Perfusion Of Guinea Pig Hearts Research Articles

The effects of levosimendan on the left ventricular function and protein phosphorylation in post-ischemic guinea pig hearts.

The widely accepted theories for the decreased function in the stunned myocardium relate to Ca2+ desensitization and free radical-mediated tissue damage of the myofilaments. The aim of the present study was to examine whether the depressed contractile function and Ca2+ responsiveness of the stunned myocardium may be restored by a new Ca2+ sensitizer (levosimendan), which has been shown to improve the Ca2+ response of the myofilaments. The effects of levosimendan on the left ventricular function and the in vivo protein phosphorylation were examined in both the non-ischemic and the stunned myocardium. Myocardial stunning was induced in Langendorff-perfused guinea pig hearts by suspending the circulation for 8 min, followed by a 20-min reperfusion period. Perfusion of post-ischemic guinea pig hearts with levosimendan (0.03-0.48 microM, 6 min) was associated with dose- and time-dependent increases in both dP/dtmax (contractility) and dP/dtmin (speed of relaxation). When the effectiveness of levosimendan was compared in non-ischemic and post-ischemic hearts, no significant differences were noted in the relative stimulatory effects on contractility and relaxation, at any given time point (time-response curve) or concentration (dose-response curve). Perfusion of the guinea pig hearts with a high (0.3 microM) levosimendan concentration did not reveal any qualitative or quantitative difference in the phosphodiesterase inhibitory potential of the compound (elevation of tissue cyclic AMP levels and characteristics of protein phosphorylation) between the non-ischemic and the post-ischemic myocardium. However, when isoproterenol was administered to induce maximal in vivo phosphorylation of cardiac phosphoproteins, an attenuation of the 32P-incorporation into troponin I was noted in the post-ischemic hearts. The decrease in isoproterenol-induced 32P-incorporation into troponin I was associated with similar alterations in the tissue level of this protein. We conclude that the Ca2+ sensitizer levosimendan exerts dose- and time-dependent positive inotropic and lusitropic effects on the post-ischemic myocardium, lending support to the hypothesis tha Ca2+ desensitization of the myofibrils is involved in myocardial stunning.

In vitro and ex vivo xanthine oxidoreductase activity in rat and guinea-pig hearts using hypoxanthine or xanthine as substrate

Through oxyradical formation xanthine oxidoreductase (XOD) could play a role in the etiology of cardiac damage. Its measurement poses problems, due to little substrate specificity, self-inactivation and endogenous inhibitors. Perfusion of guinea-pig hearts with hypoxanthine gave rise to only little xanthine release; in contrast rat hearts showed vivid xanthine production. Therefore, xanthine breakdown was hypothesized to exceed its formation in guinea-pig hearts. The kinetics of both substrates for XOD in cardiac homogenates were therefore compared with those obtained in perfused hearts. Oxypurine contents and effluent catabolites were determined by HPLC. Regardless of substrate, V max values in homogenates were about 38 and 13 mU/g for rat and guinea-pig heart, respectively. K m values were in the 3–5 μM range; therefore the hypothesis concerning the low xanthine release in guinea-pig hearts must be rejected. Activities in hearts perfused with hypoxanthine (50 μM) were 40 and 18 mU/g for rat and guinea pig, respectively; perfusion with xanthine produced < 50% of the activities observed with hypoxanthine (p < 0.002). Intracellular xanthine concentration, estimated from sorbitol distribution space and myocardial xanthine content was negative in both species, contrasting intracellular hypoxanthine levels, which approached extracellular concentrations. This disparate distribution indicates that hypoxanthine transport across the cell membrane far exceeds that of xanthine. Consequently, hypoxanthine is preferable to xanthine as substrate in perfused hearts to estimate XOD activity in situ.

Activation of pyruvate dehydrogenase by electrical stimulation, and low-Na + perfusion of guinea-pig heart

(1) Electrical stimulation (2 Hz) of guinea-pig hearts, perfused with medium containing 11 mM D-glucose plus 0.1 mM octanoate as substrate, resulted in an increase in the percentage of pyruvate dehydrogenase in the active form (PDH a) from 16 to 68%. (2) Rapid isolation of mitochondria by a technique designed to minimize net loss or gain of Ca 2+ revealed an increase in mitochondrial Ca 2+ content of the stimulated hearts, as measured with 45Ca (2.74 ± 0.27 versus 1.37 ± 0.11 nmol/mg protein; stimulated versus rested). (3) Perfusion of rested hearts with a medium containing a reduced Na + concentration (20 mM, with the remainder replaced with Li +) also gave increased values of PDH a content (30.9% versus 16% for the normal, physiological medium). This procedure is known to raise cytosol Ca 2+ concentrations and would be expected to give mitochondrial Ca 2+ loading. (4) These results are consistent with a role of mitochondrial Ca 2+ in activating pyruvate dehydrogenase in the intact heart.

Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline.

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.

Prolonged feeding of ethanol to the young growing guinea pig. II. A model to study the effects of severe ischemia on cardiac protein synthesis.

Although acute perfusion of guinea pig hearts with ethanol does not affect cardiac protein synthesis, the latter is inhibited after prolonged ingestion of ethanol when tested in an in vitro system with the working right ventricle. This study reports on the added stress of ischemia on such hearts. Hearts were removed from maturing guinea pigs after 13-16 weeks of ingesting 10% ethanol and were perfused in vitro under conditions of relative ischemia (one-sixth of normal coronary flow) with maintenance of right ventricular load and outflow resistance identical to normal pre-ischemic levels. With this degree of ischemia, there was a 4-6 fold increase in lactate production, an 80% drop in ATP, and a 90% decrease in creatine phosphate after 150 min of the ischemia. Incorporation of both labeled lysine and phenylalanine into cardiac protein was also diminished to 35% of control in the left ventricle and 55% of control in the right. This diminution of protein synthesis was the same in hearts from ethanol-drinking and matched control animals. Thus, prior prolonged ingestion of ethanol did not worsen the inhibition of protein synthesis by oxygen deprivation. There were, however, two significant differences in hemodynamic response to the ischemia by the right ventricles of hearts from ethanol-drinking guinea pigs compared to their matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ouabain and desipramine on the uptake and storage of norepinephrine and metaraminol

The uptake of 3H-dl-norepinephrine (NE) in the guinea-pig heart was measured during perfusion with 1 × 10 −4 M ouabain and 2 × 10 −5 M desipramine (DMI). The inhibition of NE uptake by ouabain was first observed after 10 min and gradually increased until, after 20 min, NE uptake was inhibited by about 50%. DMI blocked NE uptake efficiently at all times tested. When ouabain was perfused for various times before measuring uptake, it was found that inhibition of NE uptake did not begin for 8 min. After this time, inhibition increased rapidly with increasing pretreatment time until, after 15 min pretreatment, uptake was blocked by about 90%. The onset of inhibition of NE uptake elicited by ouabain could not be correlated with the ouabain induced changes in the intracellular (Na +) and (K +) of heart tissue. Perfusion of guinea-pig hearts with DMI or ouabain led to a substantial release of metaraminol when metaraminol was administered 1 hr before sacrifice but not when it was administered 24 hr before sacrifice. Similarly, 3H-NE was lost from hearts when DMI or ouabain was perfused immediately after infusion of 3H-NE. The level of endogenous NE, however, was not altered by perfusion with either DMI or ouabain. It is concluded that newly administered NE and metaraminol are not stored in the same pool as the endogenous NE.