Effects of glucocorticoids and catecholamines on maturing rat heart

1. The negative force-frequency response of normal rat heart was accentuated when the animals were adrenalectomized. Treatment of adrenalectomized animals with dexamethasone restored the normal force-frequency response. 2. Total adrenalectomy increased the sensitivity of rat heart to calcium. 3. Adrenalectomized-dexamethasone-treated hearts were more responsive to epinephrine and ouabain. 4. Total adrenalectomy caused independent myocardial disturbances in calcium handling elements (glucocorticoid effect) and beta receptors (catecholamine effect).     

Mechanical properties of developing swine myocardium

Mechanical responses of myocardium from 16 piglets were studied from 18 hr to 12 days after birth. Tension, time and velocity parameters of contraction and relaxation were determined for every contraction cycle. Increasing the frequency of stimulation in step-changes induced negative inotropy in some muscles regardless of age. Doubling extracellular calcium ion concentration induced a positive force-frequency response in all muscles. Epinephrine increased tension and velocities without affecting contraction time. The ultrastructure was immature even on the 12th postnatal day. We concluded that in newborn piglet hearts, the mechanisms for calcium delivery are not fully developed. Thus, the heart undergoes a transient phase during which at least a principal portion of calcium for the myofibers is supplied by the extracellular fluid. While receptors for catecholamines are present, the time course for their response is immature.     

Alterations in mechanical response of aged rat myocardium. Effects of dexamethasone

1. Mechanical responses of young and old rat myocardium to increasing rates of stimulation were compared. As the animals aged, we found a significant enhancement of the negative force-frequency response and a decline in the velocities of contraction and of relaxation. 2. At 6 months of age, there were no differences between rats obtained from commercial sources and a group of rats obtained from a colony maintained at the National Institute of Aging. 3. At 24 months, the negative force-frequency response was considerably greater in the former group of animals than in the latter. 4. The sensitivity to the calcium concentration in the tissue bathing solution was significantly increased in aged heart preparations. Increasing the calcium concentration reduced the negative inotropy and the decline in the velocities of contraction and relaxation. The enhancement by calcium was directly proportional to the concentration of the metal in the bathing solution. 5. When aged animals were pretreated with pharmacological doses of dexamethasone, the age-induced alterations in the mechanical responses were reversed. The aged, dexamethasone-treated myocardium also became refractory to calcium concentrations above 2.7 mM in the bathing solution. 6. It is suggested that aging induces multifocal defects and that steroid hormones play a role in the maintenance of integrity of the myocardium. The action of the steroids is on the sarcolemma, the contractile proteins and the sarcoplasmic reticulum.     

Reduced oxygen supply explains the negative force-frequency relation and the positive inotropic effect of adenosine in buffer-perfused hearts

In isolated rat hearts perfused with HEPES and red blood cell-enriched buffers, we examined changes in left ventricular pressure induced by increases in heart rate or infusion of adenosine to investigate whether the negative force-frequency relation and the positive inotropic effect of adenosine are related to an inadequate oxygen supply provided by crystalloid perfusates. Hearts perfused with HEPES buffer at a constant flow demonstrated a negative force-frequency relation, whereas hearts perfused with red blood cell-enriched buffer exhibited a positive force-frequency relation. In contrast, HEPES buffer-perfused hearts showed a concentration-dependent increase in left ventricular systolic pressure [EC50 = 7.0 +/- 1.2 nM, maximal effect (Emax) = 104 +/- 2 and 84 +/- 2 mmHg at 0.1 microM and baseline, respectively] in response to adenosine, whereas hearts perfused with red blood cell-enriched buffer showed no change in left ventricular pressure. The positive inotropic effect of adenosine correlated with the simultaneous reduction in heart rate (r = 0.67, P < 0.01; EC50 = 3.8 +/- 1.4 nM, baseline 228 +/- 21 beats/min to a minimum of 183 +/- 22 beats/min at 0.1 microM) and was abolished in isolated hearts paced to suppress the adenosine-induced bradycardia. In conclusion, these results indicate that the negative force-frequency relation and the positive inotropic effect of adenosine in the isolated rat heart are related to myocardial hypoxia, rather than functional peculiarities of the rat heart.     

Chronoinotropic reaction of the hypertrophied myocardium using an electromechanical coupling mathematical model.]

Static and dynamic chrono-inotropic responses were recorded from both normal and hypertrophic rat auricular myocardium. The slope of the static force-frequency relation from hypertrophied heart was steeper than in the control hearts. The cellular mechanisms underlying changes in the force frequency response associated with hypertrophy of the heart were studied by means of a mathematical model of excitation-contraction coupling. The characteristic features of hypertrophied heart force-frequency relations are shown to be due to the enhanced volume of the intracellular Ca-stores in contrast to the total volume of the cardiomyocyte.     

A relationship between circulating natural glucocorticoids and the mechanical responses of the heart in atricial and precocial rodents

• 1.1. A comparison was made of the mechanical performance of heart muscle from mouse, an atricial mammal, with corticosterone as glucocorticoid and spiny mouse (Acomys cahirinus), a precocial mammal, with cortisol as glucocorticoid.
• 2.2. Force-frequency responses were negative in mouse and positive in spiny mouse.
• 3.3. During recovery, there was a gradual increase and an overshoot in the mouse, while in the spiny mouse there was an initial enhanced response, diminishing gradually with time.
• 4.4. High calcium concentration inhibited contractile tension in mouse heart, while it was positively inotropic in spiny mouse heart. Changes in the concentration of calcium did not change the patterns of force-frequency response.
• 5.5. Lowering the experimental temperature increased the time course and amplitude of the tension curve. However, various parameters exhibited different temperature sensitivity.
• 6.6. There was a significant difference in the levels of circulating cortisol between male and female spiny mice.
• 7.7. It is proposed that the differences in the mechanical responses of mouse and spiny mouse hearts may be explained in terms of the effects of the specific glucocorticoid hormone on the development of the sodium-calcium exchanger.

     

Force-frequency relations in hypertrophic heart muscle: a mathematical model for excitation-contraction coupling.

Static and dynamic chrono-inotropic responses were recorded from both normal and hypertrophic rat auricular myocardium. The slope of the static force-frequency relation for hypertrophic hearts was steeper than that for control hearts. Computer experiments were designed to study the cellular mechanisms underlying the changes in the force-frequency response associated with heart hypertrophy, with the aid of a mathematical model for excitation-contraction coupling in rat heart. A set of equations was derived which permitted to study the effects on the chronoinotropic relations of both the geometrical dimensions of cardiomyocytes and the sarcoplasmic reticulum, and of the variation in activity of mechanisms for Ca movements through the sarcolemma and the sarcoreticular membrane. A comparison of data obtained from simulated and real experiments suggested that the features characteristic of force-frequency relations for hypertrophic heart are a result of an enhanced volume of intracellular Ca-stores rather than of the total volume of the cardiomyocyte.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.     

Electrical properties of developing rat heart. Effects of dexamethasone

Action potentials recorded from perinatal rat ventricles exhibited a plateau (phase 2), followed by a rapid repolarization characteristics of all mammalian ventricular cells. Within the second postnatal week, a number of distinct changes occurred in the contour of action potentials. An early slow depolarization, at the foot of the action potential, preceded the beginning of phase zero. The early slow depolarization was observed until day 12 and disappeared by day 13. A second slow depolarization occurred during the terminal phase of the rapid upstroke of the action potential, persisted through day 13 and disappeared by day 14. On day 12, what had been a homogeneous contour of action potentials seen during the first week converted into a heterogeneous contour. Occasionally, action potentials similar to those recorded from Purkinje fibres in adult heart were recorded from hearts as young as 12 days. By day 14, signs of a spike (the hallmark of action potentials from adult heart) were apparent in some fibres. Treatment of newborn rats with dexamethasone on the second day after birth prevented the disappearance of the second slow depolarization. In adult and aged rat hearts, dexamethasone treatment induced a slow depolarization and a plateau in the region of overshoot. In view of the time-dependent change of the second slow depolarization it is suggested that this phase of the action potential is influenced by the levels of circulating glucocorticoid in developing heart and by changes in calcium sensitivity observed in this species. Heterogeneity of action potentials observed on day 12 postnatal may precede structural differentiation of myofilaments.     

Neonatal murine heart slices. A robust model to study ventricular isometric contractions.

BACKGROUND/AIMS: Cardiac function is increasingly studied using murine models. However, current multicellular preparations to investigate contractile properties have substantial technical and biological limitations and are especially difficult to apply to the developing murine heart. METHODS: Newborn murine hearts were cut with a vibratome into viable tissue slices. The structural and functional integrity of the tissue was shown by histology, ATP content and sharp electrode recordings. RESULTS: Within the first 48 hours after slicing structure remained intact without induction of apoptosis. ATP concentrations and action potential parameters were comparable to those of physiological tissue. Isometric force measurements demonstrated a physiological force-frequency relationship with a ;primary-phase' negative force-frequency relationship up to 1-2 Hz and a ;secondary-phase' positive force-frequency relationship up to 8 Hz. (-)-Isoproterenol (10(-6) mol/l) increased active force to 251 +/- 35% (n=15) of baseline values and shortened relaxation times indicating a preserved beta-adrenergic regulation of contraction. Changes of the force-frequency relationship after application of ryanodine and nifedipine indicated functionality of calcium release from the sarcoplasmic reticulum and of L-type calcium channels. CONCLUSION: Generation of viable, physiological intact ventricular slices from neonatal hearts is feasible and provides a robust model to study loaded contractions.     

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain,and the ionophore R02-2985 (X537A) in the isolated,perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using ³¹P-NMR. Mechanical activity was increased by infusion of ouabain (0.9?7.0·10^?5 M), the ionophore R02-2985 (1·10^?5 M) or epinephrine (5·10^?8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ($\text{[}\text{ATP}\text{]}\text{[ADP][P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.     

Mitochondrial calcium uptake in the perfused contracting rat heart and the influence of epinephrine on calcium exchange

Perfused rat hearts were exposed to solutions containing ⁴⁵Ca²⁺ with and without epinephrine. They were subjected to differential centrifugation and the distribution of Ca and ⁴⁵Ca in mitochondria and microsomes was determined. It was found that the mitochondria contain most of the calcium of the intact heart and that the exchange of mitochondrial calcium with extracellular calcium was extremely rapid. This process was accelerated in hearts stimulated by epinephrine.     

Effect of ouabain on the innervated and noninnervated embryonic chick heart.

Attempts to assess the importance of the role played by endogenous catecholamines in the positive inotropic response to ouabain have produced contradictory results. The sympathetic nervous system is not present in the 4-day-old chicken embryo heart but is fully developed after 7 days of embryonic life. This was confirmed by the fact 4-day-old hearts do not respond to tyramine and cocaine while the usual positive inotropic and chronotropic responses were observed when these drugs were administered to 7-day-old hearts. In spite of this difference the positive inotropic response to ouabain was virtually identical at these two stages of development.     

Taurine's possible protective role in age-dependent response to calcium paradox

When hearts were reperfused with Ca++ after a short period of Ca++-free perfusion, irreversible loss of electrical and mechanical activity was observed. This phenomenon, first described by Zimmerman and Hulsmann, was termed the "calcium paradox". Chizzonite and Zak recently reported that rat hearts exhibited an age-dependent response in a calcium paradox model. The taurine (2-aminoethanesulfonic acid) content of hearts in the newborn animal is high, and decreases rapidly during the first few days of life. The present experiments were performed to test whether the myocardial taurine content was closely linked to an age-dependent response in the calcium paradox model, using post-hatched chicks. The mechanical dysfunction of the heart was much more severe in 9-day-old post-hatched chicks than in 2-day-old chicks when the hearts were subjected to the calcium paradox. Myocardial taurine content was lower in the 9-day-old chicks than in the 2-day-old chicks. The age-related response to the calcium paradox was partially protected by oral pretreatment with taurine, and there was a small increase in myocardial taurine level. It is proposed that myocardial taurine is one factor in the protection against the calcium paradox phenomenon.     

Ouabain binding sites and (Na+,K+)-ATPase activity in rat cardiac hypertrophy. Expression of the neonatal forms

The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involves several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of [3H]ouabain and ouabain-induced inhibition of (Na+,K+)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (Kd) of 1.8-3.2 X 10(-8) M and 1-8 X 10(-6) M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na+,K+)-ATPase (IC50 = 1-3 X 10(-8) M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that we observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms (Schwartz, K., Lompre, A.M., Bouveret, P., Wisnewsky, C., and Whalen, R.G. (1982) J. Biol. Chem. 23, 14412-14418), our data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor.     

Effect of chronic cocaine administration and cocaine withdrawal on coronary flow rate and heart rate responses to epinephrine and cocaine in isolated perfused rat hearts

The acute dose-dependent effects of epinephrine and cocaine on heart rate and coronary flow rate (CFR) were examined in isolated, perfused (Langendorff) rat hearts from animals: i) pretreated with daily cocaine injections (20 mg/kg/day) for 8 weeks; ii) after 2-day withdrawal from 8-week cocaine pretreatment; iii) vehicle-treated controls. Chronic cocaine (CC) hearts were significantly less sensitive to the chronotropic effects of epinephrine than control (C) or withdrawal (CW) hearts. CW hearts exhibited significantly higher heart rates in response to epinephrine than C and CC hearts. Epinephrine alone (2.5 x 10(-7) M) decreased CFR 11% (C), 9%(CC), 14%(CW) from respective baseline levels. Cocaine alone had no significant effect on CFR in C hearts but produced slight dose-dependent decrements in CFR in CC and particularly CW hearts at higher doses. Cocaine plus epinephrine markedly decreased CFR in all groups, particularly in CW hearts. The results indicate that chronic daily cocaine administration produces a functional tolerance of the heart to the chronotropic actions of epinephrine but a 2-day withdrawal from chronic cocaine results in a rebound supersensitivity to adrenergic stimulation and cocaine's sympathomimetic effects. In addition, cocaine produces only minor decrements in coronary flow in the rat heart, while cocaine acts synergisticallly with epinephrine to produce a marked decrease in CFR.     

Contractile parameters and occurrence of alternans in isolated rat myocardium at supra-physiological stimulation frequency

The cardiac refractory period prevents the heart from tetanic activation that is typically used in noncardiac striated muscle tissue. To what extent the refractory period prevents successive action potentials to activate the excitation-contraction coupling process and contractile machinery at supra-physiological rates, such as those present during ventricular fibrillation, is unknown. Using multicellular trabeculae isolated from rat hearts, we studied amplitude and kinetics of contraction at rates well above the normal in vivo rat heart range. We show that even at twice the maximal heart rate of the rat, little or no mechanical instability is observed; twitch contractions are at steady state, albeit with an elevated active diastolic force. Although the amplitude of contraction increased within in vivo heart rates (positive force-frequency response), at frequencies beyond the maximal heart rate (10-30 Hz) a steady decline of contractile amplitude is observed. Not until 30 Hz do the majority of the isolated muscle preparations show mechanical alternans, where strong and weak beats alternate. Interestingly, unlike striated limb skeletal muscle, fusing of twitch contractions did not cause a continuous increase in peak force: at frequencies of 10 Hz and above, systolic force declines with relatively little elevation in diastolic force. Contractile kinetics continued to accelerate, from 1 Hz up to 30 Hz, whereas the relative speed of contraction and relaxation remained closely coupled, reflected by a singular linear relationship between the maximal and minimal derivative of force (dF/dt). We conclude that cardiac muscle can produce mechanically stable steady-state contractions at supra-physiological pacing rates, while these contractions continue to decline in amplitude and increase in diastolic force past maximal heart rate.     

Hormonal regulation of expression of ileal bile acid binding protein in suckling rats

Ileal bile acid binding protein (IBABP) is a cytosolic protein believed to be involved in the absorption of conjugated bile acids. In rodents this protein and its mRNA have been shown to increase markedly during the third postnatal week. Because this period of ontogeny is characterized by increasing circulating concentrations of glucocorticoids and thyroxine, the goal of our study was to investigate the role of these hormones in IBABP expression in the developing rat. Administration of various doses of dexamethasone (Dex) during the second postnatal week caused a robust induction of IBABP mRNA and protein. Plateau levels of IBABP mRNA occurred at a Dex dose of 0.1 microg/g body wt, which is within the physiological range. IBABP mRNA was not appreciably induced until 24 h after treatment, suggesting that glucocorticoids influence IBABP either through a delayed primary or a secondary response mechanism. The regional pattern of IBABP mRNA elicited by Dex mimicked that seen during normal development, with appearance in distal ileum preceding proximal ileum. Thyroxine injections did not result in a significant increase of IBABP mRNA, and synergism between Dex and thyroxine was not observed. Taken together, our data suggest that maturation of IBABP expression is influenced by glucocorticoids but not by thyroxine.     

Role of endothelial cells in modulation of contractility induced by hexarelin in rat ventricle.

The synthetic growth hormone (GH) secretagogue hexarelin has important cardiac effects, that include a reduction of dysfunction in ischemic-reperfused hearts from GH-deficient rats after a chronic treatment and an increase of ejection fraction in acutely treated men. To investigate the mechanisms of its cardiac activity, we studied the effects of hexarelin (1-10 microM) on contractility of rat papillary muscles. We observed, in hexarelin treated papillary muscles, an improved recovery of contractility after anoxia. Hexarelin induced time- and frequency-dependent inotropic effects on papillary muscle. These effects were a transient increase in contractile force, abolished by propranolol (0.2 microM), followed by a reduction at low (60-240/min), but not at high (400-600/min) beating frequencies. The typical negative force-frequency relationship present in rat papillary muscles was therefore modified, and a minor increase in diastolic tension occurred after a sudden increase in stimulus frequency. Blockade of NO synthesis with 1 mM L-NAME, partially altered the response to hexarelin. MK-677 (1 microM), a non peptidyl GH secretagogue, reduced contractility, but did not alter the force-frequency relationship. The remaining effects of hexarelin were absent in papillary muscles pre-treated with indomethacin (1 microM), or after removal of endocardial endothelium with 0.5% triton X-100. The release of the prostacyclin metabolite 6-keto-PGF1alpha was increased during reoxygenation after a period of anoxia in hexarelin treated papillary muscles. Hexarelin had no significant effect on calcium transients and on I(Ca) measured in isolated ventricular cells. These findings suggest that the effects of hexarelin are mainly due to endothelium-released PGI2.     

A hypersensitivity of glycogen phosphorylase activation in hearts of diabetic rats

This study was initiated to determine whether glycogen phosphorylase activation was defective in hearts of alloxan diabetic rats. When hearts were perfused by gravity flow for 1 to 10 min with various concentrations of epinephrine, activation of glycogen phosphorylase in the diabetic was significantly greater at every time and epinephrine concentration than that seen in the normal. Cyclic AMP accumulation and protein kinase activation by epinephrine in the diabetic were not appreciably different or were lower than the normal responses to the hormone. The effects of epinephrine on cAMP and protein kinase were blocked in both normal and diabetic hearts by propranolol. While the beta blocker prevented phosphorylase activation in the normal hearts, it did not block phosphorylase activation by epinephrine in the diabetic hearts. Likewise, the alpha agonist phenylephrine activated phosphorylase in the diabetic but not in the normal hearts. While glucagon produced the same phosphorylase hypersensitivity in diabetic hearts, the cAMP and protein kinase responses were not altered by diabetes. Phosphorylase phosphatase activity was found to be unaltered by either epinephrine or diabetes, whereas phosphorylase kinase activation by epinephrine in the diabetic was double the normal response. These data are consistent with a diabetes-related unmasking of an alpha effect on cardiac phosphorylase activation and an unexplained increase in the sensitivity of phosphorylase kinase activation by protein kinase.