). Reactive oxygen species (ROS), protein carbonyl formation and apoptosis were measured. Activation of mitogen-activated protein kinase pathways (p38, c-jun N-terminal kinase [JNK] and extracellular signal-related kinase [ERK]), ER stress and apoptotic markers were evaluated using Western blot analysis. Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening and prolonged duration of relengthening in LPS-treated Fast Violet B cardiomyocytes associated with reduced intracellular Ca2+ decay. Accumulation of ROS protein carbonyl and apoptosis were elevated after LPS treatment. Western blot analysis revealed activated p38 and JNK, up-regulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2. Total protein expression of p38, JNK, and ERK was unaffected by either LPS or IGF-1. Interestingly, these LPS-induced changes in mechanical and intracellular Ca2+ properties, ROS, protein carbonyl, apoptosis, stress signal activation, and ER stress markers were effectively ablated by IGF-1. In vitro LPS exposure (1 microg mL(-1)) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting, which was alleviated by exogenous IGF-1 (50 nM). These data collectively suggested a beneficial of IGF-1 in the management of cardiac dysfunction under sepsis.

Impact of gender on basal and insulin-like growth factor I-regulated nitric oxide synthase activity in adult rat left ventricular myocytes.

Cardiovascular morbidity and mortality are far less in pre-menopausal women compared to age-matched men. Ovarian hormones are believed to be mainly responsible for this "female advantage" in cardiovascular function although the underlying mechanism has not been fully elucidated. A gender difference exists in vascular nitric oxide (NO) synthesis, which may play a key role in ventricular function and cardiac remodeling. This study was designed to compare NO production, basal NO synthase (NOS) expression and activity, as well as insulin-like growth factor I (IGF-1)-induced response on NOS activity in left ventricular myocytes from age-matched adult male and female Sprague-Dawley rats. NO production and protein expression of NOS, IGF-1 receptor (IGF-1R) and IGF binding protein-3 (IGFBP-3) were measured by Griess assay and Western blot analysis, respectively. NOS activity was evaluated by conversion of (3)H-arginine to (3)H-citrulline. Basal NO production, endothelial NOS expression and NOS activity were both significantly higher in female left ventricular myocytes than their male counterparts. However, protein expression of inducible and neuronal NOS as well as IGFBP-3 was comparable between the two genders. IGF-1R expression was less in female than male group. IGF-1 (10(-10)-10(-6) m) induced a concentration-dependent inhibition of NOS activity in male myocytes with a maximal inhibition of 22.2%. However, the IGF-1-induced inhibition in NOS activity was not present in left ventricular myocytes from female rats. These data revealed a gender difference in myocardial basal NO levels, endothelial NOS expression, basal NOS activity and IGF-1-induced inhibition on NOS activity, which may contribute to the gender-related difference of cardiac function.

Adenovirus gene transfer of recombinant endothelial nitric oxide synthase enhances contractile function in ventricular myocytes.

eNOS is expressed in cardiac myocytes and plays an important role in cardiac contractile function. This study was designed to determine whether ex vivo eNOS gene transfer in ventricular myocytes affects cardiac contractile function. Replication-incompetent adenoviral vectors encoding eNOS or marker gene beta-galactosidase (LacZ) were transduced into adult rat ventricular myocytes at an MOI of 10, 50, or 100 for 36 hours. Mechanical and intracellular Ca2+ properties of myocytes were evaluated by video-based edge detection and fura-2 fluorescence. NOS protein expression and activity were assessed by Western blot and 3H-arginine to 3H-citrulline assay. Myocytes transduced with eNOS but not LacZ displayed enhanced eNOS but not iNOS expression associated with elevated NOS activity. Myocytes transduced with eNOS exhibited significantly elevated peak shortening and velocity of shortening/relengthening associated with enhanced basal as well as electrically stimulated rise of intracellular Ca2+ compared with control or LacZ groups. The durations of shortening and relengthening were comparable in all groups. The eNOS-induced mechanical effects were paralleled with elevated phosphorylation of Akt. Furthermore, the phosphatidylinositol-3 (PI-3) kinase inhibitors wortmannin and LY294002 prevented eNOS-induced mechanical effects. These results revealed that gene transfer of eNOS directly promotes cardiomyocyte contractile function and intracellular Ca2+ handling, suggesting therapeutic potential of eNOS gene transfer.