Ricardo A. Brown

Altered inotropic response to IGF-I in diabetic rat heart: influence of intracellular Ca2+ and NO.

Normally, insulin-like growth factor I (IGF-I) exerts positive effects on cardiac growth and myocardial contractility, but resistance to its action has been reported in diabetes. This study was designed to determine whether IGF-I-induced myocardial contractile action is altered in diabetes as a result of an intrinsic alteration of contractile properties at the cellular level. Contractile responses to IGF-I were examined in left ventricular papillary muscles and ventricular myocytes from normal and short-term (5-7 days) streptozotocin-induced diabetic rats. Mechanical properties of muscles and myocytes were evaluated using a force transducer and an edge detector, respectively. Preparations were electrically stimulated at 0.5 Hz, and contractile properties analyzed include peak tension development (PTD) or peak twitch amplitude (PTA), time to peak contraction/shortening, and time to 90% relaxation/relengthening. Intracellular Ca2+ transients were measured as fura 2 fluorescence intensity changes. IGF-I (1-500 ng/ml) caused a dose-dependent increase in PTD and PTA in preparations from normal but not diabetic animals. IGF-I did not alter time to peak contraction/shortening or time to 90% relaxation/relengthening. Pretreatment with the NO synthase inhibitor N ω-nitro-l-arginine methyl ester (100 μM) attenuated IGF-I-induced increases in PTD in normal myocardium but unmasked a positive inotropic action in diabetic animals. Pretreatment with N ω-nitro-l-arginine methyl ester blocked IGF-I-induced increases in PTA in single myocytes. Consistent with its inotropic actions on muscles and myocytes, IGF-I induced a dose-dependent increase in Ca2+transients in normal but not diabetic myocytes. These results suggest that the IGF-I-induced inotropic response is depressed in diabetes because of an intrinsic alteration at the myocyte level. Mechanisms underlying this alteration in IGF-I-induced myocardial response may be related to changes in intracellular Ca2+ and/or NO production in diabetes.

Influence of age on contractile response to insulin-like growth factor 1 in ventricular myocytes from spontaneously hypertensive rats.

Evidence suggests a pathophysiological role of insulin-like growth factor 1 (IGF-1) in hypertension. Cardiac function is altered with advanced age, similar to hypertension. Accordingly, the effects of IGF-1 on cardiac myocyte shortening and intracellular Ca(2+) were evaluated in hypertension at different ages. Ventricular myocytes were isolated from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), aged 12 and 36 weeks. Mechanical and intracellular Ca(2+) properties were examined by edge-detection and fluorescence microscopy. At 12 weeks, IGF-1 (1 to 500 ng/mL) increased peak twitch amplitude (PTA) and FFI changes (DeltaFFI) in a dose-dependent manner in WKY myocytes, with maximal increases of 27.5% and 35.2%, respectively. However, IGF-1 failed to exert any action on PTA and DeltaFFI in the age-matched SHR myocytes. Interestingly, at 36 weeks, IGF-1 failed to exert any response in WKY myocytes but depressed both PTA and DeltaFFI in a dose-dependent manner in SHR myocytes, with maximal inhibitions of 40.5% and 16.1%, respectively. Myocytes from SHR or 36-week WKY were less sensitive to norepinephrine (1 micromol/L) and KCl (30 mmol/L). Pretreatment with nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) did not alter the IGF-1-induced response in 12-week WKY myocytes but unmasked a positive action in 12-week SHR and 36-week WKY myocytes. L-NAME also significantly attenuated IGF-1-induced depression in 36-week SHR myocytes. In addition, the Ca(2+) channel opener Bay K8644 (1 micromol/L) abolished IGF-1-induced cardiac depression in 36-week SHR myocytes. Collectively, these results suggest that the IGF-1-induced cardiac contractile response was reduced with advanced age as well as with hypertension. Alterations in nitric oxide and intracellular Ca(2+) modulation may underlie, in part, the resistance to IGF-1 in hypertension and advanced age.

INFLUENCE OF SEX, DIABETES AND ETHANOL ON INTRINSIC CONTRACTILE PERFORMANCE OF ISOLATED RAT MYOCARDIUM

The influence of sex on intrinsic contractile performance, diabetes-induced myocardial mechanical dysfunction and the inotropic response to clinically relevant concentrations of ethanol (ETOH) was studied using weight-matched streptozotocin-induced diabetic rats. After 8 weeks, isolated left-ventricular papillary muscles stimulated at 0.5 hz, were studied under isometric conditions at Lmax. Peak developed tension, time-topeak tension (TPT), time-to-90% relaxation (RT90) and the maximum velocities of tension development and rate of tension decay were assessed at baseline and in response to changes in stimulation frequency and varying extracellular calcium concentrations. In male but not in female rats, body weight and heart size were significantly correlated with glycemic status. In both sexes, diabetes was associated with prolongation of baseline TPT and RT90 values. However, diabetes-induced prolongations of contraction and relaxation duration were greater in papillary muscles obtained from male than in those from female animals. The negative staircase effect of increasing the frequency of stimulation was not influenced by sex or the diabetic state. Similarly, neither the positive inotropic effect of increasing extracellular calcium nor the negative inotropic effect of ethanol was modified by sex or experimental diabetes. Our results suggest that: 1) myocardium from female rats is resistant to diabetes-induced myocardial dysfunction and 2) neither baseline developed tension, calcium-mediated nor ethanol-induced inotropic responsiveness are influenced by sex or experimental diabetes.

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.

Effects of acetaldehyde on the isolated papillary muscle of diabetic rats.

The effects of acetaldehyde (ACA) were examined in isolated electrically driven papillary muscle preparations from normal and streptozotocin-treated diabetic rats. Muscles from diabetic rats developed greater tension than those from normal rats. In muscles from both groups, ACA caused concentration-dependent negative inotropic effects that were independent of cholinergic or purinergic mechanisms and were not attributable to nitric oxide (NO) release. ACA was three to five times more potent with regard to its negative inotropic effect in diabetic than in normal rat muscles. A propranololsensitive, sympathetically mediated positive inotropic effect occurred at certain concentrations. Decreasing [Ca2+ ]0 from 2.7 to 0.5 mM reduced basal developed force to a significantly greater extent in muscles from normal rats than in those from diabetic rats. In low [Ca2+ ]0, concentration-response curves to CaCl2 in diabetic muscles were displaced to the left of that in normal muscles, suggesting that diabetic muscles are more sensitive to the positive inotropic effect of added CaCl2 at low [Ca2+ ]0, whereas at higher [Ca2+ ]0 (>1 mM), normal muscles developed more force in response to added CaCl2. ACA 10 and 30 mM more readily inhibited CaCl2-induced positive inotropic effect in normal than in diabetic muscles. Force-frequency curves, (negative staircase response) were recorded in both normal and diabetic muscles. In diabetic muscles, the curve exhibited a positive component at the lowest frequencies applied and was displaced to the right of that in normal muscle. ACA concentration-dependently inhibited force development, and diabetic muscles were more susceptible to the negative inotropic effect of ACA, when the stimulation frequency was increased. Ouabain 30 μM induced a positive inotropic effect of 21% in normal rat muscles, but increased basal developed force by 7% in diabetic preparations (p < 0.01). The magnitude of the negative inotropic effect of ACA with or without ouabain were similar in papillary muscles from both groups. In the diabetic myocardium, in which resting intracellular Ca2+ levels reportedly are increased, the negative inotropic effect of ACA is attenuated by increasing the [Ca2+ ]0, but not by increasing the [Ca2+ ]i with ouabain. The mechanism(s) involved in the negative inotropic effect of ACA are intimately related to the intracellular Ca2+ levels, which may not be identical in normal and diabetic myocardium, and that ACA reduces contractility by preventing the release of activator Ca2+.

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.

Inotropic effects of ethanol on the isolated papillary muscle of the diabetic rat

The present study was designed to characterize the inotropic effects of ethanol (ETOH) on diabetic rat heart. Left-ventricular papillary muscles, from normal and diabetic (streptozotocin, 50 mg/kg IV; 6 weeks) rats, were superfused with Tyrodes solution at 30 oC while driven at 0.5 Hz. Developed tension, time-to-peak tension (TPT), time-to-90% relaxation (RT90), maximum rate of tension developed (+VT) and maximum rate of tension fall (-VT) were determined in the absence and presence of clinically relevant concentrations of ETOH (80-240 mg/dl, i.e., 1.7-5.2 mM). Ethanol decreased developed tension, TPT, RT90, and VT in papillary muscles from both groups. Ethanol 80 mg/dl reduced tension, +VT and -VT in preparations from diabetic animals. However, this concentration had no effect on normal muscles. An intermediate concentration of ETOH (120 mg/dl), decreased tension and +VT in preparations from both groups, whereas a higher dose (240 mg/dl) decreased tension, TPT, RT90, VT, and -VT, and induced spontaneous contractures in both groups. The negative inotropic effects of ethanol were generally concentration-dependent and the diabetic myocardium maybe more sensitive to inotropic effects of clinically relevant concentrations of ETOH. Ethanol-induced diminution in contractile tension in normal and diabetic myocardium is associated with reduced contraction and relaxation velocity as well as their respective duration.

Influence of Age on the Inotropic Response to Acute Ethanol Exposure in Spontaneously Hypertensive Rats

Acute ethanol exposure depresses cardiac electromechanical function, whereas chronic ethanol consumption leads to the development of a specific myopathic state. Chronic hypertension and aging have similar effects in the impairment of myocardial function. However, little is known about the effects of ethanol on cardiac mechanical function in hypertension. We studied the effect of age on baseline mechanical properties and the inotropic response to clinically relevant concentrations of ethanol (18 to 71 mmol/L) using papillary muscles from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) at 10 and 25 weeks of age. Mechanical parameters measured were peak tension developed, time to peak tension, time to 90% relaxation, and maximal velocities of tension development and tension decline. SHR exhibited elevated systolic pressure and body weight as well as cardiomegaly and hepatomegaly at 10 and 25 weeks of age. Baseline mechanical properties were similar in SHR and WKY muscles at 10 weeks, whereas at 25 weeks, SHR muscles developed less tension, and both maximal velocities of tension development and tension decline were markedly depressed. Ethanol exposure produced concentration-dependent negative inotropic effects in both groups at both ages. Ethanol (> 18 nmol/L) decreased peak tension developed in both groups at 10 weeks, although higher concentrations were required at 25 weeks. The negative inotropic effect of ethanol resulted in the shortening of time to 90% relaxation in both groups at 10 weeks and was associated with a slowing of maximal velocities of both tension development and tension decline. The results suggest that aging depresses baseline mechanical properties when coupled with hypertension. In addition, the magnitude of the negative inotropic effect of ethanol was attenuated in both groups at 25 weeks of age.

Acute and chronic effects of ethanol on papillary muscles from spontaneously hypertensive rats

The effects of chronic ethanol ingestion (12 weeks) on the mechanical properties of hypertrophied papillary muscle and the in vitro effects of ethanol (80-640 mg/dl) was studied. Papillary muscles from spontaneously hypertensive rats (SHRs) and their normotensive controls, the Wistar-Kyoto rat (WKY), were used in this study. Peak-developed tension was significantly less in muscles obtained from SHR compared with WKY even when normalized for muscle cross-sectional area. Chronic ethanol ingestion resulted in a significant shortening of both contraction and relaxation duration in muscles from SHR and WKY. In muscles from SHR and WKY, acute in vitro ethanol exposure produced concentration-dependent negative inotropic effects that were associated with a reduction in the duration of contraction and relaxation and marked slowing in the maximum velocities of tension development and decay. These findings suggest that the contractile response to ethanol exposure, in vitro, is not modified by either chronic ethanol ingestion or hypertension.

Differential effects of chronic calcium channel blocker treatment on the inotropic response of diabetic rat myocardium to acute ethanol exposure

Cardiomyopathy is a consistent feature of diabetic myocardium as well as in prolonged alcohol consumption. Diabetes-induced myocardial dysfunction has been attributed, in part, to calcium overload within individual myocytes. The present study compares the effectiveness of the calcium channel blocker nifedipine (dihydropyridine-type) with verapamil (phenylalkylamine-type) in reversing myocardial dysfunction and diminishing the negative inotropic effect of ethanol on diabetic rat myocardium. Wistar rats were made diabetic with streptozotocin (55 mg/kg, i.v.) and isolated electrically stimulated papillary muscles were studied under isometric conditions in the absence and presence of clinically relevant concentrations of ethanol (80-240 mg/dl, i e., 17.4-52.1 mM). Subgroups of diabetic and normal animals received daily injections of verapamil or nifedipine 2 weeks after induction of diabetes for 8 weeks. Untreated diabetic animals exhibited hyperglycemia, hyperlipidemia, reduced growth, cardiomegaly, and hepatomegaly. Compared to verapamil chronic nifedipine treatment normalized or reversed the effects of diabetes on myocardial mechanical function. The negative inotropic effect of ethanol was attenuated only in muscles from verapamil-treated diabetic animals. Thus, chronic nifedipine treatment may be more effective than verapamil in reducing hyperglycemia, attenuating both cardiac and liver enlargement, and restoring myocardial mechanical function, in experimental diabetes. However, chronic verapamil therapy is more effective in diminishing the negative inotropic effect of ethanol on diabetic myocardium. These findings may have clinical significance among diabetic patients who consume alcoholic beverages while receiving long-term calcium blocker therapy.