RAGE upregulation and nuclear factor-kappaB activation associated with ageing rat cardiomyocyte dysfunction

Evidence suggests that ageing is a major risk factor for cardiac dysfunction. Interactions between advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE) are known to cause chronic cellular activation, including activation of nuclear factor-kappaB (NF-kappaB), which has been implicated as a causal factor in the ageing process. To assess whether cardiomyocyte contractile function and the interaction of AGEs with RAGE in the heart are altered in ageing, 25- and 2-month-old male rats were compared. Mechanical properties were assessed in ventricular myocytes using an edge-detection system, including peak twitch amplitude (PTA), time-to-PTA (TPS), time-to-75% relengthening (TR75) and maximal velocity of shortening/relengthening (+/-dL/dt) in ventricular myocytes. AGEs were detected by using a fluorescence assay. The expression of RAGE and NF-kappaB was assessed through a Western blot analysis. Compared with young myocytes, aged myocytes displayed a prolonged TR75 at 1 Hz. With increasing stimulus frequency (from 2 to 4 Hz), aged myocytes' PTA was significantly reduced relative to young myocytes. Aged rat hearts displayed high level of AGEs, RAGE upregulation and NF-kappaB activation. These findings demonstrate impaired cardiomyocyte relaxation and reduced tolerance to increased stimulus frequency in aged rats, which might be associated with enhanced AGEs, RAGE expression, and NF-kappaB activation.     

Metallothionein antagonizes aging-induced cardiac contractile dysfunction: role of PTP1B, insulin receptor tyrosine phosphorylation and Akt

Aging is often accompanied by reduced insulin sensitivity and cardiac dysfunction. However, the causal relationship between the two remains poorly understood. This study was designed to determine the impact of cardiac-specific overexpression of antioxidant metallothionein (MT) on aging-associated cardiac dysfunction and impaired insulin signaling. Contractile and intracellular Ca(2+) properties were evaluated in left ventricular myocytes including peak shortening (PS), maximal velocity of shortening/relengthening (+/- dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), fura-2 fluorescence intensity change (DeltaFFI) and intracellular Ca(2+) decay rate. Expression of insulin receptor, protein-tyrosine phosphatase 1B (PTP1B), phosphorylation of insulin receptor (Tyr1146) and Akt were evaluated by Western blot analysis. Aged wild-type FVB and MT transgenic mice (26-28 months old) displayed glucose intolerance and hyperinsulinemia. Cardiomyocytes from aged FVB mice exhibited prolonged TR(90) and intracellular Ca(2+) decay associated with normal PS, +/- dL/dt, TPS and DeltaFFI compared with those from young (2-3 months old) mice. Western blot analysis revealed reduced Akt expression and insulin (5 mU g(-1))-stimulated Akt phosphorylation, elevated PTP1B expression and diminished basal insulin receptor tyrosine phosphorylation associated with comparable insulin receptor expression in aged FVB mouse hearts. All of these aging-related defects in cardiac contractile function and insulin signaling (although not hyperinsulinemia and glucose intolerance) were significantly attenuated or ablated by MT transgene. These data indicate that enhanced antioxidant defense is beneficial for aging-induced cardiac contractile dysfunction and alteration in insulin signaling.     

Influence of ovariectomy on ventricular myocyte contraction in simulated diabetes

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a 'diabetes-simulated high-glucose' environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR(90), slower +/- dL/dt, lower fluorescence intensity and higher tau (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR90, reduced fluorescence intensity, higher tau and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR90 and tau), reversed (PS and NOS) or lost (+/- dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.     

Cardiac-specific overexpression of insulin-like growth factor 1 attenuates aging-associated cardiac diastolic contractile dysfunction and protein damage

Aging is associated with hepatic growth hormone resistance resulting in a fall in serum insulin-like growth factor 1 (IGF-1) level. However, whether loss of IGF-1 contributes to cardiac aging is unclear. This study was designed to examine the effect of cardiac overexpression of IGF-1 on cardiomyocyte contractile function in young (3 mo) and old (26-28 mo) mice. Cardiomyocyte contractile function was evaluated, including peak shortening (PS), time to 90% PS, time to 90% relengthening (TR(90)), and maximal velocity of shortening/relengthening (+/-dL/dt). Levels of advanced glycation end product, protein carbonyl, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban, and Na(+)/Ca(2+) exchanger were assessed by Western blot analysis. SERCA activity was measured by (45)Ca(2+) uptake. Aging induced a decline in plasma IGF-1 levels. Aged cells exhibited depressed +/-dL/dt, prolonged TR(90), and a steeper PS decline in response to increasing stimulus frequency compared with those in young myocytes. IGF-1 transgene alleviated aging-induced loss in plasma IGF-1 and aging-induced mechanical defects with little effect in young mice. The beneficial effect of IGF-1 transgene on aging-associated cardiomyocyte contractile dysfunction was somewhat mimicked by short-term in vitro treatment of recombinant IGF-1 (500 nM). Advanced glycation end product and protein carbonyl levels were higher in aged mice, which were not affected by IGF-1. Expression of SERCA2a (but not Na(+)/Ca(2+) exchanger and phospholamban) and SERCA activity were reduced with aging, which was ablated by the IGF-1 transgene. Collectively, our data suggest a beneficial role of IGF-1 in aging-induced cardiac contractile dysfunction, possibly related to improved Ca(2+) uptake.     

Comparison of cardiac excitation-contraction coupling in isolated ventricular myocytes between rat and mouse

Transgenic animals offer many advantages for physiological study. The mouse is the most extensively utilized mammalian model for gene modification. Isolated ventricular myocytes are pivotal for assessment of cardiac function by allowing direct cellular and environmental manipulation without interference from compensatory mechanisms that may exist in vivo. This study was designed to compare the basic excitation-contraction coupling properties of mouse and rat ventricular myocytes. Cardiac myocytes were isolated from age- and gender-matched mice (FVB and C57BL/6) and rats (Sprague-Dawley (SD) and Wistar). Mechanical and intracellular Ca2+ properties were measured with an IonOptix SoftEdge system, including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening and relengthening (+/-dL/dt), and intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Resting cell length was variable among the different species or strains. PS from FVB group was significantly higher than the SD group. TPS and TR(90) were significantly shorter in mice. +dL/dt was similar among all groups whereas -dL/dt was significantly faster in the C57BL/6 group compared to the rat groups. Resting intracellular Ca2+ was lower in mice than in rats, and Ca2+-induced Ca2+ release was variable among the four groups. Intracellular Ca2+ decay was slower in Wistar compared to all other groups. The myocytes from C57BL/6 did not respond to increases in extracellular Ca2+. Myocytes from the FVB group exhibited a lesser reduction in PS in response to elevated stimulus frequency. These data suggest that inherent differences between strains or species should be taken into consideration when comparing results from these different animal models.     

Characterization of cardiomyocyte excitation-contraction coupling in the FVB/N-C57BL/6 intercrossed "chocolate" brown mice

Mice are extensively used for gene modification research and isolated cardiomyocytes are essential for evaluation of cardiac function without interference from non-myocyte contribution. This study was designed to characterize cardiomyocyte excitation-contraction coupling in FVB/N-C57BL/6 intercrossed brown mice. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix softedge system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca(2+) rise and decay rate. Resting cell length was longer in age- and gender-matched C57BL/6 and brown mice compared to FVB strain. PS and +/- dL/dt were significantly lower in brown mice compared to FVB/N and C57BL/6 groups. TPS was shortened in C57BL/6 mice and TR(90) was prolonged in brown mice compared to other groups. Resting intracellular Ca(2+) level and single exponential intracellular Ca(2+) decay constant were comparable among all three mouse lines. Rise in intracellular Ca(2+) in response to electrical stimulus was higher in C57BL/6 mouse myocytes whereas bi-exponential intracellular Ca(2+) decay was faster in brown mice. Myocytes from all three groups exhibited similar fashion of reduction in PS in response to increased stimulus frequency. These data suggest that inherent differences in cardiomyocyte excitation-contraction coupling exist between strains, which may warrant caution when comparing data from these mouse lines.     

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.     

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 (TR90), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10(-9)-10(-5) M) depressed PS, prolonged TR90, and decreased electrically stimulated intracellular Ca2+ rise in a concentration-dependent manner. TPS,+/-dL/dt, resting intracellular Ca2+ level, and intracellular Ca2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 microM) and the p38 MAP kinase inhibitor SB203580 (1 microM). 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.     

Basal and ethanol-induced cardiac contractile response in lean and obese zucker rat hearts

Obesity plays a pivotal role in metabolic and cardiovascular diseases. Certain types of obesity may be related to alcohol ingestion, which itself leads to impaired cardiac function. This study analyzed basal and ethanol-induced cardiac contractile response using left-ventricular papillary muscles and myocytes from lean and obese Zucker rats. Contractile properties analyzed include: peak tension development (PTD), peak shortening amplitude (PS), time to PTD/PS (TPT/TPS), time to 90% relaxation/relengthening (RT(90)/TR(90)) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT and +/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes and fluorescence decay time (FDT). In papillary muscles from obese rats, the baseline TPT and RT(90) were significantly prolonged accompanied with low to normal PTD and +/-VT compared to those in lean rats. Muscles from obese hearts also exhibited reduced responsiveness to postrest potentiation, increase in extracellular Ca(2+) concentration, and norepinephrine. By contrast, in isolated myocytes, obesity reduced PS associated with a significant prolonged TR(90), normal TPS and +/-dL/dt. Intracellular Ca(2+) recording revealed decreased resting Ca(2+) levels and prolonged FDT. Acute ethanol exposure (80-640 mg/dl) caused comparable concentration-dependent inhibitions of PTD/PS and DeltaFFI, associated with reduced +/-VT in both groups. Collectively, these results suggest altered cardiac contractile function and unchanged ethanol-induced depression in obesity.     

Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA

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(90)) and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) was evaluated as Ca(2+)-induced Ca(2+) release [difference in fura 2 fluorescent intensity (Delta FFI)] and fluorescence decay rate (tau). Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a, phospholamban (PLB), Na(+)-Ca(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, +/-dL/dt, and Delta FFI associated with prolonged TPS, TR(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.     

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 C2-dihydroceramide (25 microM). Contractile and intracellular Ca2+ 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), intracellular Ca2+ rise (Delta[Ca2+]) and intracellular Ca2+ decay. While ceramide did not elicit any effect on cell mechanics and intracellular Ca2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, +/-dL/dt, Delta[Ca2+] and prolonged TR90. With ceramide co-incubation, 5 nM leptin depressed PS, +/-dL/dt, Delta[Ca2+] and prolonged TR90 whereas 50 nM leptin-elicited effects on PS, +/-dL/dt, Delta[Ca2+] and TR90 were significantly potentiated in addition to slowing intracellular Ca2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction.     

Impact of estrogen replacement on ventricular myocyte contractile function and protein kinase B/Akt activation

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(2)) replacement (40 microg. kg(-1). day(-1)) for 8 weeks. Mechanical and intracellular Ca(2+) properties were evaluated including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt), fura 2 fluorescence intensity (FFI), and decay rate. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), and Akt were assessed by Western blot. Ovx promoted body weight gain associated with reduced serum E(2) and uterine weight, all of which were abolished by E(2). Ovx depressed PS and +/-dL/dt, prolonged TPS, TR(90), and decay rate, and enhanced resting FFI, all of which, with the exception of TPS, were restored by E(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(2). E(2) enhanced PS and +dL/dt in vitro, which was abolished by the E(2) receptor antagonist ICI-182780. Ovx reduced myocyte Ca(2+) responsiveness and lessened stimulating frequency-induced decline in PS, both ablated by E(2). These data suggest that mechanical and protein functions of ventricular myocytes are directly regulated by E(2).     

Particulate matter exposure exacerbates high glucose-induced cardiomyocyte dysfunction through ROS generation

Diabetes mellitus and fine particulate matter from diesel exhaust (DEP) are both important contributors to the development of cardiovascular disease (CVD). Diabetes mellitus is a progressive disease with a high mortality rate in patients suffering from CVD, resulting in diabetic cardiomyopathy. Elevated DEP levels in the air are attributed to the development of various CVDs, presumably since fine DEP (<2.5 μm in diameter) can be inhaled and gain access to the circulatory system. However, mechanisms defining how DEP affects diabetic or control cardiomyocyte function remain poorly understood. The purpose of the present study was to evaluate cardiomyocyte function and reactive oxygen species (ROS) generation in isolated rat ventricular myocytes exposed overnight to fine DEP (0.1 μg/ml), and/or high glucose (HG, 25.5 mM). Our hypothesis was that DEP exposure exacerbates contractile dysfunction via ROS generation in cardiomyocytes exposed to HG. Ventricular myocytes were isolated from male adult Sprague-Dawley rats cultured overnight and sarcomeric contractile properties were evaluated, including: peak shortening normalized to baseline (PS), time-to-90% shortening (TPS(90)), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (±dL/dt), using an IonOptix field-stimulator system. ROS generation was determined using hydroethidine/ethidium confocal microscopy. We found that DEP exposure significantly increased TR(90), decreased PS and ±dL/dt, and enhanced intracellular ROS generation in myocytes exposed to HG. Further studies indicated that co-culture with antioxidants (0.25 mM Tiron and 0.5 mM N-Acetyl-L-cysteine) completely restored contractile function in DEP, HG and HG+DEP-treated myocytes. ROS generation was blocked in HG-treated cells with mitochondrial inhibition, while ROS generation was blocked in DEP-treated cells with NADPH oxidase inhibition. Our results suggest that DEP exacerbates myocardial dysfunction in isolated cardiomyocytes exposed to HG-containing media, which is potentially mediated by various ROS generation pathways.     

Altered cardiac excitation-contraction coupling in ventricular myocytes from spontaneously diabetic BB rats

Cardiac excitation-contraction (E-C) coupling abnormalities in chemically induced diabetes have been well defined. Heart dysfunction has also been reported in diabetes of genetic origin. The purpose of this study was to determine whether heart dysfunction in genetically predisposed diabetes is attributable to impaired E-C coupling at the cellular level. Myocytes were isolated from 1-yr-old BioBreed (BB) spontaneously diabetic-prone (BB/DP) rats and their diabetic-resistant littermates (BB/DR). Mechanical properties were evaluated by use of a video edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), and maximal velocities of shortening and relengthening (+/-dL/dt). Intracellular Ca(2+) handling was evaluated with fura 2 fluorescent dye. Myocytes from spontaneously diabetic hearts exhibited a depressed PS, prolonged TPS and TR(90), and reduced +/-dL/dt. Consistent with the mechanical response, myocytes from the BB/DP group displayed reduced resting and peak intracellular Ca(2+) concentration associated with a slowed Ca(2+)-transient decay. Furthermore, myocytes from BB/DP hearts were less responsive to increases in extracellular Ca(2+) and norepinephrine and equally responsive to increases in stimulation frequency and KCl compared with those from the BB/DR group. These results suggest that the genetic diabetic state produces altered cardiac E-C coupling, in part, because of abnormalities of the myocyte, similar to that demonstrable after chemically induced diabetes or during human diabetes.     

Metallothionein alleviates cardiac dysfunction in streptozotocin-induced diabetes: role of Ca2+ 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 beta-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.     

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

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

Hypertension augments ethanol-induced depression of cell shortening and intracellular Ca(2+) transients in adult rat ventricular myocytes.

Ethanol, a risk factor for myocardial dysfunction, depresses myocardial contraction. This study was to determine whether ethanol-induced myocardial depression is affected by hypertension. Mechanical properties of ventricular myocytes isolated from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were evaluated using a video edge-detection system. Myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include peak twitch amplitude (PTA), time-to-PTA (TPS), time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes. Acute ethanol exposure (80-640 mg/dl) caused a concentration-dependent inhibition of PTA and DeltaFFI in both WKY and SHR myocytes. The extent of maximal inhibition of PTA and FFI was significantly greater in SHRs (53.7 and 38.9%) compared to the WKY group (21.0 and 25.4%). Ethanol did not affect TPS but shortened TR(90) and slowed +/-dL/dt at high concentration ranges. Interestingly, the augmented ethanol-induced inhibition of cell shortening in hypertension was greatly attenuated by Ca(2+) channel opener BayK 8644 (1 microM). These results suggest that ethanol-induced myocardial depression may be augmented in hypertension, possibly due to mechanism(s) involving sarcolemmal Ca(2+) channels.     

High-fat diet-induced obesity leads to resistance to leptin-induced cardiomyocyte contractile response

Levels of the obese gene product leptin are often elevated in obesity and may contribute to obesity-induced cardiovascular complications. However, the role of leptin in obesity-associated cardiac abnormalities has not been clearly defined. This study was designed to determine the influence of high-fat diet-induced obesity on cardiac contractile response of leptin. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix system in cardiomyocytes from adult rats fed low- and high-fat diets for 12 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were examined including peak shortening, duration and maximal velocity of shortening/relengthening (TPS/TR(90), +/-dl/dt), Fura-2-fluorescence intensity change (DeltaFFI), and intracellular Ca(2+) decay rate (tau). Expression of the leptin receptor (Ob-R) was evaluated by western blot analysis. High-fat diet increased systolic blood pressure and plasma leptin levels. PS and +/-dl/dt were depressed whereas TPS and TR(90) were prolonged after high-fat diet feeding. Leptin elicited a concentration-dependent (0-1,000 nmol/l) inhibition of PS, +/-dl/dt, and DeltaFFI in low-fat but not high-fat diet-fed rat cardiomyocytes without affecting TPS and TR(90). The Janus kinase 2 (JAK2) inhibitor AG490, the mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the nitric oxide synthase (NOS) inhibitor L-NAME abrogated leptin-induced cardiomyocyte contractile response in low-fat diet group without affecting the high-fat diet group. High-fat diet significantly downregulated cardiac expression of Ob-R. Elevation of extracellular Ca(2+) concentration nullified obesity-induced cardiomyocyte mechanical dysfunction and leptin-induced depression in PS. These data indicate presence of cardiac leptin resistance in diet-induced obesity possibly associated with impaired leptin receptor signaling.