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Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, estrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and estrogen replacement on ventricular myocyte contractile function and PKB/Akt activation. Nulliparous female rats were subjected to bilateral ovariectomy (Ovx) or sham operation (sham). A subgroup of Ovx rats received estrogen ([E.sub.2]) replacement (40 [micro]g*[kg.sup.-1]* [day.sup.-1]) for 8 weeks. Mechanical and intracellular [Ca.sup.2+] properties were evaluated including peak shortening (PS), time to PS (TPS), time to 90% relengthening (T[R.sub.90]), maximal velocity of shortening/relengthening ([+ or -]dL/dt), fura 2 fluorescence intensity (FFI), and decay rate. Levels of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA2a), phospholamban (PLB), and Akt were assessed by Western blot. Ovx promoted body weight gain associated with reduced serum [E.sub.2] and uterine weight, all of which were abolished by [E.sub.2]. Ovx depressed PS and [+ or -] dL/dt, prolonged TPS, T[R.sub.90], and decay rate, and enhanced resting FFI, all of which, with the exception of TPS, were restored by [E.sub.2]. Ovx did not alter the levels of SERCA2a, PLB, and total Akt, but significantly reduced Akt activation [phosphorylated Akt (pAkt)], pAkt/Akt, and the SERCA2a-to-PLB ratio. These alterations in protein expression were restored by [E.sub.2]. [E.sub.2] enhanced PS and +dL/dt in vitro, which was abolished by the [E.sub.2] receptor antagonist ICI-182780. Ovx reduced myocyte [Ca.sup.2+] responsiveness and lessened stimulating frequency-induced decline in PS, both ablated by [E.sub.2]. These data suggest that mechanical and protein functions of ventricular myocytes are directly regulated by [E.sub.2]. ovariectomy; cardiac myocyte; contraction, intracellular [Ca.sup.2+]

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (T[R.sub.90]) and maximal velocity of shortening/relengthening ([+ or -]dL/dt). Intracellular [Ca.sup.2+] was evaluated as [Ca.sup.2+]-induced [Ca.sup.2+] release [difference in fura 2 fluorescent intensity ([DELTA]FFI)] and fluorescence decay rate ([tau]). Sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA)2a, phospholamban (PLB), [Na.sup.+]-[Ca.sup.2+] exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, [+ or -]dL/dt, and ([DELTA]FFI associated with prolonged TPS, T[R.sub.90], and [tau]. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and [DELTA]FFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA. diabetic mouse; ventricular myocyte; excitation-contraction coupling; insulin-like growth factor I; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase; sodium-calcium exchanger

Alcoholic cardiomyopathy is characterized by impaired ventricular function although its toxic mechanism is unclear. This study examined the impact of cardiac overexpression of alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde (ACA), on ethanol-induced cardiac contractile defect. Mechanical and intracellular [Ca.sup.2+] properties were evaluated in ventricular myocytes from ADH transgenic and wild-type (FVB) mice. ACA production was assessed by gas chromatography. ADH myocytes exhibited similar mechanical properties but a higher efficiency to convert ACA compared with FVB myocytes. Acute exposure to ethanol depressed cell shortening and intracellular [Ca.sup.2+] in the FVB group with maximal inhibitions of 23.3% and 23.4%, respectively. Strikingly, the ethanol-induced depression on cell shortening and intracellular [Ca.sup.2+] was significantly augmented in the ADH group, with maximal inhibitions of 43.7% and 40.6%, respectively. Pretreatment with the ADH inhibitor 4-methylpyrazole (4-MP) or the aldehyde dehydrogenase inhibitor cyanamide prevented or augmented the ethanol-induced inhibition, respectively, in the ADH but not the FVB group. The ADH transgene also substantiated the ethanol-induced inhibition of maximal velocity of shortening/relengthening and unmasked an ethanol-induced prolongation of the duration of shortening/relengthening, which was abolished by 4-MP. These data suggest that elevated cardiac ACA exposure due to enhanced ADH expression may play an important role in the development of alcoholic cardiomyopathy. transgene; acetaldehyde; shortening; intracellular calcium ion transient

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, estrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and estrogen replacement on ventricular myocyte contractile function and PKB/Akt activation. Nulliparous female rats were subjected to bilateral ovariectomy (Ovx) or sham operation (sham). A subgroup of Ovx rats received estrogen (E[sub 2]) replacement (40 μg·kgsup -1]. day[sup -1]) for 8 weeks. Mechanical and intracellular Ca[sup 2+] properties were evaluated including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR[sub 90]), maximal velocity of shortening/relengthening (±dL/dt), fura 2 fluorescence intensity (FFI), and decay rate. Levels of sarco(endo)plasmic reticulum Ca[sup 2+]-ATPase (SERCA2a), phospholamban (PLB), and Akt were assessed by Western blot. Ovx promoted body weight gain associated with reduced serum E[sub 2] and uterine weight, all of which were abolished by E[sub 2]. Ovx depressed PS and ±dL/dt, prolonged TPS, TR[sub 90], and decay rate, and enhanced resting FFI, all of which, with the exception of TPS, were restored by E[sub 2]. Ovx did not alter the levels of SERCA2a, PLB, and total Akt, but significantly reduced Akt activation [phosphorylated Akt (pAkt)], pAkt/Akt, and the SERCA2ato-PLB ratio. These alteration in proton expression were restored by E[sub 2]. E[sub 2] enhanced PS and +dL/dt in vitro, which was abolished by the E[sub 2] receptor antagonist ICI-182780. Ovx reduced myocyte Ca[sup 2+] responsiveness and lessened stimulating frequency-induced decline in PS, both ablated by E[sub 2]. These data suggest that mechanical and proton functions of ventricular myocytes are directly regulated by E[sub 2]. [ABSTRACT FROM AUTHOR]

Objective: Cardiac resistance to IGF-1 occurs in diabetes and is attributed to cardiac dysfunction in diabetes. However, the mechanism of action responsible for cardiac IGF-1 resistance is still unknown. This study was designed to examine the impact of high glucose on IGF-1-induced contractile response and activation of serine-threonine kinase Akt as well as extracellular signal-regulated kinase (ERK1/2) in cardiac myocytes. Methods: Isolated adult rat ventricular myocytes were cultured for 12–18 h in a serum-free medium containing either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose. Mechanical properties were evaluated using an IonOptix MyoCam® system. Myocytes were electrically stimulated at 0.5 Hz and contractile properties analyzed included peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR90). Intracellular Ca2+-induced Ca2+ release was measured as fura-2 fluorescence intensity change (ΔFFI). Protein levels of total and phosphorylated Akt and ERK1/2, indicators of Akt and ERK1/2 activation, IGF-1 receptors (pro-IGF-1R and IGF-1Rα) as well as the glucose transporter GLUT4 were assessed by Western blot. Results: IGF-1 (10−10–10−6 M) elicited a dose-dependent increase in PS and ΔFFI in myocytes maintained in NG medium. However, IGF-1 induced a negative response on PS and ΔFFI in HG myocytes. The IGF-1-induced responses in NG or HG myocytes were blunted by the IGF-1 receptor antagonist H-1356. Western blot analysis revealed that IGF-1Rα but not pro-IGF-1R was reduced in HG myocytes. While IGF-1 (10−6 M) upregulated total Akt protein levels in both NG and HG myocytes, it only induced a significant activation of Akt in NG but not HG myocytes. IGF-1 elicited comparable ERK1/2 activation in both NG and HG myocytes. Conclusion: These results suggest that the cardiac IGF-1 resistance in diabetes is likely attributed, at least in part, to reduced IGF-1R and attenuated IGF-1-induced Akt phosphorylation under elevated extracellular glucose. [Copyright &y& Elsevier]

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR[SUB90]) and maximal velocity of shortening/relengthening (±dL/dt). Intracellular Ca[SUP2+] was evaluated as Ca[SUP2+]-induced Ca[SUP2+] release [difference in fura 2 fluorescent intensity (ΔFFI)] and fluorescence decay rate (τ). Sarco(endo)plasmic reticulum Ca[SUP2+]-ATPase (SERCA)2a, phospholamban (PLB), Na[SUP+]-Ca[SUP2+] exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, ± dL/dt, and ΔFFI associated with prolonged TPS, TR[SUB90], and τ. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and AFFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA. [ABSTRACT FROM AUTHOR]

— To assess the teratogenic action of ethanol on cardiac contractile function in offspring exposed to ethanol in utero, pregnant Sprague–Dawley rats were fed with ethanol during gestation. Left-ventricular papillary muscles and myocytes were isolated from the offspring of the ethanol-ingesting and control pregnant rats. Mechanical parameters measured were peak tension development (PTD, indicating the myocardial force-generating capacity), peak cell shortening (PS), time-to-PTD/PS (TPT/TPS), time-to-90% relaxation/re-lengthening (RT90/TR90), and maximal velocities of contraction/shortening and relaxation/re-lengthening (±VT and ±dL/dt). Intracellular Ca2+ levels and apoptosis were evaluated with fura-2 fluorescent dye and Caspase-3 activation assay, respectively. Offspring of the ethanol group displayed decreased heart weight associated with comparable body, liver and kidney weight, and papillary muscle weight/size, compared to the control group. However, prenatal ethanol exposure depressed myocardial PTD and ±VT. The myocardium from the ethanol group also exhibited slightly but significantly shortened TPT, accompanied with normal RT90. Muscles from both groups exhibited comparable responses to post-rest potentiation, increasing extracellular Ca2+ concentration, noradrenaline and acute ethanol challenge. Ventricular myocytes from both the control and ethanol groups possessed similar PS, TPS, TR90 and ±dL/dt. Both resting and peak intracellular Ca2+ levels were elevated in myocytes from the ethanol group. Additionally, acute ethanol application depressed caffeine-induced intracellular Ca2+ rise in myocytes from both groups. Myocytes from the ethanol group displayed an enhanced Caspase-3 activation, compared to control myocytes. These results suggest that prenatal ethanol exposure alters myocardial contractile function and may contribute to the development of postnatal cardiac dysfunction through, in part, increased intracellular Ca2+ loading and apoptosis. [ABSTRACT FROM PUBLISHER]

Acute ethanol exposure depresses ventricular contractility and contributes to alcoholic cardiomyopathy in both men and women chronically consuming ethanol. However, a gender-related difference in the severity of myopathy exists with female being more sensitive to ethanol-induced tissue damage. Acetaldehyde (ACA), the major oxidized product of ethanol, has been implicated to play a role in the pathogenesis and gender-related difference of alcoholic cardiomyopathy, possibly due to its direct cardiac effect and interaction with estrogen. This study was designed to compare the effects of cardiac overexpression of alcohol dehydrogenase (ADH), which converts ethanol into ACA, on the cardiac contractile response to ethanol in ventricular myocytes isolated from age-matched adult male and female transgenic (ADH) and wild-type (FVB) mice. Mechanical properties were measured with an IonOptix SoftEdge system. ACA production was assessed by gas chromatography. The ADH myocytes from both genders exhibited similar mechanical properties but a higher efficacy to produce ACA compared to FVB myocytes. Exposure to ethanol (80–640 mg/dl) for 60 min elicited concentration-dependent decrease of cell shortening in both FVB and ADH groups. The ethanol-induced depression on cell shortening was significantly augmented in female but not male ADH group. ADH transgene did not exacerbate the ethanol-induced inhibition of maximal velocity of shortening/relengthening in either gender. In addition, neither ethanol nor ADH transgene affect the duration of shortening and relengthening in male or female mice. These data suggest that females may be more sensitive to ACA-induced cardiac contractile depression than male, which may attribute to the gender-related difference of alcoholic cardiomyopathy. [Copyright &y& Elsevier]

Insulin-like growth factor-I (IGF-1) ameliorates cardiac dysfunction in diabetes although the mechanism of action remains poorly understood. This study examined the role of PI-3 kinase/Akt/mammalian target of rapamycin (mTOR) and calcineurin pathways in cardiac effects of IGF-1 against glucose toxicity. Adult rat ventricular myocytes were cultured for 8 h with either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose, in the presence or absence of IGF-1 (10-500 nM), the PI-3 kinase/Akt inhibitor LY294002 (10 microM), the mTOR inhibitor rapamycin (20 microM) or the calcineurin inhibitors cyclosporin A (5 microM) or FK506 (10 mg/l). Mechanical properties were evaluated using an IonOptix MyoCam system. HG depressed peak shortening (PS), reduced maximal velocity of shortening/relengthening (+/- dl/dt) and prolongs time-to-90% relengthening (TR90), which were abolished by IGF-1 (100 and 500 nM). Interestingly, the IGF-1-elicited protective effect against HG was nullified by either LY294002 or rapamycin, but not by cyclosporine A or FK506. None of the inhibitors affected cell mechanics. Western blot analysis indicated that HG and IGF-1 stimulated phosphorylation of Akt and mTOR. HG also activated p70s6k and suppressed GSK-3beta phosphorylation. However, the HG-induced alterations in phosphorylation of Akt, mTOR, p70s6k and GSK-3beta were significantly reversed by IGF-1. Protein expression of Akt, mTOR, p70s6k, GSK-3beta, SERCA2a and phospholamban was unaffected by HG, IGF-1 or rapamycin. Rapamycin significantly enhanced Akt phosphorylation whereas it inhibited mTOR phosphorylation. Collectively, our data suggest that IGF-1 may provide cardiac protection against glucose in part through a PI-3 kinase/Akt/mTOR/ p70s6k-dependent and calcineurin-independent pathway.

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.

Background: Alcoholic cardiomyopathy is manifested as ventricular dysfunction, although its specific toxic mechanism remains obscure. This study was designed to examine the impact of enhanced acetaldehyde exposure on cardiac function via cardiac-specific overexpression of alcohol dehydrogenase (ADH) after alcohol intake., Methods: ADH transgenic and wild-type FVB mice were placed on a 4% alcohol or control diet for 8 weeks. Mechanical and intracellular Ca2+ properties were evaluated in cardiac myocytes. Levels of acetaldehyde, lipid peroxidation, and protein carbonyl formation were determined., Results: FVB and ADH mice consuming ethanol exhibited elevated blood ethanol/acetaldehyde, cardiac acetaldehyde, and cardiac hypertrophy compared with non-ethanol-consuming mice. However, the levels of cardiac acetaldehyde and hypertrophy were significantly greater in ADH ethanol-fed mice than FVB ethanol-fed mice. ADH transgene itself did not affect mechanical and intracellular Ca2+ properties with the exception of reduced resting intracellular Ca2+ and Ca2+ re-sequestration at low pace frequency. Myocytes from ethanol-fed mice showed significantly depressed peak shortening, velocity of shortening/relengthening, rise of intracellular Ca2+ transients, and sarco(endo)plasmic reticulum Ca2+ load associated with similar duration of shortening/relengthening compared with myocytes from control mice. Strikingly, the ethanol-induced mechanical and intracellular Ca2+ defects were exacerbated in ADH myocytes compared with the FVB group except velocity of shortening/relengthening. The lipid peroxidation end products malondialdehyde and protein carbonyl formation were significantly elevated in both livers and hearts after chronic ethanol consumption, with the cardiac lipid and protein damage being exaggerated by ADH transgene., Conclusion: These data suggest that increased cardiac acetaldehyde exposure due to ADH transgene may play an important role in cardiac contractile dysfunctions associated with lipid and protein damage after alcohol intake.