Diabetic type of cardiomyopathy in food-restricted rats.

We have demonstrated that food restriction that is associated with weight loss can produce a type of cardiac dysfunction similar to that produced by diabetes. As in diabetic atria, the food-restricted atria had a 2-fold increase in contraction force, rate of force development, and rate of force decline compared with controls. Both food-restricted and diabetic atria could tolerate anoxia better than controls. The contractile function of the whole perfused heart from the food-restricted rat was reduced, as in the case of the diabetic heart. As the left ventricular volume was increased, the left ventricular developed pressure and the rate of rise and fall in pressure were significantly reduced in both food-restricted and diabetic hearts, compared with those of age- and weight-matched controls. The positive inotropic responses of atria and whole perfused heart to increasing concentrations of extracellular calcium were similarly altered in food-restricted and diabetic hearts. The possible molecular mechanisms of these findings and some of the differences observed between food-restricted and diabetic hearts are discussed.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats.

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.     

Ectopic pacing at physiological rate improves postanoxic recovery of the developing heart

Recently, rapid and transient cardiac pacing was shown to induce preconditioning in animal models. Whether the electrical stimulation per se or the concomitant myocardial ischemia affords such a protection remains unknown. We tested the hypothesis that chronic pacing of a cardiac preparation maintained in a normoxic condition can induce protection. Hearts of 4-day-old chick embryos were electrically paced in ovo over a 12-h period using asynchronous and intermittent ventricular stimulation (5 min on-10 min off) at 110% of the intrinsic rate. Sham (n = 6) and paced hearts (n = 6) were then excised, mounted in vitro, and subjected successively to 30 min of normoxia (20% O(2)), 30 min of anoxia (0% O(2)), and 60 min of reoxygenation (20% O(2)). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout the experiment. Reoxygenation-induced chrono-, dromo-, and inotropic disturbances, incidence of arrhythmias, and changes in electromechanical delay (EMD) in atria and ventricle were systematically investigated in sham and paced hearts. Under normoxia, the isolated heart beat spontaneously and regularly, and all baseline functional parameters were similar in sham and paced groups (means +/- SD): heart rate (190 +/- 36 beats/min), P-R interval (104 +/- 25 ms), mechanical atrioventricular propagation (20 +/- 4 mm/s), ventricular shortening velocity (1.7 +/- 1 mm/s), atrial EMD (17 +/- 4 ms), and ventricular EMD (16 +/- 2 ms). Under anoxia, cardiac function progressively collapsed, and sinoatrial activity finally stopped after approximately 9 min in both groups. During reoxygenation, paced hearts showed 1) a lower incidence of arrhythmias than sham hearts, 2) an increased rate of recovery of ventricular contractility compared with sham hearts, and 3) a faster return of ventricular EMD to basal value than sham hearts. However, recovery of heart rate, atrioventricular conduction, and atrial EMD was not improved by pacing. Activity of all hearts was fully restored at the end of reoxygenation. These findings suggest that chronic electrical stimulation of the ventricle at a near-physiological rate selectively alters some cellular functions within the heart and constitutes a nonischemic means to increase myocardial tolerance to a subsequent hypoxia-reoxygenation.     

Contrasting response of protein degradation to starvation and insulin as measured by release of N tau-methylhistidine or phenylalanine from the perfused rat heart.

An isotope-dilution method is described for the measurement of N tau-methylhistidine release from the perfused rat heart. We argue that release of N tau-methylhistidine is indicative of cardiac actin degradation. N tau-Methylhistidine release is compared with phenylalanine release in the presence of cycloheximide (phenylalanine release being a measure of degradation of mixed proteins). In hearts perfused with glucose plus acetate, the rate of actin degradation was increased by starvation and was not inhibited by insulin. In contrast, the rate of mixed-protein degradation was decreased by starvation and was inhibited by insulin. The fractional rate of degradation of mixed proteins in hearts from fed or starved rats was greater than that for actin. It is suggested that there are at least two pools of intracellular protein, the degradation rates of which differ in terms of their response to insulin and starvation.     

The role of amino acid catabolism in the formation of the tricarboxylic acid cycle intermediates and ammonia in anoxic rat heart

Amino acid catabolism, the tricarboxylic acid cycle intermediates and ammonia formation were studied in isolated perfused rat heart under anoxia. The total net anaplerosis due to amino acid degradation in anoxia was equal to that in oxygenation (6.29 and 6.09 mumol/g dry weight per h, respectively) as a result of the increased transamination of glutamic and aspartic acids. During anoxic perfusion, the rate of catabolism of glutamic and aspartic acids was 1.5-times higher than in normoxia, while depletion of branched-chain amino acids, lysine, proline, arginine and methionine, was inhibited. Alanine was the product of excessive degradation of glutamic and aspartic acids. Under anaerobic conditions, in spite of inhibition of amino acid deamination, ammonia formation was increased 2.7-fold as compared to oxygenation. The principal amount of ammonia (96%) was produced at degradation of adenine nucleotides. A 2.5-fold increase in the pool of the tricarboxylic acid cycle intermediates under anoxia was associated mainly with accumulation of succinate. The data suggest that the coupling of alanine- and aspartate amino transferases is a mechanism controlling the tricarboxylic acid cycle pool size in anoxic heart.     

Relative synthesis of cardiac contractile proteins. Evidence for synthesis from the same precursor pool.

The relative molar synthesis of cardiac contractile proteins has been measured in the perfused heart under control haemodynamic conditions. This synthesis, of myosin heavy chains, individual light chains (1 and 2), actin and tropomyosin, was determined from isolated guinea-pig hearts perfused for 3h simultaneously with constant specific radioactivities and concentrations of [3H]lysine and [3H]phenylalanine.The data strongly suggest that all of the proteins studied were synthesized from the same precursor pools of lysine and phenylalanine, since the ratio of the specific activities of the two labels was the same in all of the proteins. Measurement of molar synthesis of each contractile protein was the same with either labelled amino acid. Under control haemodynamic-perfusion conditions, the relative molar synthesis of the contractile proteins was actin greater than heavy chains greater than light chain 2 greater than light chain 1 greater than tropomyosin.     

Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart.

Leucine, but not isoleucine or valine, inhibited protein degradation and accelerated protein synthesis in hearts perfused with buffer that contained glucose (15 mM) and normal plasma levels of other amino acids, except for the branched chain compounds. Products of leucine, isoleucine, and valine metabolism also inhibited protein degradation and stimulated protein synthesis. These compounds included the transamination and decarboxylation products, as well as acetate, acetoacetate, and propionate. In some, but not all instances, inhibition of degradation and acceleration of synthesis were accompanied by an increase in intracellular leucine. When insulin was added to the perfusate, the rate of degradation was reduced by 40%, but addition of leucine was ineffective in the presence of the hormone. Insulin, leucine (2 mM) and a mixture of branched chain amino acids at normal plasma levels increased latency of cathepsin D in hearts that were perfused with buffer containing glucose. A combination of leucine and insulin increased latency more than either substance alone. These studies indicate that leucine as well as a variety of substrates that are oxidized in the citric acid cycle are involved in regulation of protein turnover in heart muscle.     

Extraneuronal accumulation of isoproterenol in atria and ventricle of perfused rat heart

Extraneuronal accumulation of isoproterenol in atria and ventricle of perfused rat heart was investigated. Rat hearts were perfused with various concentrations of 3H-isoproterenol for 30 min in the absence and the presence of catechol-O-methyltransferase (COMT) inhibitor (tropolone). When COMT was intact, the accumulation of 3H-isoproterenol in both atria and ventricle after perfusion with low concentration of 3H-isoproterenol (0.01 to 1 mumol/l) was less than that of perfusing concentration; the tissue/medium ratio (T/M) of isoproterenol for artia was lower than that for ventricle. The T/M of isoproterenol after perfusion with 10 and 20 mumol/l of 3H-isoproterenol were 0.94 and 1.76 for atria and 3.25 and 2.95 for ventricle, respectively. When COMT was inhibited by tropolone, the T/M increased 6.3-9.0 folds for atria and 5.1-6.7 folds for ventricle after perfusion with 3H-isoproterenol (0.01 to 1 mumol/l). From these results, it was concluded that both atria and ventricle of the rat heart have an extraneuronal O-methylating system as reported in rat whole heart, and was suggested that there might be different capacities of extraneuronal uptake and COMT between them.     

HSP25 in isolated perfused rat hearts: Localization and response to hyperthermia

Recent investigations concentrate on the correlation between the myocardial expression of the inducible 70-kDa heat shock protein (HSP70i) by different stress conditions and its possible protective effects. Only few studies have focused on the involvement of small heat shock proteins in this process. We analyzed the location of the small heat shock protein HSP25 in isolated cardiomyocytes as well as its location and induction in isolated perfused hearts of rats. By immunofluorescence microscopy HSP25 was found to colocalize with actin in the I-band of myofibrils in cardiomyocytes of isolated perfused hearts as well as in isolated neonatal and adult cardiomyocytes. Hyperthermic perfusion of isolated hearts for 45 min resulted in modulation of different parameters of heart function and in induction of HSP25 and HSP70i. Temperatures higher than 43°C (44–46°C) were lethal with respect to the contractile function of the hearts. Compared to control hearts perfused at 37°C, significant increases during hyperthermic perfusion at 42°C and 43°C were obtained for heart rate, contraction velocity and relaxation velocity. In response to hyperthermia at 43°C and after subsequent normothermic perfusion for 135 min at 37°C, left ventricular pressure, contraction velocity and relaxation velocity remained significantly elevated. However, heart rate returned to control values immediately after the period of heat treatment. HSP25 is constitutively expressed even in normothermic perfused hearts as shown by Western blotting. Hyperthermia increased the content of HSP25 only in the left ventricular tissue. In contrast, HSP70i was strongly induced in all analyzed parts of the myocardium (left ventricle, right ventricle, septum). Our findings suggest a differential regulation of HSP25 and HSP70i expression in response to hyperthermia in isolated perfused hearts. The constitutively expressed HSP25 seems to be located adjacent to the myofibrils which implies a specific role of this protein even under unstressed conditions for the contractile function of the myocardium.     

Absence of purinoceptors in the heart of the turtle (Emys orbicularis)

The effects of adenosine, adenosine 5'-triphosphate (ATP), a slowly degradable ATP analogue beta,gamma-methylene ATP (APPCP) and a degradation resistant ATP analogue alpha,beta-methylene ATP (APCPP) were examined on the turtle heart. Adenosine, ATP, APPCP and APCPP had no effect on the rate or force of contraction of either the atrium or ventricle. The effects of acetylcholine and noradrenaline were also examined on the turtle heart. Acetylcholine decreased the force and rate of contraction of turtle atria in a concentration-dependent manner. Noradrenaline increased the rate of contraction but caused a slight decrease in the force of contraction of the atrium. Neither acetylcholine nor noradrenaline produced an inotropic effect on the ventricle.     

Expression of heat shock proteins in turtle and mammal hearts: relationship to anoxia tolerance

Heat shock proteins (HSPs) may play a cardioprotective role during hypoxia or ischemia. We hypothesized that cardiac tissue from hypoxia-tolerant animals might have high levels of specific HSPs. We measured myocardial HSP60 and HSP72/73 in painted and softshell turtles during normoxia and anoxia (12 h) and after recovery (12 or 24 h). We also measured myocardial HSPs in normoxic rats and rabbits. During normoxia, hearts from the most highly anoxia-tolerant species, the painted turtle, expressed the highest levels of HSP60 (22.6+/-2.0 mg/g total protein) followed by softshells (11.5+/-0.8 mg/g), rabbits (6.8+/-0.9 mg/g), and rats (4.5+/-0.5 mg/g). HSP72/73 levels, however, were not significantly different. HSP60 levels in hearts from both painted and softshell turtles did not deviate significantly from control values after either 12 h of anoxia or 12 or 24 h of recovery. The pattern of changes observed in HSP72/73 was quite different in the two turtle species. In painted turtles anoxia induced a significant increase in myocardial HSP72/73 (from 2.8+/-0.1 mg/g normoxic to 3.9+/-0.2 mg/g anoxic, P<0.05). By 12 h of recovery, HSP72/73 had returned to control levels (2.7+/-0.1 mg/g) and remained there through 24 h (2.6+/-0.2 mg/g). In softshell turtles, HSP72/73 decreased significantly after 12 h of anoxia (from 2.4+/-0.4 mg/g normoxic to 1.3+/-0.2 mg/g anoxic, P<0.05). HSP72/73 levels were still slightly below control after 12 h of recovery (2.1+/-0.1 mg/g) and then rose to significantly above control after 24 h of recovery (4.1+/-0.7 mg/g, P<0.05). We also conclude that anoxia-tolerant and anoxia-sensitive turtles exhibit different patterns of myocardial HSP changes during anoxia and recovery. Whether these changes correlate with their relative degrees of anoxia tolerance remains to be determined.     

Effects of increasing extracellular pH on protein synthesis and protein degradation in the perfused working rat heart.

Increasing the extracellular pH over the range pH 7.4-8.9 stimulated protein synthesis by about 60% in the rat heart preparation anterogradely perfused in vitro. Protein degradation was inhibited by this pH increase. The magnitudes of the effects at pH 8.9 on protein synthesis and degradation were similar to those of high concentrations of insulin. Cardiac outputs were increased, as were cardiac phosphocreatine contents, indicating that the alterations in extracellular pH did not adversely affect the physiological viability of the preparation. ATP contents were unaltered. The creatine kinase equilibrium was used to assess the magnitude of the change in intracellular pH induced by these treatments. The increase in intracellular pH was about 0.2 for a 1-unit increase in extracellular pH. Thus small changes in intracellular pH have dramatic effects on cardiac protein turnover.     

The effects of lactate, acetate, glucose, insulin, starvation and alloxan-diabetes on protein synthesis in perfused rat hearts.

Compared with glucose, lactate + acetate stimulated ventricular protein synthesis in anterogradely perfused hearts from fed or 72 h-starved rats. Stimulation was greater on a percentage basis in starved rats. Atrial protein synthesis was not detectably stimulated by lactate + acetate. Insulin stimulated protein synthesis in atria and ventricles. The stimulation of protein synthesis by lactate + acetate and insulin was not additive, the percentage stimulation by insulin being less in the ventricles of lactate + acetate-perfused hearts than in glucose-perfused hearts. Perfusion of hearts from 72 h-starved or alloxan-diabetic rats with glucose + lactate + acetate + insulin did not increase protein-synthesis rates or efficiencies (protein synthesis expressed relative to total RNA) to values for fed rats, implying there is a decrease in translational activity in these hearts. In the perfused heart, inhibition of protein synthesis by starvation and its reversal by re-feeding followed a relatively prolonged time course. Synthesis was still decreasing after 3 days of starvation and did not return to normal until after 2 days of re-feeding.     

Role of bradykinin in the antihypertr. ophic effects of enalapril in the newborn pig heart

Rapid growth of the left ventricle of the newborn pig heart can be restrained by treating piglets with the angiotensin converting enzyme inhibitor, enalapril maleate. This reduced rate of growth is reflected in vitro by reduced rates of ribosome formation and protein synthesis, and may be due to decreased availability of angiotensin II (All), a potentially hypertrophic agent; decreased numbers of All receptors; increased availability of bradykinin, a potentially antihypertrophic agent; or reduced hemodynamic load on the left ventricle. Because enalapril decreases degradation of bradykinin, the role of bradykinin as an inhibitor of cardiac growth in the newborn heart was investigated. Addition of 1 × 10^–5 M bradykinin and 1 × 10^–6 Menalapril to the perfusate of isolated hearts from 2 day old piglets did not significantly alter heart rate, contents of ATP or creatine phosphate or rates of ribosome formation or protein synthesis during 1 h of perfusion. Similarly, exposure of myocytes isolated from the left ventricular free wall of piglets to 5 × 10^–6 M bradykinin for 72 h did not alter the rate of [³H]-phenylalanine incorporation into total protein. The reduced rate of left ventricular growth in vivo caused by enalapril administration was not reversed by simultaneous treatment with the specific bradykinin receptor antagonist, HOE 140. HOE 140 alone did not alter ventricular growth as compared to hearts from untreated piglets. In summary, these results demonstrate that the reduced rate of left ventricular growth in vivo and the reduced rate of ribosome formation and protein synthesis in the left ventricle in vitro after enalapril treatment of piglets is not the result of an inhibitory effect of bradykinin on cardiac growth.     

Cryoprotective effect of methanol during cooling of frog hearts

It has been shown that frog hearts, perfused with gradually increasing concentrations of ethylene glycol (to 11 m) as the temperature was gradually lowered to ?55 °C and then cooled abruptly to ?78 °C, resumed spontaneous contractions when rewarmed. The thin-walled sinus venosus and atria showed significantly better recovery than the thick-walled ventricle. It was suggested that the difference in recovery of the various parts of the heart might be related to the degree of penetration of the glycol into the tissue. In an attempt to achieve better penetration during perfusion, in particular at subzero temperatures, methanol was substituted for glycol in the perfusate. Hearts equilibrated at room temperature in nontoxic concentrations of methanol were perfused with gradually increasing concentrations as the specimen was gradually cooled to various temperatures. The hearts were gradually rewarmed, and during the rewarming the concentrations of methanol in the perfusate was gradually reduced. All hearts resumed spontaneous rhythmic contractions providing they were not cooled to below ?30 °C or perfused with methanol solutions exceeding 10 m concentration. Cooling to lower temperatures and exposure to higher concentrations of methanol did not permit recovery. These results show that at temperatures as low as ?30 °C methanol in concentrations up to 10 m is comparable to ethylene glycol in its ability to protect hearts from cryoinjury. Its failure to protect at lower temperatures may be related to the development of toxic concentrations when water is removed in the form of ice.     

Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen

In the absence of glucose, insulin stimulated the incorporation of (14)C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on (14)C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.     

The effect of prolonged anoxia at 3°C on tissue high energy phosphates and phosphodiesters in turtles: A 31P-NMR study

Selected tissues (skeletal muscle, heart ventrical, and liver), sampled from turtles (Chrysemys picta bellii) at 3°C either under normoxic conditions or after 12 weeks of anoxic submergence were quantiaatively analysed for intracellular pH and phosphorus metabolites using ³¹P-NMR. Plasma was tested for osmolality and for the concentrations of lactate, calcium, and magnesium to confirm anoxic stress. We hypothesized that, in the anoxic animals, tissue ATP levels would be maintained and that the increased osmolality of the body fluids of anoxic turtles would be accounted for by a corresponding increase in the concentrations of phosphodiesters. The responses observed differed among the three tissues. In muscle, ATP was unchanged by anoxia but phosphocreatine was reduced by 80%; in heart, both ATP and phosphocreatine fell by 35–40%. The reduction in phosphocreatine in heart tissue at 3°C was similar to that observed in isolated, perfused working hearts from turtles maintained at 20°C but no decrease in ATP occurred in the latter tissues. In liver, although analyses of several specimens were confounded by line-broadening, neither ATP nor phosphocreatine was detectable in anoxic samples. Phosphosdiesters were detected in amounts sufficient to account for 30% of normoxic cell osmotic concentration in heart and 11% and 12% in liver and muscle, respectively. The phosphodiester levels did not change in anoxia. Heart ventricular phosphodiester levels in turtles at 3°C were significantly higher than those determined for whole hearts from turtles at 20°C. ¹H, ¹³C and ³¹P NMR analyses of perchloric acid extracts of heart and skeletal muscle from 20°C turtles con firmed that the major phosphodiester observed by NMR in these tissues is serine ethanolamine phosphate. We conclude that the three types of tissues studied differ substantially in their ability to maintain levels of ATP during anoxia, and that liver may continue to function despite NMR-undetectable levels of this metabolite. In addition, we conclude that phosphodiesters do not serve as regulated osmolytes during anoxia, and that the functional significance of their high concentrations in turtle tissues remains uncertain.