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.     

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.     

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.     

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.     

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 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.     

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.     

Leptin and Its Relation to Obesity and Insulin in the SHR/N-corpulent Rat,A Model of Type II Diabetes Mellitus

The spontaneously hypertensive/NIH-corpulent (SHR/N-cp) rat is a genetic animal model that exhibits obesity, metabolic features of hyperinsulinemia, hyperglycemia, and hyperlipidemia, which are characteristic of type II diabetes and mild hypertension. To determine the role of leptin, the protein product of the ob gene, in the development of obesity and diabetes in this model, we measured steady-state circulating levels of leptin in obese and lean SHR/N-cp rats and examined the relation between plasma leptin levels and metabolic variables at the stage of established obesity in these animals. Mean fasting plasma leptin concentration was 8-fold higher in obese than in lean rats (p<0.01). This was associated with a 6-fold elevation in plasma insulin in the obese group. Fasting levels of plasma glucose, cholesterol, and triglyceride were all significantly higher in obese rats than in lean controls. Spearman correlation analysis showed a significant positive correlation between plasma leptin concentration and body weight among the animals (r=0.73, p<0.01). Similarly, plasma insulin concentration was significantly correlated with BW in all animals (r=0.54, p<0.05). There was also a significant positive.correlation between plasma leptin and plasma insulin in the entire group (r=0.70, p<0.01). However, this relationship was significant only for lean rats but not for obese rats (r=0.59, p<0.05 for lean rats, and r=0.23, p=NS, for obese rats). Plasma leptin also correlated positively with fasting plasma glucose (r=0.75, p<0.05), total cholesterol (r=0.63, p<0.05), and triglyceride (r=0.67, p <0.05). The marked elevation of plasma leptin in obese SHR/N-cp rats suggests that obesity in this animal model is related to up-regulation of the ob gene. Circulating leptin appears to be one of the best biological markers of obesity and that hyperleptinemia is closely associated with several metabolic risk factors related to insulin resistance in the diabesity syndrome.     

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.     

Overexpression of alcohol dehydrogenase exacerbates ethanol-induced contractile defect in cardiac myocytes

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(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(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(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.     

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.     

Augmentation of the inotropic response to insulin in diabetic rat hearts.

Insulin participates in the modulation of myocardial function, but its inotropic action in diabetes mellitus is not fully clear. In the present study, we examined contractile responses to insulin in left-ventricular papillary muscles and ventricular myocytes isolated from hearts of normal or short-term (5-7 days) streptozotocin-induced (65 mg/kg) diabetic rats. Mechanical properties of papillary muscles and ventricular myocytes were evaluated using a force transducer and an edge-detector, respectively. Contractile properties of papillary muscles or cardiac myocytes, electrically stimulated at 0.5 Hz, were analyzed in terms of peak tension development (PTD) or peak twitch amplitude (PTA), time-to-peak contraction (TPT) and time-to-90% relaxation (RT90). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity change (deltaFFI). Insulin (1-500 nM) had no effect on PTD in normal myocardium, whereas it produced a positive inotropic response in preparations from diabetic animals, with a maximal increase of 11%. Insulin did not modify TPT or RT90 in either group. Further studies revealed that insulin enhanced cell shortening in diabetic but not normal myocytes, with a maximal increase of 21%. Consistent with its action on the mechanical properties of papillary muscles and cardiac myocytes, insulin also induced a dose-dependent increase in the intracellular Ca2+ transient in diabetic but not normal myocytes. Collectively, these data suggest that the myocardial contractile response to insulin may be altered in 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.     

Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats

Obesity, insulin resistance, dyslipidemia, and hypertension are components of the pathophysiological state known as metabolic syndrome. Adrenergic vasoconstriction is mediated through increases in cytosolic Ca2+ and the myofilaments' sensitivity to Ca2+. In many pathophysiological states, there is an enhanced role for Rho kinase (ROK)-mediated increases in Ca2+ sensitivity of the contractile apparatus. Thus we hypothesized that there is a greater role for ROK-mediated increases in Ca2+ sensitivity in alpha1-adrenergic vasoconstriction in arteries from obese Zucker (OZ) rats. Therefore, small gracilis muscle arteries from 11- to 12-wk-old and 16- to 18-wk-old lean and OZ rats were isolated, cannulated, and pressurized to 75 mmHg. For some experiments, vessels were loaded with fura 2-AM. Changes in luminal diameter and vessel wall Ca2+ concentration ([Ca2+]) were measured in response to phenylephrine (PE), the thromboxane mimetic U-46619, and KCl. alpha1-Adrenergic vasoconstriction was similar between 11- to 12-wk-old lean and obese animals and greater in older obese animals compared with controls. PE-induced increases in vascular smooth muscle cell [Ca2+] were blunted in OZ animals compared with lean controls in both age groups of animals. KCl and U-46619 elicited similar vasoconstriction and vascular smooth muscle cell [Ca2+] in both groups. ROK inhibition attenuated PE vasoconstriction to a greater degree in arteries from 11- to 12-wk-old OZ rats compared with lean animals; ROK inhibition in arteries from older rats right shifted both concentration-response curves to the same point. Total RhoA and ROKalpha protein expressions were similar between groups. These results suggest an enhanced role for the ROK pathway in alpha1-adrenergic vasoconstriction in metabolic syndrome.     

Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes.

Intermedin (IMD), also called adrenomedullin-2, is a 47-amino acid peptide from the calcitonin gene-related peptide (CGRP)/adrenomedullin family of peptides. Recent studies suggest that IMD may participate in the regulation of cardiovascular function and fluid and electrolyte homeostasis. To evaluate the role of IMD on cardiomyocyte contractile function, electrically paced murine ventricular myocytes were acutely exposed to IMD, and the following indexes were determined: peak shortening (PS), time to PS, time-to-90% relengthening, and maximal velocity of shortening and relengthening. Intracellular Ca(2+) was assessed using fura 2-AM fluorescent microscopy. Our results revealed that IMD (10 pM to 10 nM) significantly increased PS and maximal velocity of shortening and relengthening in ventricular myocytes, the maximal effect of which (approximately 46%) was somewhat comparable to those elicited by CGRP (1 nM) and adrenomedullin (100 nM). Exposure of IMD significantly shortened time-to-90% relengthening without affecting time to PS, similar to CGRP and adrenomedullin. IMD also enhanced intracellular Ca(2+) release, with a maximal increase of approximately 50%, and facilitated the intracellular Ca(2+) decay rate. The IMD-induced effects were abolished by the protein kinase C inhibitor chelerythrine (1 microM), downregulation of protein kinase C using phorbol 12-myristate 13-acetate (1 microM), and the protein kinase A inhibitor H89 (1 microM). Our data suggest that IMD acutely augments cardiomyocyte contractile function through, at least in part, a protein kinase C- and protein kinase A-dependent mechanism.     

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.     

Hepatic reduction of insulin-like growth factor (IGF)-I and IGF binding protein-3 that results from fasting is not attenuated in genetically obese rats

Fasting or caloric restriction causes substantial reductions in serum IGF-I in normal weight humans and animals, and reductions of liver IGF-I and IGFBP-3 mRNAs in animals. Obese humans, however, have attenuated and delayed decrements in IGF-I in serum when subjected to caloric restriction. Obese Zucker rats show a clear tendency to preserve body protein during fasting. To determine whether obesity opposes the effects of fasting on IGF-I and IGFBP-3, and thereby contributes to preservation of lean tissue, we have examined the effect of 72 h of fasting on IGF-I and IGFBP-3 in lean and obese Zucker rats. We observe that between lean and obese animals, fasting for 72 h produces similar decrements in body weight, serum IGF-I, liver IGF-I mRNA, serum IGFBP-3 and liver IGFBP-3 mRNA. Our finding that the reduction of IGF-I and IGFBP-3 in liver that results from 72 h of fasting is not attenuated in obese Zucker rats raises the possibility that conservation of lean tissue in these animals during fasting is not related to the hepatic production of IGF-I and IGFBP-3.     

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Chronic treatment with insulin-like growth factor I (IGF-I) improves contractile function in congestive heart failure and ischemic cardiomyopathy. The present study investigated the effect of chronic treatment with IGF-I on intrinsic myocyte function and the role of the phosphatidylinositol (PI)3-kinase-Akt-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a signaling cascade in these responses. Myocytes were isolated from 23 adult rats and cultured with and without IGF-I (10(-6) M). After 48 h of treatment, myocyte function was evaluated. IGF-I increased contractile function (percent contraction, 7.7 +/- 0.3% vs. 4.5 +/- 0.3%; P < 0.01) and accelerated relaxation time (time for 70% relengthening, 81 +/- 4 vs. 106 +/- 5 ms; P < 0.05) compared with untreated myocytes [control (Con)]. The enhanced function was associated with an increase in Ca(2+) transients assessed by fura-2 (340/380 nm; IGF-I, 0.42 +/- 0.02 vs. Con, 0.25 +/- 0.01; P < 0.01). The PI3-kinase inhibitor LY-249002 (10(-9) M) abolished the enhanced function caused by IGF-I. IGF-I increased both Akt and SERCA2a protein levels 2.5- and 4.8-fold, respectively, compared with those of Con (P < 0.01); neither phospholamban nor calsequestrin was affected. To evaluate whether the SERCA2a protein was directly mediated by Akt-SERCA2a signaling, IGF-I-induced changes in the SERCA2a protein were compared in myocytes transfected with adenovirus harboring either constitutively active Akt [multiplicity of infection (MOI), 15] or dominant negative Akt (dnAkt; MOI, 15). The ability of IGF-I to upregulate the SERCA2a protein in myocytes transfected with active Akt was absent in dnAkt myocytes. Taken together, our findings indicate that chronic treatment with IGF-I enhances intrinsic myocyte function and that this effect is due to an enhancement in intracellular Ca(2+) handling, secondary to the activation of the PI3-kinase-Akt-SERCA2a signaling cascade.