The effects of 17 alpha-methyltestosterone on myocardial function in vitro.

Testosterone analogs have been used as performance enhancers by athletes for more than 40 yr. We asked whether the anabolic steroid 17 alpha-methyl-4-androstene-17-ol-3-one (17 alpha-MT) would affect intrinsic contractile function of the heart. Male Sprague-Dawley rats, 125-150 g, were treated with 17 alpha-MT either parenterally or orally for up to 8 wk. Intrinsic contractile function of the hearts was assessed utilizing both the isolated working heart and isovolumic perfused heart preparations. Isolated working hearts from 17 alpha-MT-treated rats had a 45% decrease in heart work attributable largely to a similarly decreased stroke volume. Isovolumic perfused hearts from treated animals had elevated left ventricular systolic and diastolic pressures at similar interventricular volumes compared to controls. Rates of ventricular pressure development (+dP/dT) or relaxation (-dP/dT) were unchanged as a result of the treatment. However, static elastance was reduced in potassium-arrested hearts from the 17 alpha-MT treatment (63% increase in interventricular pressure), consistent with a limitation being imposed on stroke volume by a decreased myocardial compliance. Hydroxyproline content of the hearts was not altered by 17 alpha-MT treatment suggesting that increased stiffness was not a consequence of collagen proliferation. Treatment of the steroid rats with beta-aminopropionitrile, a compound that inhibits lysyl oxidase, restored the left ventricular volume-pressure relationship (elastance curve) to that of control hearts. Thus, chronic treatment with anabolic steroids appears to reduce left ventricular compliance, possibly related to an enhanced activity of lysyl oxidase, and results in increased crosslink formation between collagen strands in the extracellular matrix.

The effect of clenbuterol and recombinant erythropoietin on tumor growth and the anemia caused by the Walker 256 carcinosarcoma.

In patients with advanced cancer, anemia is a common complication indicative of a poor prognosis. Attempts to alleviate this have met with mixed success and interventions including erythropoietin often fail to elicit an appropriate response. We have used rats implanted with the Walker 256 carcinosarcoma as a model of non-responsive anemia. This study demonstrates that the provision of recombinant erythropoietin in the presence of clenbuterol, a beta2 agonist, attenuates both the cancer induced anemia and the growth of the tumor in this model. We hypothesize that this treatment relieves the tumor induced inhibition of hematopoiesis, which allows for not only an increase in hematocrit but an increased immunosurveillance resulting in tumor suppression.

Myocardial effects of experimental neoplastic disease.

The purpose of this study was to determine the effects of the 7288ctc Morris hepatoma on heart size and performance. Hearts from tumor bearing and control animals were perfused in the working configuration one to three weeks post implantation. As tumor growth progressed there was an inverse linear relationship between tumor size and heart weight. When intrinsic heart work (defined as the product of the cardiac output and peak systolic pressure) was assessed in vitro over a range of physiologic preloads, significant differences were found between tumor-bearing and non-tumor-bearing animals. The hearts from the tumor-bearing animals (tumor weight 10-20 grams) developed only 76% of the heart work of control animals at maximal left atrial filling pressure (25 cm H2O). Hearts excised from rats with tumor masses from 30-65 grams developed 43% of the myocardial work as controls at the same (25 cm) preload. At the time of sacrifice resting blood pressures, blood glucose, insulin, and tumor necrosis factor (TNF) levels were similar in both groups. Blood from tumor bearing animals were negative for both aerobic and anaerobic bacteria. The data suggests that the decrements in cardiac size and performance are independent of several vectors known to influence heart size and performance and occur in the absence of detectable bacteremia.

Impairment of insulin action on peripheral glucose uptake and hepatic glucose production in tumor-bearing rats.

The present study was performed to determine the time-course for the development of peripheral and hepatic insulin resistance in rats as a result of an increasing tumor burden. Animals were inoculated with Yoshida ascites hepatoma, and studies were conducted during the early phase of tumor growth (day 4) at which time there was no change in food intake and at a later time point (day 8) when the tumor burden was increased and rats demonstrated anorexia. In vivo insulin action was accessed under euglycemic hyperinsulinemic conditions, in which insulin was infused at rates sufficient to produce arterial insulin levels that represent high physiological (3.5 ng/ml) or maximally stimulating values (180 ng/ml). On day 4, tumor-bearing (TB) rats were euglycemic, and whole body glucose turnover was elevated 32%. Insulin-mediated glucose uptake (IMGU) in TB rats was similar to control values at the low insulin infusion rate but reduced by 53% under maximally stimulating conditions. The insulin-induced suppression of glucose production was similar in TB and control animals at this time point. In contrast, on day 8, TB rats were hypoglycemic and glucose turnover was reduced 35%. The impairment in IMGU was more severe than seen earlier, with glucose uptake being reduced 39 and 61% at both levels of hyperinsulinemia. At this time point, the ability of insulin to inhibit glucose production was also impaired. These results indicate that the insulin resistance induced by the Yoshida hepatoma was manifested initially by a reduction in IMGU by peripheral tissues. As the tumor burden increased peripheral insulin resistance became more severe and an impairment in hepatic insulin action was observed.

Myocardial metabolic effects of in vivo hydralazine treatment of streptozotocin-diabetic rats.

We determined myocardial pumping capacity, glucose oxidation, and mechanical response to ischemia in streptozotocin-diabetic rats treated for 4 wk with or without hydralazine (0.5 mg/g of chow). Plasma triglycerides and cholesterol were decreased 73 and 50%, respectively, in the treated animals. Blood glucose levels were greater than 400 mg/100 g in both groups. Hearts were perfused in the working configuration with buffer containing 5 mM [U-14C]glucose. Starling curves were constructed by increasing left atrial filling pressure from 5 to 20 cm of water. Diabetic heart mechanical function was depressed compared with control and hydralazine treatment restored function to normal. Oxidation of [U-14C]glucose was comparably depressed in the treated and untreated diabetics. The provision of 1 mM dichloroacetate in the perfusate increased glucose oxidation in the hearts from hydralazine-treated rats, however. Twenty minutes of global ischemia resulted in 65% decrease in mechanical function in the hearts of hydralazine-treated group vs. 15% for hearts from nontreated diabetics. The data suggest that measures to normalize lipid metabolism may not normalize myocardial glucose oxidation or permit better mechanical recovery after ischemia in the diabetic myocardium.

The effect of dichloroacetate on the isolated no flow arrested rat heart.

Ischemic dysfunction, including contracture, has been attributed to lack of ATP, although previous work has not been consistent with this concept. We describe here a model of no flow ischemic arrest, characterized by depressed levels of mechanical function upon reperfusion and high energy phosphate stores within normal limits. The decreased mechanical function bears an inverse relationship to myocardial lactate levels after twenty-minutes of reperfusion in the absence or presence of dichloroacetic acid (DCA). Post-ischemic non-DCA treated hearts attained peak work of only 25% of that of controls, while those treated with DCA following ischemia performed almost as well as controls. ATP and CP levels remained high in both DCA treated and non-DCA treated hearts. Lactate levels were high in hearts immediately following ischemia, but were reduced to control levels in post-ischemic hearts perfused with DCA within twenty minutes, whereas those not treated with DCA had lactate levels two to three times that of controls within the same time period. Pyruvate dehydrogenase (PDH) activity was reduced in non-DCA treated post ischemic hearts after twenty minutes reperfusion but was elevated above controls in hearts reperfused with DCA. The data indicates that DCA increases mechanical performance of the isolated post-ischemic rat heart and the proposed mechanism for this increase is the oxidative removal of lactate resulting from an increase in PDH activity.

Inotropic interactions of dichloroacetate with amrinone and ouabain in isolated hearts from endotoxin-shocked rats.

The inotropic effects of ouabain and amrinone singly and in combination with dichloroacetate (DCA) were assessed using isolated working perfused hearts from endotoxin-shocked (LD50/6 h) rats, with glucose and free fatty acids as substrates. Amrinone (2.7 X 10(-4) M) and ouabain (10(-5) M) alone improved myocardial mechanical performance from 25 to 75%, depending on the preload. Amrinone was more effective than ouabain at all left atrial filling pressures tested. DCA enhanced the inotropic effect of both compounds, improving myocardial work greater than 65% in combination with ouabain (at low filling pressures) to 125% with amrinone (at high filling pressures). Glucose oxidation rose two- to threefold when DCA was present with either drug. Ouabain was without effect on pyruvate dehydrogenase (PDH) activity; however, when present with DCA, PDH activity increased fourfold. Amrinone alone augmented PDH activity 2.5 times as compared with controls, and 4.5 times when combined with DCA. All three compounds individually elevated myocardial ATP levels, but in contrast to the inotropic effects, when used in combination caused no further increase in ATP. Myocardial cyclic AMP (cyclic AMP) levels were augmented five times control values in the presence of amrinone. The simultaneous presence of DCA and amrinone did not further augment the myocardial concentration of cAMP. DCA alone enhanced the myocardial oxidation of glucose in isolated myocardial cells from endotoxin-shocked animals. In contrast, ouabain and amrinone did not affect cellular glucose oxidation. These data indicate that the provision of DCA can enhance the inotropic effect of amrinone and ouabain on the isolated working heart.(ABSTRACT TRUNCATED AT 250 WORDS)

T3 treatment does not prevent myocardial dysfunction in chronically diabetic rats.

The isolated working heart preparation was used to investigate the effect of continuous triiodothyronine (T3) administration on cardiac function and metabolism of rats rendered diabetic for a period of 4 wk with streptozocin (STZ). T3 controlled-release pellets were implanted 1 wk after STZ (70 mg/kg) injection. Rats injected with citrate buffer without STZ received T3 pellets 1 and/or 2 wk later. A comparable number of rats received placebo pellets. Untreated diabetic rats exhibited a decrease in spontaneous heart rate and myocardial cytochrome c concentrations concurrent with depressed plasma T3 values compared with untreated controls. T3 treatment did not improve in vitro cardiac performance (assessed as cardiac output times peak systolic pressure per gram dry heart weight) in hearts from diabetic rats perfused with glucose alone. Addition of octanoate reversed this depression and improved cardiac function to a greater extent in treated than in untreated diabetic animals. However, these differences between treated and untreated diabetic animals disappeared when heart rate was controlled by cardiac pacing. Furthermore, T3 treatment of controls and diabetics did not alter the oxidation of octanoate or the cardiac responsiveness to isoproterenol. These results suggest that experimental diabetic cardiomyopathy is partly attributable to a substrate deficiency and is not due entirely to hypothyroidism.

Mechanism of cardiac dysfunction in hearts from endotoxin-treated rats.

Myocardial performance was assessed in isolated perfused working hearts 3 hr after in vivo endotoxin administration (LD50-6 hr) to rats. Hearts removed from endotoxin-treated rats developed approximately 70% of the peak systolic pressure and 50% of the cardiac output (45% of the aortic flow and 50% of the coronary flow) at 25 cm H2O left atrial filling pressure (LAFP) compared to hearts from vehicle-injected controls. Hearts from endotoxin-treated rats were also characterized by decreased mechanical responsiveness to isoproterenol challenge at a high LAFP. These data suggest that myocardial dysfunction associated with endotoxin shock persists in vitro under conditions in which flow returning to the heart is not a limiting factor. Cyclic AMP accumulation was reduced in myocytes from endotoxin rats in response to 1 microM isoproterenol (29% of control values) and 1 microM forskolin (35% of control values) in both the absence and presence of phosphodiesterase inhibition with 0.2 mM isobutylmethylxanthine. Adenylate cyclase activity in crude membrane preparations of ventricular tissue from endotoxin-treated rats was unresponsive to challenge by isoproterenol and was reduced to 65% of control values by 100 micron GppNHp and by 10 mM NaF. Maximal specific binding of (-)[3H]dihydroalprenolol was decreased in membranes from endotoxin (56 +/- 2 fmol/mg) compared to control (64 +/- 2 fmol/mg) rats (P less than 0.05) but with similar antagonist affinities. These results suggest a decreased adrenergic responsiveness in myocytes and ventricular membrane preparations from endotoxin rats that may be linked to the reduction in myocardial adrenergic responsiveness during endotoxemia.

The effect of in vivo endotoxin on myocardial function in vitro.

Isolated perfused working hearts from male adult Sprague Dawley rats were used to examine myocardial performance following acute in vivo endotoxin administration (LD50-6-hour). Three hours after endotoxin administration, cardiac output and peak systolic pressure in the isolated perfused working heart were depressed 25-50% over a range of left atrial filling pressures (preload) from 10 to 30 cm of water. Linear regression analysis of the relationship between myocardial work indices, oxygen uptake, and glucose oxidation indicated that the hearts from the endotoxin-treated animals required more oxygen and glucose to perform the same amount of work. Levels of cyclic 3'5' adenosine monophosphate were 72% higher in ventricular tissue from the endotoxin-treated group compared to the hearts of controls. Isoproterenol (10(-9) mol/min) raised levels of the nucleotide to the same final concentration in both groups of hearts, whereas myocardial pressure work in hearts from endotoxin-treated rats was only 70% that of control. Provision of isoproterenol, while increasing mechanical work by the hearts in the endotoxin-treated group, did not induce an increase to the same performance levels as that of control hearts not treated with isoproterenol. These data are consistent with the concept that endotoxemia produces an intrinsic defect in myocardial mechanical performance.

Catecholamine-stimulated lipolysis in adipocytes of spontaneously hypertensive rats.

We investigated the catecholamine-stimulated lipolytic response of perirenal adipocytes isolated from spontaneously hypertensive (SHR) and normotensive (C) rats of the Sprague-Dawley (SD) and Wistar-Kyoto (WKY) strain. Younger rats (10-17 weeks) were matched with respect to age and body weight. Age-matched SHR rats were smaller than their C counterparts, had equal-size adipocytes, and demonstrated lipolytic responses equal to C cells. Weight-matched SHR rats were older than normotensive controls, had larger adipocytes, and showed depressed norepinephrine (NE)-stimulated responses with a rightward shift in the dose-response curve. Rates of lipolysis of SHR and C cells were not different in the simultaneous presence of norepinephrine and theophylline. Nine- to ten-month-old rats were of comparable body weight and adipocyte size regardless of blood pressure status; however, SHR cells still showed a significantly blunted response to catecholamine stimulation. We conclude that (1) the NE-stimulated lipolytic response of adipocytes of SHR rats is significantly less than that elicited from C cells; (2) this function difference seems unrelated to a size difference between cells of younger, SHR and C rats, thus implicating the adrenergic system; and (3) whole body growth (as reflected by body weight) and perirenal adipocyte growth do not proceed in parallel in actively growing SHR rats.

Myocardial dysfunction in a nonlethal, nonshock model of chronic endotoxemia.

Escherichia coli endotoxin (ET) was administered to adult rats by continuous IV infusion from a subcutaneously implanted osmotic pump (Alzet). Myocardial function was assessed after 6 and 30 hr of ET infusion and compared with control rats which received a saline infusion and were fasted to match the anorexia of ET rats. Cardiac output (CO) and coronary blood flow, measured by the radiolabeled microsphere method, and mean arterial blood pressure, heart rate, total peripheral resistance, and stroke volume, were determined in vivo. Treatment differences were limited to a 13% lower arterial pressure in ET rats after 30 hr of infusion. Myocardial function was evaluated in vitro in similarly treated rats with the isolated perfused working heart preparation; preload was altered by raising the left atrial filling pressure (LAFP) from 10 to 30 cm water. After both 6 and 30 hr of infusion, hearts from ET rats exhibited a significantly lower peak systolic pressure (PSP), CO, and coronary flow in response to increasing LAFP, and a greater oxygen consumption per unit of myocardial work (CO X PSP). Reduced in vitro work performance of hearts from endotoxemic rats was demonstrated early in the course of chronic endotoxemia and in the absence of in vivo evidence of cardiac dysfunction. Myocardial dysfunction, masked in vivo by compensatory mechanisms used to maintain adequate cardiovascular function, may be an important feature in the pathogenesis of both experimental endotoxemia and clinical gram-negative sepsis.

Culture of the terminally differentiated ventricular cardiac muscle cell. Characterization of exogenous substrate oxidation and the adenylate cyclase system.

Oxidation of several exogenous substrates by cultured adult rat ventricular cardiac muscle cells has been assessed. Unlike freshly isolated cardiac muscle cells which oxidize glucose preferentially, the cultured cells more closely resemble metabolically the in situ heart and the isolated perfused heart, in that their preference for exogenous substrates is in the order of fatty acid greater than glucose. This switch in metabolic preference from glucose to fatty acid is complete within 12 h after placing freshly isolated cells in culture. Glucose oxidation is stimulated by insulin and isoproterenol and inhibited by beta-hydroxybutyrate and octanoate. The adenylate cyclase system has also been examined in these cultured cells. Isoproterenol, norepinephrine and epinephrine stimulate the accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) in a concentration-dependent manner. The order of potency is isoproterenol greater than norepinephrine approximately equal to epinephrine. This stimulation is potentiated by 1-isobutyl-3-methylxanthine and inhibited by 1-propranolol.

Effect of burn shock on myocardial function in guinea pigs.

The effect of burn shock on myocardial function was studied using an isolated working heart preparation. Guinea pig hearts were studied 2-, 4-, and 8-hr following burn injury as well as in time-matched controls. The hearts were excised from both burned (35% body surface area) and unburned unresuscitated animals, and ventricular function curves were generated on each heart. The 4- and 8-hr postburn hearts, particularly the 4-hr group, exhibited depression in cardiac output, stroke volume, coronary flow, peak systolic pressure, mean aortic pressure, estimated myocardial work, stroke work, and oxygen consumption when compared to controls. The ventricular function curve for the 4-hr postburn hearts was shifted to the right indicating a decrease in contractility, which complements a decrease seen in compliance. These pronounced changes seen in the 4-hr postburn hearts were not observed 2 hr after burn injury. The data suggest that intrinsic myocardial function following burn injury is depressed and the degree of dysfunction is influenced by the time from thermal injury.

Effect of thyroxine treatment on exogenous myocardial lactate oxidation.

The effect of thyroxine treatment on myocardial lactate oxidation was examined by use of an isolated, working rat heart preparation. Thyroxine treatment, both acute and chronic, was associated with a decrease in lactate oxidation, when the heart was perfused with a physiological blend of substrates (free fatty acids, lactate, and glucose). This decrease in lactate oxidation was not caused by a generalized impairment in mitochondrial oxidation of acetyl coenzyme A (CoA), as oxygen consumption was normal and fatty acid oxidation was elevated in the treated animals. The block in lactate oxidation was localized to the conversion of pyruvate to acetyl CoA, as indicated by the depressed oxidation of pyruvate and lactate. Thyroxine treatment was associated with a decrease in pyruvate dehydrogenase activity. The decrease in pyruvate dehydrogenase activity was reversible and was attributed to the enhanced myocardial oxidation of free fatty acids.

Myocardium in hypertrophy: oxygen consumption by isolated cardiac myocytes and working hearts from spontaneously hypertensive rats.

Oxygen consumption was measured on suspensions of calcium tolerant myocytes obtained from hearts of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar Kyoto Rats (WKY). Oxygen consumptions of the isolated cells were not significantly different from each other either in the presence or absence of added calcium (1.5 mM). Additionally, there was excellent agreement between the oxygen consumption of the isolated cells and estimates of basal oxygen consumption between work and myocardial oxygen utilization in isolated perfused working hearts. At any given workload there was no significant difference in oxygen consumption between SHR hearts and WKY hearts. The mechanical performance of the SHR hearts was lower compared to that of the WKY hearts at low preloads. At high preloads and high afterloads the SHR hearts developed higher pressures than did hearts obtained from WKY rats. The data suggest that: (1) basal oxygen consumption of the two hearts are similar and (b) the contractile defects in the SHR heart are not the result of hypoxia.

Effect of ouabain on exogenous substrate oxidation by isolated cardiac myocytes.

Ouabain (10(-4) M) stimulated glucose oxidation and inhibited octanoate oxidation by isolated cardiac myocytes. Omission of potassium or calcium from the buffer abolished the ouabain stimulation of glucose oxidation. The inhibition of octanoate oxidation by ouabain persisted in the calcium deficient media.

Exogenous substrate utilization in Ca2+-tolerant myocytes from adult rat hearts.

A preparation of isolated noncontracting adult rat heart myocytes can be obtained in which viability is high, yield is adequate, and the cells are not sensitive to the deleterious effects of Ca2+ at physiological levels. Myocytes are enzymatically dispersed and maintained in a medium containing amino acids and 1% bovine serum albumin at 37 degrees C. The initial viability of 77% fell to 70% after a 60-min incubation in the presence of 1.5 mM Ca2+. The initial cellular ATP content of the myocytes was 27 nmol/mg protein. Cellular ATP content fell to 21 and 17 nmol/mg protein when cells were incubated for 60 min with and without 1.5 mM Ca2+, respectively. The oxidation of glucose and lactate was increased by 50 and 30%, respectively, by the addition of Ca2+. A decrease in the oxidation rate of these two substrates was noted when the myocytes were incubated with medium containing ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. No changes in the rate of palmitate oxidation were observed. The significant increase in glucose and lactate oxidation due to ca2+ provides further evidence that ca2+ influences myocardial substrate utilization independent of its effects on mechanical performance of the myocardium.

Amino acid stimulation of oxygen and substrate utilization by cardiac myocytes.

The inclusion of plasma levels of the natural amino acids plus 2.5 mM glutamate and 2.5 mM malate (PAAGM) raised the oxygen consumption and glucose oxidation of isolated cardiac myocytes in phosphate buffered saline. The addition of calcium (1.25 mM) and magnesium (0.66 mM) potentiated the stimulatory effect of PAAGM on glucose oxidation and oxygen consumption, PAAGM did not alter the shape of the dose-response curve for glucose oxidation by the isolated cardiac myocyte preparation. It did increase the amount of glucose oxidation at any given media glucose concentration up to 20 mM. PAAGM also increased the rate of lactate oxidation by the isolated cardiac myocyte preparation. PAAGM did not stimulate the oxidation of octanoate unless there was glucose present in the incubation media as well. Measurements of the concentrations of free amino acids indicated higher levels in myocytes incubated in PAAGM than in myocytes incubated in phosphate buffered saline. The data suggest that substrate metabolism by the isolated cardiac myocyte preparation can be influenced by the presence of plasma constituents that would be available to the myocardium in vivo.