time-to-PS Research Articles

High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis

Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague-Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (+/- dl/dt). Intracellular Ca properties were assessed using fluorescent microscopy. High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, +/- dl/dt, prolonged TPS/TR90 reduced intracellular Ca release and Ca clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.

Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide

Uncorrected obesity is often accompanied by ventricular contractile dysfunction, elevation of the lipotoxic mediator ceramide and the obesity gene product leptin. Both ceramide and leptin participate in the regulation of cardiac function and are speculated to play roles in obesity-related cardiac dysfunctions. The purpose of this study was to examine the effect of ceramide on leptin-elicited cardiac contractile response. Adult rat left ventricular myocytes were incubated for 24 h with low (5 nM) or high (50 nM) concentration of leptin in the absence or presence of the active ceramide analog C 2-dihydroceramide (25 μM). Contractile and intracellular Ca 2+ properties were evaluated using an IonOptix MyoCam ® system including peak shortening (PS), maximal velocity of shortening/relengthening (±d L/d t), time-to-PS (TPS), time-to-90% relengthening (TR 90), intracellular Ca 2+ rise (Δ[Ca 2+]) and intracellular Ca 2+ decay. While ceramide did not elicit any effect on cell mechanics and intracellular Ca 2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca 2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, ±d L/d t, Δ[Ca 2+] and prolonged TR 90. With ceramide co-incubation, 5 nM leptin depressed PS, ±d L/d t, Δ[Ca 2+] and prolonged TR 90 whereas 50 nM leptin-elicited effects on PS, ±d L/d t, Δ[Ca 2+] and TR 90 were significantly potentiated in addition to slowing intracellular Ca 2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction.

Metallothionein alleviates cardiac dysfunction in streptozotocin-induced diabetes: Role of Ca 2+ cycling proteins, NADPH oxidase, poly(ADP-Ribose) polymerase and myosin heavy chain isozyme

Diabetic cardiomyopathy contributes to high morbidity and mortality in diabetic populations. It is manifested by compromised ventricular contraction and prolonged relaxation attributable to multiple causative factors including oxidative stress. This study was designed to examine the effect of cardiac overexpression of the heavy metal scavenger metallothionein (MT) on cardiac contractile function, intracellular Ca 2+ cycling proteins, stress-activated signaling molecules and the myosin heavy chain (MHC) isozyme in diabetes. Adult male wild-type (FVB) and MT transgenic mice were made diabetic by a single injection of streptozotocin (STZ). Contractile properties were evaluated in cardiomyocytes including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR 90), maximal velocity of shortening/relengthening (±dL/dt) and intracellular Ca 2+ fluorescence. Diabetes significantly depressed PS, ±dL/dt, prolonged TPS, TR 90 and intracellular Ca 2+ clearing, elevated resting intracellular Ca 2+, reduced caffeine-induced sarcoplasmic reticulum Ca 2+ release and dampened stress tolerance at high stimulus frequencies. MT itself exhibited little effect on myocyte mechanics but it significantly alleviated STZ-induced myocyte contractile dysfunctions. Diabetes enhanced expression of the AT 1 receptor, phospholamban, the p47 phox NADPH oxidase subunit and poly(ADP-ribose) polymerase (PARP), depressed the level of SERCA2a, Na +-Ca 2+ exchanger and triggered a β-MHC isozyme switch. All of these STZ-induced alterations with the exception of depressed SERCA2a and enhanced phospholamban were reconciled by MT. Collectively, these data suggest a beneficial effect of MT in the therapeutics of diabetic cardiomyopathy, possibly through a mechanism related to NADPH oxidase, PARP and MHC isozyme switch.

Interaction between tumor necrosis factor-α and leptin-induced inhibition of cardiac contractile function in isolated ventricular myocytes

Pro-inflammatory factors such as the adipokine leptin and cytokine tumor necrosis factor-α (TNFα) have been implicated in the onset of myocardial dysfunction in ischemia–reperfusion injury, sepsis, heart failure, viral myocarditis and cardiac allograft rejection. Although circulating TNFα and leptin levels are both elevated under a variety of inflammatory conditions, it remains unknown whether TNFα and leptin depress cardiac contractile function independently or synergistically. We examined the effect of acute (30 min) and short-term (24 h) exposure of TNFα, leptin or both on cardiac contractile function in adult rat ventricular myocytes. Contractile properties were evaluated using an Ionoptix Softedge system including peak shortening (PS), maximal velocity of shortening/relengthening (±ⅆ L/ⅆ t), time-to-PS (TPS) and time-to-90% relengthening (TR 90). Both TNFα (0.5–500 pg/ml) and leptin (1–100 nm) exerted concentration-dependent inhibitions in PS and ±ⅆ L/ⅆ t following a 30-min exposure. TNFα but not leptin prolonged TR 90. Interestingly, TNFα-induced depression of cell shortening was masked by leptin and vice versa. Following a 24-h incubation, both TNFα and leptin significantly inhibited PS and ±ⅆ L/ⅆ t without affecting TPS and TR 90. There was no additive or synergistic response by the two pro-inflammatory factors. The nitric oxide synthase inhibitor l-NMMA abolished depression of myocyte shortening elicited by TNFα, leptin or both. In summary, this study demonstrated that the inhibitory effect on cardiac contraction by TNFα and leptin may mask each other and share a common mechanism(s), probably dependent on NO.

High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus

Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.

Increased contractility of cardiomyocytes from copper-deficient rats is associated with upregulation of cardiac IGF-I receptor

Hearts from severely Cu-deficient rats show a variety of pathological defects, including hypertrophy and, in intact hearts, depression of contractile function. Paradoxically, isolated cardiomyocytes from these rats exhibit enhanced contractile properties. Because hypertrophy and enhanced contractility observed with other pathologies are associated with elevation of insulin-like growth factor-I (IGF)-I, this mechanism was examined for the case of dietary Cu deficiency. Male, weanling Sprague-Dawley rats were provided diets that were deficient (approximately 0.5 mg Cu/kg diet) or adequate (approximately 6 mg Cu/kg diet) in Cu for 5 wk. IGF-I was measured in serum and hearts by an ELISA method, cardiac IGF-I and IGF-II receptors and IGFBP-3 were measured by Western blotting analysis, and mRNAs for cardiac IGF-I and IGF-II were measured by RT-PCR. Contractility of isolated cardiomyocytes was assessed by a video-based edge-detection system. Cu deficiency depressed serum and heart IGF-I and heart IGFBP-3 protein levels and increased cardiac IGF-I receptor protein. Cardiac IGF-II protein and mRNA for cardiac IGF-I and IGF-II were unaffected by Cu deficiency. A Cu deficiency-induced increase in cardiomyocyte contractility, as indicated by increases in maximal velocities of shortening (-dL/dt) and relengthening (+dL/dt) and decrease in time to peak shortening (TPS), was confirmed. These changes were largely inhibited by use of H-1356, an IGF-I receptor blocker. We conclude that enhanced sensitivity to IGF-I, as indicated by an increase in IGF-I receptor protein, accounts for the increased contractility of Cu-deficient cardiomyocytes and may presage cardiac failure.

Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification

Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24-26-month) mice using a MyoCam system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, +/- dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was approximately 2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4-7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs.

Inhibition of Sarco(endo)plasmic Reticulum Ca<SUP>2+</SUP>-ATPase Differentially Regulates Contractile Function in Cardiac Myocytes From Normotensive and Spontaneously Hypertensive Rats: Role of Ca<SUP>2+</SUP> Regulatory Proteins

Hypertension leads to impaired contractile function. This study examined the impact of inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin or cyclopiazonic acid (CPA) on cardiac contractile function in ventricular myocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Mechanical properties were examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ transients were evaluated as fura-2 fluorescent intensity (FFI), excitation-induced change in FFI (DeltaFFI = peak-basal), and fluorescence decay rate (tau). Expression of Ca2+ regulatory proteins SERCA2a, Na+-Ca2+ exchanger (NCX), and phospholamban (PLB) were assessed by reverse transcriptase polymerase chain reaction and Western blot. SHR rats exhibited elevated blood pressure. SHR myocytes displayed decreased PS +/- dL/dt, peak FFI, and DeltaFFI; shortened TPS; prolonged tau with normal TR90; and basal FFI compared with WKY myocytes. Inhibition of SERCA with thapsigargin (5 microM) or CPA (10 microM) significantly depressed PS +/- dL/dt, baseline FFI, and DeltaFFI, and prolonged TPS, TR90, and tau in WKY myocytes. However, SHR myocytes were relatively insensitive to thapsigargin or CPA with only TPS and TR90 prolonged. Both mRNA and protein expressions of NCX and PLB were significantly enhanced, whereas SERCA2a protein abundance was reduced in SHR rats compared with the WKY group. Our data suggest that inhibition of SERCA function differentially affected cardiac contractile function in ventricular myocytes from normotensive and hypertensive rats possibly through reduced SERCA2a, elevated PLB, and NCX expression under hypertension.

Doxorubicin induces cardiomyocyte dysfunction via a p38 MAP kinase-dependent oxidative stress mechanism

Doxorubicin, an anthracycline used for cancer therapy, is known to elicit an irreversible cardiotoxicity. Several mechanisms were postulated for its cardiac toxicity including generation of reactive oxygen species (ROS). This study was designed to determine the acute effect of doxorubicin on cardiac mechanical and intracellular Ca 2+ properties in isolated ventricular myocytes. Contractile properties of male adult rat ventricular myocytes were analyzed including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90) and maximal velocity of shortening/relengthening (±d L/d t). Intracellular Ca 2+ transients and generation of ROS were measured with fura-2 and fluoroprobe 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, respectively. Acute (5 min) incubation of myocytes with doxorubicin (10 −9–10 −4 M) significantly prolonged TPS, TR 90 and intracellular Ca 2+ transient decay rate without affecting PS, ±d L/d t, resting intracellular Ca 2+ levels and electrically triggered intracellular Ca 2+ rise. Interestingly, the doxorubicin-induced prolongation of TPS and TR 90 was ablated by treatment of the antioxidant Vitamin C (100 μM) or the p38 MAP kinase inhibitor SB203580 (10 μM). Both Vitamin C and SB203580 unmasked a doxorubicin-induced positive response in PS. Vitamin C itself enhanced basal ±d L/d t, whereas, SB203580 unmasked a doxorubicin-induced positive response of ±d L/d t. The doxorubicin-induced response of intracellular Ca 2+ transients was essentially unaffected by Vitamin C. The role of ROS in doxorubicin-induced cardiac contractile response was confirmed with the ability of doxorubicin to enhance ROS generation, which was prevented by Vitamin C and SB203580. These data provide evidence that doxorubicin impairs cardiac contractile property in single myocytes through an oxidative stress-mediated pathway.

Korean anti-inflammatory compound allergina enhances cardiac contractile function in isolated ventricular cardiomyocytes.

The herbal compound Allergina has a long history in the clinical treatment of allergic inflammatory diseases in some countries including Korea. However, the direct effect of Allergina on ventricular contractile function has not been defined. This study was designed to investigate the impact of Allergina on ventricular contractile function. Ventricular contractile function was examined in single isolated left ventricular cardiomyocytes. Isolated cardiomyocytes from adult male Sprague-Dawley rats were electrically stimulated to contract at 0.5 Hz. Mechanical and intracellular Ca2+ transients properties of cardiomyocytes were evaluated using an IonOptix Myocam (IonOptix Inc., Milton, MA) system and fura-2 fluorescent dye. Contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (+/- dL/dt). Intracellular Ca2+ transients were evaluated as the fura-fluorescence intensity change (DeltaFFI) and fluorescence decay time. Allergina (10(-7) - 10(-3) mg/mL) significantly augmented PS in a dose-dependent manner with a maximal response of 50.4%. Allergina did not elicit any effect on TPS, TR(90), and +/-dL/dt. Intracellular Ca2+ transients displayed consistent findings in that Allergina enhanced the electrically-stimulated increase in change of intracellular Ca2+ transients (DeltaFFI) and slowed intracellular Ca2+ fluorescence decay without affect the resting intracellular Ca2+ levels. Collectively, these data demonstrated a direct cardiac stimulatory property of Allergina on cardiac contraction and intracellular Ca2+ transients in isolated left ventricular cardiomyocytes.

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90 ), and maximal velocities of shortening/relengthening (± d L /d t ). Intracellular Ca 2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10 −9 –10 −5 M) depressed PS, prolonged TR 90 , and decreased electrically stimulated intracellular Ca 2+ rise in a concentration-dependent manner. TPS, ± d L /d t , resting intracellular Ca 2+ level, and intracellular Ca 2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2 ′ ,7 ′ -Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), and maximal velocities of shortening/relengthening (± d L/d t). Intracellular Ca 2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10 −9–10 −5 M) depressed PS, prolonged TR 90, and decreased electrically stimulated intracellular Ca 2+ rise in a concentration-dependent manner. TPS, ± d L/d t, resting intracellular Ca 2+ level, and intracellular Ca 2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2 ′,7 ′-Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.

Phytoestrogenic Isoflavones Daidzein and Genistein Reduce Glucose‐Toxicity‐Induced Cardiac Contractile Dysfunction in Ventricular Myocytes

Epidemiological evidence suggests a reduction in the incidence of coronary heart disease, cancer and osteoporosis in populations with a high dietary intake of plant estrogen or phytoestrogen. The clinical benefit of phytoestrogens in cereals, vegetables and medicinal plants is attracting increasing attention for the general public. In the present study, we examined the effect of phytoestrogenic isoflavones daidzein and genistein on glucose toxicity‐induced cardiac mechanical malfunction simulating diabetic cardiomyopathy. Adult rat ventricular myocytes were isolated and maintained for 24 hours in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) medium in the absence or presence of isoflavones daidzein (50 µM) or genistein (20 µM). Cardiac contractile indices were evaluated using an IonOptix® MyoCam system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time‐to‐PS (TPS) and time‐to‐90% relengthening (TR90). Myocytes maintained in HG medium displayed altered mechanical function simulating in vivo diabetes including reduced PS, ± dL/dt and prolonged TR90 associated with normal TPS compared to those from NG myocytes. Interestingly, these HG‐induced mechanical dysfunctions were abolished by co‐incubation of daidzein or genistein. However, daidzein but not genistein itself depressed PS in NG myocytes. Neither daidzein nor genistein affected any other mechanical parameters tested in NG myocytes. Collectively, these data suggest that the phytoestrogenic isoflavones daidzein and genistein may reduce glucose toxicity‐induced cardiac mechanical dysfunction and thus possess therapeutic potential against diabetes‐associated cardiac defects.

Transgenic overexpression of insulin-like growth factor I prevents streptozotocin-induced cardiac contractile dysfunction and beta-adrenergic response in ventricular myocytes

Diabetic cardiomyopathy is characterized by cardiac dysfunction and altered level/function of insulin-like growth factor I (IGF-I). Both endogenous and exogenous IGF-I have been shown to effectively alleviate diabetes-induced cardiac dysfunction and oxidative stress. This study was designed to examine the effect of cardiac overexpression of IGF-I on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse myocytes. Both IGF-I heterozygous transgenic mice and their wild-type FVB littermates were made diabetic with a single injection of STZ (200 mg/kg, i.p.) and maintained for 2 weeks. The following mechanical indices were evaluated in ventricular myocytes: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Intracellular Ca2+ was evaluated as resting and peak intracellular Ca2+ levels, Ca2+-induced Ca2+ release and intracellular Ca2+ decay rate (tau). STZ led to hyperglycemia in FVB and IGF-I mice. STZ treatment prolonged TPS and TR90, reduced Ca2+-induced Ca2+ release, increased resting intracellular Ca2+ levels and slowed tau associated with normal PS and +/- dL/dt. All of which, except the elevated resting intracellular Ca2+, were prevented by the IGF-I transgene. In addition, myocytes from STZ-treated FVB mice displayed an attenuated contractile response to the beta-adrenergic agonist isoproterenol, which was restored by the IGF-I transgene. Contractile response to the alpha-adrenergic agonist phenylephrine and angiotensin II was not affected by either STZ treatment or IGF-I. These results validate the beneficial role of IGF-I in diabetic cardiomyopathy, possibly due to an improved beta-adrenergic response.

The Oxygen Radical Generator Pyrogallol Impairs Cardiomyocyte Contractile Function Via a Superoxide and p38 MAP Kinase-Dependent Pathway: Protection by Anisodamine and Tetramethylpyrazine

Oxygen-derived free radicals have been demonstrated to contribute to the pathogenesis of myocardial dysfunction, although the underlying mechanism remains not fully understood. This study was designed to examine the role of the superoxide generator pyrogallol on cardiac contractile function and possible intervention with herbal medicines anisodamine and tetramethylpyrazine (TMP) on pyrogallol-induced cardiac contractile response. Adult rat ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an IonOptix system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), and maximal velocity of shortening/relengthening (+/-dL/dt). A 10-min exposure of pyrogallol (0 to 10(-2) M) did not affect cardiac contractile mechanics. However, longer duration of pyrogallol exposure (1, 3, and 6 h) significantly shortened resting cell length, reduced PS and +/-dL/dt, and prolonged TPS and TR90 in time- and concentration-dependent manners. The pyrogallol (10(-4) M with 6-h incubation)-induced mechanical defects were prevented by the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (1 microM) and superoxide dismutase (SOD, 500 U/mL) with the exception that pyrogallol-induced PS depression was unaffected by SOD. Interestingly, incubation of herbal antioxidants anisodamine (10(-7) M) and TMP (10(-7) M) effectively attenuated the pyrogallol-induced cardiac mechanical defects with the exception of PS unaffected by TMP. Our data demonstrate a direct inhibitory effect of pyrogallol on cardiac contraction, probably in a superoxide- and p38 MAP kinase-dependent manner. The antioxidant medicines anisodamine and TMP may be useful in the treatment of oxygen free radical-induced myocardial dysfunction.

Tetramethylpyrazine elicits disparate responses in cardiac contraction and intracellular Ca 2+ transients in isolated adult rat ventricular myocytes

Tetramethylpyrazine (TMP) is the biologically active ingredient isolated from a popular Chinese medicinal plant, Ligusticum wallichil franchat, which has been used effectively since the 1970s to treat ischemic heart disease, cerebrovascular and thrombotic vascular diseases. The direct action of TMP on cardiac contractile function, however, is largely unclear. This study was designed to examine the effect of TMP on ventricular contractile function at the single cardiac myocyte level. Adult rat ventricular myocytes were isolated and stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca 2+ properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR 90), maximal velocity of shortening/relengthening (±dl/dt), resting intracellular Ca 2+ level, Ca 2+-induced Ca 2+ release (CICR) and decay. TMP (10 −10–10 −5 M) exhibited an increase in PS with a maximal increase of 30.9%. TMP had no effect on ±dl/dt, TPS/TR 90 or CICR but lowered resting intracellular Ca 2+ level and slowed intracellular Ca 2+ decay. Pretreatment with either the nonspecific nitric oxide synthase (NOS) inhibitor Nω-nitro- l-arginine methyl ester ( l-NAME, 100 μM) or inducible NOS inhibitor W1400 effectively abolished the positive effect of TMP on myocyte shortening. Our data demonstrate a direct positive inotropic effect of TMP in cardiac myocytes, which may be related, at least in part, to NO production.

The cyclooxygenase-2 product prostaglandin E 2 modulates cardiac contractile function in adult rat ventricular cardiomyocytes

Prostaglandin E 2 (PGE 2), a product of the cyclooxygenase-2 pathway, has been shown to increase cardiac output and modulate cardiac contractile function. However, whether the cardiac contractile response of PGE 2 is due to its action on single ventricular myocytes has not been elucidated. To assess the mechanical effect of PGE 2 at the cellular level, adult rat ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical and intracellular Ca 2+ properties were evaluated using an IonOptix Myocam ® analog-to-digital optical detection system. Contractile and intracellular Ca 2+ properties were evaluated as peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), maximal velocity of shortening or relengthening (±d L/d t) and Ca 2+-induced intracellular Ca 2+ fluorescence release (CICR), baseline intracellular Ca 2+ levels and intracellular Ca 2+ decay rate ( τ). PGE 2 (10 −8 to 10 −3 M) elicited an augmentation in PS but had no effect on TPS, TR 90, ±d L/d t, CICR and τ. High concentration of PGE 2 (10 −5 M or higher) reduced the baseline intracellular Ca 2+ levels. These data indicate that the myocardial contractile response of PGE 2 may be due to its direct cardiac contractile action at the single ventricular myocyte level, probably through a mechanism independent of intracellular Ca 2+ release.

Insulin resistance induces hyperleptinemia, cardiac contractile dysfunction but not cardiac leptin resistance in ventricular myocytes.

Insulin resistance is a metabolic syndrome commonly seen in obesity. Leptin, the obese gene product, plays a role in the regulation of cardiac function. Elevated leptin levels have been demonstrated under insulin-resistant states such as obesity and hypertension, although their role in cardiac dysfunction is unknown. This study was designed to determine the impact of prediabetic insulin resistance on leptin levels and leptin-induced cardiac contractile response. Whole-body insulin resistance was generated with a 10-week dietary sucrose feeding. Contractile and intracellular Ca(2+) properties were evaluated in ventricular myocytes using an IonOptix system. The contractile indices analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt), fura-fluorescence intensity change (deltaFFI) and decay rate (tau). Sucrose-fed rats displayed significantly elevated body weight and plasma leptin levels, depressed PS, +/-dL/dt, shortened TPS, prolonged TR(90) and tau, as well as reduced deltaFFI compared to the starch-fed control group. Leptin (1-1000 nM) elicited a concentration-dependent depression of PS and deltaFFI in myocytes from both starch and sucrose groups. Leptin-induced contractile depression was abolished by the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyle ester, elevation of the extracellular Ca(2+) concentration, the Janus activated kinase 2 inhibitor AG-490 or the mitogen activated protein kinase inhibitor SB203580 in myocytes from both sucrose and starch groups. Moreover, AG-490 and SB203580 unmasked a positive response of PS in myocytes from both groups. These data indicate that insulin resistance directly induces hyperleptinemia and cardiac contractile dysfunction, without affecting leptin-mediated cardiac contractile function at the myocyte level.

Malondialdehyde inhibits cardiac contractile function in ventricular myocytes via a p38 mitogen-activated protein kinase-dependent mechanism.

(1) Increased oxidative stress plays a significant role in the etiology of cardiovascular disease. Lipid peroxidation, initiated in the presence of hydroxy radicals resulting in the production of malondialdehyde, directly produces oxidative stress. This study was designed to examine the direct impact of malondialdehyde on ventricular contractile function at the single cardiac myocyte level. Ventricular myocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening amplitude (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dLdt), and Ca(2+)-induced intracellular Ca(2+) fluorescence release (CICR) and intracellular Ca(2+) decay (tau). p38 mitogen-activated protein (MAP) kinase phosphorylation was assessed with Western blot. (2) Our results indicated that malondialdehyde directly depressed PS, +/-dLdt and CICR in a concentration-dependent manner and shortened TPS without affecting TR(90) and tau. Interestingly, the malondialdehyde-induced cardiac mechanical effect was abolished by both the p38 MAP kinase inhibitor SB203580 (1 and 10 micro M) and the antioxidant vitamin C (100 micro M). Western blot analysis confirmed direct phosphorylation of p38 MAP kinase by malondialdehyde. (3) These findings revealed a novel role of malondialdehyde and p38 MAP kinase in lipid peroxidation and oxidative stress-associated cardiac dysfunction.

AT1 blockade prevents glucose-induced cardiac dysfunction in ventricular myocytes: role of the AT1 receptor and NADPH oxidase.

Enhanced tissue angiotensin (Ang) II levels have been reported in diabetes and might lead to cardiac dysfunction through oxidative stress. This study examined the effect of blocking the Ang II type 1 (AT1) receptor on high glucose-induced cardiac contractile dysfunction. Rat ventricular myocytes were maintained in normal- (NG, 5.5 mmol/L) or high- (HG, 25.5 mmol/L) glucose medium for 24 hours. Mechanical and intracellular Ca2+ properties were assessed as peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), and intracellular Ca2+ decay (tau). HG myocytes exhibited normal PS; decreased +/-dL/dt; and prolonged TPS, TR90, and tau. Interestingly, the HG-induced abnormalities were prevented with the AT1 blocker L-158,809 (10 to 1000 nmol/L) but not the Janus kinase-2 (JAK2) inhibitor AG-490 (10 to 100 micromol/L). The only effect of AT1 blockade on NG myocytes was enhanced PS at 1000 nmol/L. AT1 antagonist-elicited cardiac protection against HG was nullified by the NADPH oxidase activator sodium dodecyl sulfate (80 micromol/L) and mimicked by the NADPH oxidase inhibitors diphenyleneiodonium (10 micromol/L) or apocynin (100 micromol/L). Western blot analysis confirmed that the protein abundance of NADPH oxidase subunit p47phox and the AT1 but not the AT2 receptor was enhanced in HG myocytes. In addition, the HG-induced increase of p47phox was prevented by L-158,809. Enhanced reactive oxygen species production observed in HG myocytes was prevented by AT1 blockade or NADPH oxidase inhibition. Collectively, our data suggest that local Ang II, acting via AT1 receptor-mediated NADPH oxidase activation, is involved in hyperglycemia-induced cardiomyocyte dysfunction, which might play a role in diabetic cardiomyopathy.