CK flux or direct ATP transfer: versatility of energy transfer pathways evidenced by NMR in the perfused heart.

How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes (myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR. To get insight in the energy transfer pathway in the perfused rat heart, we developed a combined analysis of several protocols of magnetization transfer associated with biochemical data and quantitatively evaluated which scheme of energetic exchange best describes the NMR data. This allows to show the kinetic compartmentation of subcellular CKs and to quantify their fluxes. Interestingly, we could show that the energy transfer pathway shifts from the phosphocreatine shuttle in the oxygenated perfused heart to a direct ATP diffusion from mitochondria to cytosol under moderate inhibition of ATP synthesis. Furthermore using NMR measured fluxes and the known kinetic properties of the enzymes, it is possible to model the system, estimate local ADP concentrations and propose hypothesis for the versatility of energy transfer pathway. In the normoxic heart, a 3-fold ADP gradient was found between mitochondrial intermembrane space, cytosol and ADP in the vicinity of ATPases. The shift from PCr to ATP transport observed when ATP synthesis decreases might result from a balance in the activity of two populations of ANT, either coupled or uncoupled to CK. We believe this NMR approach could be a valuable tool to reinvestigate the control of respiration by ADP in the whole heart reconciling the biochemical knowledge of mitochondrial obtained in vitro or in skinned fibers with data on the whole heart as well as to identify the implication of bioenergetics in the pathological heart.

Modeling the energy transfer pathways. creatine kinase activities and heterogeneous distribution of ADP in the perfused heart.

The exchange scheme of high energy phosphate transport in a whole heart relies on a system of CK functioning in different ways. This suggests that the CKs are able to act both like a shuttle and like a buffer for the energy transfer. The challenge is to understand how these two functions are balanced in the CK system. One key of this balance is the knowledge of the local concentrations of the ADP nucleotide. These concentrations cannot be directly measured, but they may be derived by computation. In the present report we introduce the known properties of the enzymes catalyzing the exchange of high energy phosphate into the model of flux pathways derived from NMR experiments to compute both the maximum activity of each enzyme and the local concentrations of all the substrates. We show that the ADP distribution must be heterogeneous for the system to work. Its concentration is 50% higher in the vicinity of ATPase sites and 50% lower in the intermembrane space of the mitochondria than in the cytosol. Another result of this analysis is that the apparent large unbalance of the CKmito pathway is imposed by the adenosine nucleotide transferase fluxes. This analysis proves that it is possible to deduce biochemistry the local concentrations of a substrate by combining data originating from NMR, and enzymology into a common model.

Identification of subcellular energy fluxes by P NMR spectroscopy in the perfused heart: contractility induced modifications of energy transfer pathways.

The identification of subcellular fluxes of exchange of ATP, phosphocreatine (PCr) and Pi between mitochondria, cytosol and ATPases and pathways of energy transfer in a whole organ is a challenge specially in the myocardium where 50% of creatine kinases (CK) are found in close vicinity of ATP producing (mito-CK) and utilizing (MM-bound CK) reactions. To dissect their contribution in cardiac energy transfer we recently developed a new experimental 31P NMR spectroscopy approach. This led to identify three kinetically different subcellular CKs and to evidence experimentally the CK shuttle in a rat heart perfused in isovolumy. Here we show that a decreased energy demand alters energetic pathways : two CKs (cytosolic and MM-bound) functioning at equilibrium and a non mitochondrial ATP<-->Pi exchange was sufficient to describe NMR data. Mito-CK fluxes was not detected anymore. This confirms the dependence of energy pathways upon cardiac activity. Indeed the subcellular localization and activity of CKs may have important bioenergetic consequences for the in vivo control of respiration at high work: free ADP estimated from global CK equilibrium might not always adequately reflect its concentration at the ANT.

Discrimination of cardiac subcellular creatine kinase fluxes by NMR spectroscopy: a new method of analysis.

A challenge in the understanding of creatine kinase (CK) fluxes reflected by NMR magnetization transfer in the perfused rat heart is the choice of a kinetic model of analysis. The complexity of the energetic pathways, due to the presence of adenosine triphosphate (ATP)-inorganic phosphate (Pi) exchange, of metabolite compartmentation and of subcellular localization of CK isozymes cannot be resolve from the sole information obtained from a single NMR protocol. To analyze multicompartment exchanges, we propose a new strategy based on the simultaneous analysis of four inversion transfer protocols. The time course of ATP and Phosphocreatine (PCr) magnetizations computed from the McConnell equations were adjusted to their experimental value for exchange networks of increasing complexity (up to six metabolite pools). Exchange schemes were selected by the quality of their fit and their consistency with data from other sources: the size of mitochondrial pools and the ATP synthesis flux. The consideration of ATP-Pi exchange and of ATP compartmentation were insufficient to describe the data. The most appropriate exchange scheme in our normoxic heart involved the discrimination of three specific CK activities (cytosolic, mitochondrial, and close to ATPases). At the present level of heart contractility, the energy is transferred from mitochondria to myofibrils mainly by PCr.

Cardiac creatine kinase metabolite compartments revealed by NMR magnetization transfer spectroscopy and subcellular fractionation.

In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstrated by an apparent discrepancy between the forward (F(f)) and reverse (F(r)) CK flux if this compartment was neglected in the analysis [Joubert et al. (2000) Biophys. J. 79, 1-13]. To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or gamma ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F(f)/F(r) ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP). Under conditions masking the influence of the ATP-P(i) exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.

Evidence for myocardial ATP compartmentation from NMR inversion transfer analysis of creatine kinase fluxes.

The interpretation of creatine kinase (CK) flux measured by (31)P NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P(i) exchange on the forward (F(f): PCr --> ATP) and reverse (F(r)) CK fluxes derived from complete analysis of inversion transfer. Although F(f) should equal F(r) because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P(i) was masked by saturation of P(i) (sat-P(i)), F(f)/F(r) significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F(f)/F(r) for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P(i) might not result only from the masking of ATP-P(i) exchange.

Cardiac performance and creatine kinase flux during inhibition of ATP synthesis in the perfused rat heart.

To study the relation among mitochondrial energy supply, cardiac performance, and energy transfer through creatine kinase (CK), two acute models of inhibition of ATP synthesis were compared in the isovolumic acetate-perfused rat heart. Similar impairments of mechanical performance (rate-pressure product, RPP) were achieved by various stepwise decreases in O(2) supply (PO(2) down to 20% of control) or by infusing CN (0.15-0.25 mM). The forward CK flux measured by saturation-transfer (31)P NMR spectroscopy was 6.1 +/- 0. 4 mM/s in control hearts. Only after severe hypoxia (PO(2) < 40% of control) did CK flux drop (to 1.9 +/- 0.2 mM/s at PO(2) = 25% of control) together with impaired systolic activity and a rise in end-diastolic pressure. In contrast, in mild hypoxia CK flux remained constant and similar to control (5.3 +/- 0.5 mM/s, not significant) despite a twofold reduction in systolic activity. Similarly in all CN groups, constant CK flux was maintained for a threefold reduction in RPP, showing the absence of a relation between cardiac performance and global NMR-measured CK flux during mild ATP synthesis inhibition.

Chronic exposure of rats to hypoxic environment alters the mechanism of energy transfer in myocardium.

We have investigated the effect of chronic exposure of rats to an hypoxic environment (10% O2; 3 weeks), on the first step of the intracellular energy transfer process in the myocardium, i.e. the transfer at mitochondrial level of high energy bonds from ATP to creatine. In the left ventricles from rats adapted to normobaric hypoxia, we observed, using the permeabilized fiber technique, that the stimulatory effect of creatine on the mitochondrial respiration in presence of a low ADP concentration (0.1 mM) was attenuated when compared to control. Furthermore, the creatine-induced decrease of the apparent K(m) for ADP of the mitochondrial respiration, which is observed in control, was significantly reduced. Both the basal and maximal respiratory rates of the fibers were unchanged by the hypoxic exposure of the rats. A significant decrease of the total creatine kinase activity from 755 to 630 IU/g wet weight (for control and hypoxic rats, respectively) was detected and was accompanied by a 25% decrease in mitochondrial isoform activity (mitoCK) and in the mitoCK/citrate synthase ratio. In the right ventricles, identical alterations in the effect of creatine on apparent K(m) for ADP were observed while we did not detect any changes in CK activity. The decrease in mitoCK activity and the fall in the reactivity of respiration to creatine could be interpreted as a mechanism for downregulating oxygen demand during chronic hypoxia. The consequences of such alterations on energy metabolism of cardiomyocytes under conditions of reduced oxygen supply are discussed.

13C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts.

A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.

Kinetics of creatine kinase in an experimental model of low phosphocreatine and ATP in the normoxic heart.

To study the dependence of the forward flux of creatine kinase (CK) on its substrates and products we designed an acute normoxic model of steady-state depletion of phosphocreatine (PCr) and adenylate in the isovolumic acetate-perfused rat heart. Various concentrations of PCr and ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presence of insulin. The apparent rate constant (k(f)) and the forward CK flux were measured under metabolic and contractile steady state by progressive saturation-transfer 31P nuclear magnetic resonance (NMR). At high adenylate content CK flux was constant for a twofold reduction in PCr concentration ([PCr]); CK flux was 6.3 +/- 0.6 mM/s (vs. 6.5 +/- 0.2 mM/s in control) because of a doubling of k(f). Although, at the lowest ATP concentration and [PCr], CK flux was reduced by 50%, it nevertheless always remained higher than ATP synthesis estimated by parallel oxygen consumption measurement. NMR-measured flux was compared with the flux computed under the hypothesis of CK equilibrium. CK flux could not be fully predicted by the concentrations of CK metabolites. This is discussed in terms of metabolite and CK isozyme compartmentation.

Magentization transfer study of creatine kinase in myocardium. Potential clinical interest of in vivo enzymology.

Since myocardial contractility relies on the continuous balance of ATP synthesis and ATP degradation, a dynamic estimation of the high energy phosphates (HEP) turnover by magnetization transfer 31P NMR spectroscopy appears to be a more valuable index of heart function than the static evaluation of HEP concentration. The theory of the main magnetization transfer techniques (saturation transfer, inversion transfer) is described as well as a critical evaluation of their application to the determination of creatine kinase (CK) fluxes in myocardium. The determinants of CK flux in vivo is evaluated (total CK activity, isozymic CK expression and concentrations of CK substrates and products). CK flux is shown to vary in relation to contractility during short term stress (hypoxia, ischemia) and in long term adaptation or pathology of the myocardium. The dynamic estimation of energetic flux in vivo is proposed as a non-invasive tool of diagnosis in myocardial pathologies.

Adaptation of cardiac myosin and creatine kinase to chronic hypoxia: role of anorexia and hypertension.

The effects of chronic hypobaric hypoxia (CHH, 28 days, simulated altitude 5,500 m) on the cardiac expression of myosin heavy chain (MHC) and creatine kinase (CK) was studied in rat left (LV) and right (RV) ventricle. To separate the effects of hypoxia from its associated perturbations, anorexia and pulmonary hypertension (resulting in RV hypertrophy), CHH animals were compared with normoxic controls (C) and with rats restricted in food supply (pair fed, PF). In RV, the increased proportion of beta-MHC in CHH (20 +/- 3%) vs. C (7 +/- 2%, P < 0.01) and vs. PF (12 +/- 2%, P < 0.05) rats was mainly attributed to hypertension. In contrast, the higher beta-MHC of CHH (23 +/- 2%) vs. C (13 +/- 2%, P < 0.05) in LV was mainly ascribed to anorexia (PF = 21 +/- 3%, not significant). A major contribution of anorexia was also evidenced in the isozymic profile of CK; anorexia accounted for a 25% decrease in mito-CK specific activity in LV, whereas hypertension partly accounted for the threefold increase in BB-CK in RV. CHH specifically induced a twofold rise in LV BB-CK. This suggests that both the expression of slow myosin, improving the economy of contraction, and the changes in CK isozymic profile could provide a biochemical basis for the CHH resistance to ischemia.

A simple mathematical model and practical approach for evaluating citric acid cycle fluxes in perfused rat hearts by 13C-NMR and 1H-NMR spectroscopy.

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C-NMR and 1H-NMR spectroscopy. We demonstrate that 13C-NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments. Wistar rat hearts (five beating and four KCl-arrested hearts) were aerobically perfused with 100% enriched [2-(13)C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 +/- 0.003 min(-1) for beating hearts and 0.0741 +/- 0.004 min(-1) for KCl-arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl-arrested hearts are 1.06 +/- 0.06 micromol x min(-1) x mg(-1) and 0.21 +/- 0.02 micromol x min(-1) x g(-1), respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 +/- 1.7% and 8.8 +/- 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2-oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half-time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.

Myofibrillar creatine kinase and cardiac contraction.

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.

Compartmentation of creatine kinases during perinatal development of mammalian heart.

Maturation of the cardiac cell is characterized by increasing diversity of isozymic expression of creatine kinases. Expression of the M-CK isozyme always precedes that of mitochondrial isozyme (mi-CK), however the expression of an isoform does not inform about its localization or cellular function. The functional role of isozymes binding to sites of energy utilization and production characteristic of the adult myocardium can be evidenced by the functional coupling of M-CK to myofibrillar ATPase and mito-CK to translocase in Triton X-100 and saponin skinned fibers. Functional activity of M-CK and mito-CK were investigated during perinatal development. Both functional activities appear during late fetal life in species mature at birth like guinea pig, and in the first postnatal weeks in immature species like rat or rabbit. Thus, the functional activity of bound CK isozymes is not associated with birth per se but with the general process of cell maturation. Localization of CK in the cytosol appears optimal for the transfer of glycolytic production of ATP to sites of utilization in an immature heart. During cell maturation, the increasing contribution of oxidative phosphorylation to ATP production, the apparition and binding of mi-CK to mitochondria, the binding of M-CK to myofibrils, turn the cell in a compartmentalized system of energy production. This provides the cellular basis for energy transfer by the PCr-Cr-CK system between sites of ATP production and utilization. Compartmentation of both Ca handling and energy turnover leads to a highly structured cell organization and could be essential for the efficiency of heart function.

Functional development of the creatine kinase system in perinatal rabbit heart.

The functional development of the creatine kinase system has been studied in rabbit heart during perinatal growth. Fiber bundles were obtained from left ventricles of fetal rabbits at the 30th day of gestation, newborn rabbits aged 1, 3, 8, and 17 days, and adult rabbits. Total creatine kinase activity was constant during perinatal development, whereas myofibrillar bound creatine kinase activity increased 15-fold during the first postnatal week. Functional activity of myofibrillar creatine kinase was assayed in Triton X-100-skinned fibers by its ability to induce active tension in the absence of ATP or to relax rigor tension. It was very low in 1-day-old newborns and increased during the first 2 weeks to reach adult levels 17 days after birth. Functional activity of mitochondrial creatine kinase was determined in saponin-skinned fibers. Creatine-stimulated respiration appeared only after birth and increased gradually between 1 and 17 days after birth. The results show that, although the two creatine kinase isoforms (mitochondrial and myofibrillar) are expressed at different stages during development, their functional activities appear in parallel in mitochondria and myofibrils. Early postnatal development is characterized by binding of creatine kinase isoenzymes to intracellular organelles. Such compartmentation participates in the postnatal cardiac cellular maturation.

Sustained function of normoxic hearts depleted in ATP and phosphocreatine: a 31P-NMR study.

A model of high-energy phosphate depletion was developed in the normoxic isovolumic rat heart perfused with acetate, 2-deoxy-D-glucose (2DG), and insulin. Intracellular phosphorylation of 2DG abstracts phosphorus from its normal pathways. This results in a decrease of high-energy phosphates without any increase in Pi. During the first 15 min of 2DG phosphorylation, the changes in ATP, Pi, and intracellular pH (pHi) were slight, and work was unaltered, although phosphocreatine (PCr) concentration dropped by 50%. After 45 min, the heart reached a new steady state characterized by a drastic reduction in both PCr and ATP: PCr was 15% of control, and in most hearts ATP became invisible on the nuclear magnetic resonance (NMR) spectra. Nevertheless, the heart still developed 65% of its original systolic pressure, whereas diastolic pressure was unchanged. Oxygen consumption per unit work remained constant during 2DG perfusion. This is, to our knowledge, the first experimental model of sustained cardiac contractility at such low contents of both ATP and PCr. However, our results are compatible with present knowledge of the cytosolic energy transfer by PCr and of the control of force in myofilaments.

A phosphorus-31 nuclear magnetic resonance study of the metabolic, contractile, and ionic consequences of induced calcium alterations in the isovolumic rat heart.

Isolated adult rat hearts perfused in an isovolumic mode were used to study the effects of sodium-potassium pump inhibition and sodium-calcium exchange alterations on the tissue content of adenosine triphosphate, phosphocreatine, inorganic phosphate, and intracellular pH, all measured by phosphorus-31 nuclear magnetic resonance spectroscopy. Rates of oxygen consumption, contractile function, and the cell contents of calcium, sodium, and potassium also were determined. The inhibition of sodium-potassium adenosine triphosphatase, either by the reduction in perfusate potassium from 5.9 to 1 millimolar or less, or by the addition of 10(-4) molar ouabain, transiently increased systolic pressure. This was followed by a decrease in systolic pressure, an increase in diastolic pressure, and eventual inexcitability. This contractile profile was accompanied by a persistent increase in oxygen consumption, a monotonic decline in cellular adenosine triphosphate and phosphocreatine content, the development of marked intracellular acidosis, a gain in cell sodium and calcium content, and a reduction in cell potassium. Quite similar metabolic changes were also observed when cell calcium was increased after a reduction in perfusate sodium. These metabolic and contractile effects could be prevented or reversed by decreasing perfusate calcium. The results emphasize the profound role of calcium in modulating cell oxygen consumption, energy balance, pH, excitability, and force production. These data are discussed in light of changes in the myocardial energy supply/demand balance, as well as from the viewpoint of the known competition between mechanisms for mitochondrial calcium transport vs. high-energy phosphate production.

Load sensitivity of relaxation in the foetal and newborn rabbit heart.

Differences in the mechanism of cardiac relaxation and the influence of changes in the stimulation frequency were studied in foetal and newborn rabbit hearts. In the foetal rabbit heart which lacks a well developed sarcoplasmic reticulum, load sensitivity of relaxation was investigated and compared with that observed in the newborn. Load sensitivity was studied by measuring force and shortening length in twitches with increasing afterloads and also when load clamp steps were rapidly imposed during the twitch. Quantification of the load sensitivity was achieved by the measurement of the time to relaxation "tRi" which was linearly related to the relative developed force. The slope (S) of this linear relation quantifies the load sensitivity: the higher the slope, the more load sensitive is the relaxation. At a frequency of 24 beats X min-1, S was respectively 0.24 in the foetal heart and 0.36 in the newborn heart showing at both ages the existence of a load sensitivity and its significant increase at birth. No further increase in load sensitivity was observed from 1 day to 21 days after birth. Reducing the stimulation frequency from 24 to 10 beats X min-1 abolished the load sensitivity in foetal hearts (S = 0.05) while, in the newborn, a significant load sensitivity could still be observed (S = 0.25). Thus, in rabbit myocardium, the load sensitivity of cardiac relaxation depended upon the age and the stimulation frequency showing a perinatal development of the structures involved in the control of myocardial relaxation.

Participation of the sarcolemma in the control of relaxation of the mammalian heart during perinatal development.

Stereological measurement of cell diameter and cell surface-to-volume ratio (S/V) were compared in fetal, newborn, and adult rabbit and frog hearts. In the frog heart, S/V was high (1.32 micron-1), comparable to that of the fetal rabbit heart (1.03 micron-1). Only 8 days after birth, cell diameter increased while S/V progressively decreased towards the adult value of 0.30 micron-1. The possible implications of the S/V decrease were investigated on the relaxation of cardiac contraction. Unlike the relaxation of the adult mammalian heart, but like that of the frog, relaxation of the fetal heart was sensitive to a reduction of Na+-Ca2+ exchange by a low-Na+ medium. With perinatal development, the participation of Na+-Ca2+ exchange in relaxation decreased and was progressively masked by the sarcoplasmic reticulum and/or other systems.