Jacqueline Hoerter

Cardiac Specific Increase in Aldosterone Production Induces Coronary Dysfunction in Aldosterone Synthase–Transgenic Mice

Background—Elevated circulating aldosterone level is associated with impaired cardiovascular function. Although the mechanisms are not fully understood, aldosterone antagonists decrease total and cardiovascular mortality in heart failure and myocardial infarction. Aldosterone induces cardiac fibrosis in experimental models, and it is synthesized locally in rat heart. These observations suggest pathological effects of aldosterone in heart that remain unclear. Methods and Results—Transgenic mice (TG) that overexpress the terminal enzyme of aldosterone biosynthesis, aldosterone synthase (AS), in heart have been raised by gene targeting with the &agr;-myosin heavy chain promoter. AS mRNA increased 100-fold and aldosterone concentration 1.7-fold in hearts of male TG mice relative to wild-type. No structural or myocardial alterations were evidenced, because ventricle/body weight, AT1 and AT2 receptor binding, and collagen content were unchanged in TG. No alteration in cardiac function was evidenced by echocardiography, isolated perfused heart, or whole-cell patch clamp experiments. In contrast, coronary function was impaired, because basal coronary flow was decreased in isolated perfused heart (−55% of baseline values), and vasodilatation to acetylcholine, bradykinin, and sodium nitroprusside was decreased by 75%, 60%, and 75%, respectively, in TG mice compared with wild-type, showing that the defect was not related to NO production. Conclusions—Increased cardiac aldosterone production in male mice induces a major coronary endothelium-independent dysfunction with no detectable alterations in cardiac structure and function. However, coronary dysfunction may be harmful for coronary adaptation to increased flow demand.

Mitochondrial Uncoupling Protein 1 Expressed in the Heart of Transgenic Mice Protects Against Ischemic-Reperfusion Damage

Background—Mitochondrial respiration is the main source of energy in aerobic animal cells and is adapted to the energy demand by respiratory coupling. Uncoupling proteins (UCPs) perturb respiratory coupling by inducing a proton leak through the mitochondrial inner membrane. Although this could lead to deleterious energy waste, it may prevent the production of oxygen radicals when the rate of phosphorylation of ADP into ATP is low, whereas oxygen and substrate availability to mitochondria is high. The latter conditions are encountered during cardiac reperfusion after ischemia and are highly relevant to heart infarction. Methods and Results—Heart function of 6 transgenic mice expressing high amounts of UCP1 and of 6 littermate controls was compared in isolated perfused hearts in normoxia, after 40-minute global ischemia, and on reperfusion. In normoxia, oxygen consumption, contractility (quantified as the rate-pressure product), and their relationship (energetic yield) were similar in controls and transgenic mice. Although UCP1 expression did not alter the sensitivity to ischemia, it significantly improved functional recovery on reperfusion. After 60 minutes of reperfusion, contractility was 2-fold higher in transgenic mice than in controls. Oxygen consumption remained significantly depressed in controls (53±27% of control), whereas it recovered strikingly to preischemic values in transgenic mice, showing uncoupling of respiration by UCP1 activity. Glutathione and aconitase, markers of oxidative damage, indicated lower oxidative stress in transgenic mice. Conclusions—UCP1 activity is low under normoxia but is induced during ischemia-reperfusion. The presence of UCP1 mitigates reperfusion-induced damage, probably because it lowers mitochondrial hyperpolarization at reperfusion.

Perinatal growth of the rabbit cardiac cell: Possible implications for the mechanism of relaxation

Abstract Stereological analyses were performed on the right papillary muscle of fetal, newborn and adult rabbit heart and on the ventricular trabeculum of frog heart. Cell surface to volume ratio ( S V ) was high in the fetus (1.03 μm −1 ) comparable to that observed in the frog heart (1.32 μm −1 ) and remained stable in the newborn up to 8 days post-partum. At these ages there were no T-tubules. From 8 days post-partum onwards, cell diameter increased and S V ratio decreased progressively towards the adult value (0.30 μm −1 and 0.47 μm −1 including T-tubular area). A possible implication of the marked decrease of S V on cardiac mechanical activity was investigated. Unlike the relaxation of adult mammalian heart, the relaxation of the fetal heart was found to be sensitive to a reduction of NaCa exchange by low-Na solution as already shown in frog heart relaxation. During growth, the involvement of NaCa exchange in the relaxation of perinatal heart decreased and was progressively masked by a faster Ca-sequestration by the sarcoplasmic reticulum and/or other systems.

Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8)

Hearts from AC8TG mice develop a higher contractility (LVSP) and larger Ca2+ transients than NTG mice, with (surprisingly) no modification in L‐type Ca2+ channel current (ICa,L) (1). In this study, we examined the cardiac response of AC8TG mice to β‐adrenergic and muscarinic agonists and IBMX, a cyclic nucleotide phosphodiesterase (PDE) inhibitor. Stimulation of LVSP and ICa,L by isoprenaline (ISO, 100 nM) was twofold smaller in AC8TG vs. NTG mice. In contrast, IBMX (100 µM) produced a twofold higher stimulation of ICa,L in AC8TG vs. NTG mice. IBMX (10 µM) increased LVSP by 40% in both types of mice, but contraction and relaxation were hastened in AC8TG mice only. Carbachol (10 µM) had no effect on basal contractility in NTG hearts but decreased LVSP by 50% in AC8TG mice. PDE assays demonstrated an increase in cAMP‐PDE activity in AC8TG hearts, mainly due to an increase in the hydrolytic activity of PDE4 and PDE1 toward cAMP and a decrease in the activity of PDE1 and PDE2 toward cGMP. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L‐type Ca2+ channels and protects the cardiomyocytes from Ca2+ overload.—Georget, M., Mateo, P., Vandecasteele, G., Lipskaia, L., Defer, N., Hanoune, J., Hoerter, J., Lugnier, C., Fischmeister, R. Cyclic AMP compartmentation due to increased cAMP‐phosphodiesterase activity in transgenic mice with a cardiac‐directed expression of the human adenylyl cyclase type 8 (AC8). FASEB J. 17, 1380–1391 (2003)

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.

AMP-Activated Protein Kinase α2 Deficiency Affects Cardiac Cardiolipin Homeostasis and Mitochondrial Function

AMP-activated protein kinase (AMPK) plays an important role in controlling energy homeostasis and is envisioned as a promising target to treat metabolic disorders. In the heart, AMPK is involved in short-term regulation and in transcriptional control of proteins involved in energy metabolism. Here, we investigated whether deletion of AMPKα2, the main cardiac catalytic isoform, alters mitochondrial function and biogenesis. Body weight, heart weight, and AMPKα1 expression were similar in control littermate and AMPKα2−/− mice. Despite normal oxygen consumption in perfused hearts, maximal oxidative capacity, measured using saponin permeabilized cardiac fibers, was ∼30% lower in AMPKα2−/− mice with octanoate, pyruvate, or glutamate plus malate but not with succinate as substrates, showing an impairment at complex I of the respiratory chain. This effect was associated with a 25% decrease in mitochondrial cardiolipin content, the main mitochondrial membrane phospholipid that is crucial for complex I activity, and with a 13% decrease in mitochondrial content of linoleic acid, the main fatty acid of cardiolipins. The decrease in cardiolipin content could be explained by mRNA downregulation of rate-limiting enzymes of both cardiolipin synthesis (CTP:PA cytidylyltransferase) and remodeling (acyl-CoA:lysocardiolipin acyltransferase 1). These data reveal a new role for AMPKα2 subunit in the regulation of cardiac muscle oxidative capacity via cardiolipin homeostasis.

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 three 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.

Augmentation of cardiac contractility with no change in L-type Ca2+ current in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8)

The β‐adrenergic cascade is severely impaired in heart failure (HF), in part because of a reduction in the activity of the two dominant cardiac adenylyl cyclase (AC) isoforms, AC5 and AC6. Hence, cardiac‐directed AC overexpression is a conceivable therapeutic strategy in HF. In this study, we explored the consequences at the cellular and organ level of a cardiac‐directed expression of the human AC8 in the transgenic mouse line AC8TG. Unlike AC5 and AC6, which are inhibited by intracellular Ca2+, AC8 is stimulated by Ca2+‐calmodulin. Langendorff perfused hearts from AC8TG mice had a twofold higher left ventricular systolic pressure, a 40% faster heart rate, a 37% faster relaxation, and a 30% higher sensitivity to external Ca2+ than nontransgenic control mice (NTG). Cell shortening measured in isolated ventricular myocytes developed 22% faster and relaxed 43% faster in AC8TG than in NTG mice. Likewise, Ca2+ transients measured in fluo‐3 AM‐loaded myocytes were 30% higher and relaxed 24% faster in AC8TG compared with NTG mice. In spite of the large increase in Ca2+ transients and contraction, expression of AC8 had no effect on the whole‐cell L‐type Ca2+ current (ICa,L) amplitude. Moreover, ICa,L was unchanged even when AC8 was activated by raising intracellular Ca2+. Thus, cardiac expression of AC8 leads to an increase in cAMP that activates specifically Ca2+ uptake into the sarcoplasmic reticulum but not Ca2+ influx at the sarcolemma, suggesting a strong compartmentation of the cAMP signal.

Developmental Changes in Effects of Halothane and Isoflurane on Contractile Properties of Rabbit Cardiac Skinned Fibers

Immature hearts of various animal species and humans have been demonstrated to be more sensitive than adult hearts to the myocardial depressant effects of volatile anesthetics. To further investigate the mechanisms involved, the calcium sensitivity and maximal activated tension of detergent-treated left ventricular fibres of fetuses (30 days), newborn (1-day-old), immature (3-, 8-, and 17-day-old), and adult rabbits were determined by stepwise exposure to increasing Ca2+ concentrations. Responses were measured prior to and after exposure to equianesthetic concentrations of halothane (1%) or isoflurane (1.5%) applied in a random order. In control conditions maximal developed tension was the lowest in fetuses (11.1 +/- 0.6 mN.mm-2), intermediate in newborn and immature rabbits, and highest in adults (25.6 +/- 2.9 mN.mm-2). There were also age-related changes in calcium sensitivity; pCa (= -log10[Ca2+]) for half-activation (pCa50) was significantly less in 1-, 3-, and 8-day-old rabbits (5.444 +/- 0.036, 5.425 +/- 0.017, and 5.385 +/- 0.019, respectively) than in adults (5.517 +/- 0.010), whereas it was not different in fetuses (5.521 +/- 0.017). During anesthetic exposure both calcium sensitivity and maximal developed tension decreased significantly in all age groups of animals, with both anesthetics having a similar effect in animals of identical age. However, calcium sensitivity decreased significantly more in newborn animals (0.192 and 0.196 pCa unit for halothane and isoflurane, respectively) compared with adults (0.122 and 0.137 pCa units, respectively). By contrast, fetuses were less sensitive to the myocardial depressant effects of anesthetics than were newborn animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of energy transfer pathways between mitochondria and myofibrils by changes in performance of perfused heart.

In the heart, the energy supplied by mitochondria to myofibrils is continuously and finely tuned to the contraction requirement over a wide range of cardiac loads. This process is mediated both by the creatine kinase (CK) shuttle and by direct ATP transfer. The aim of this study was to identify the contribution of energy transfer pathways at different cardiac performance levels. For this, five protocols of 31P NMR inversion and saturation transfer experiments were performed at different performance levels on Langendorff perfused rat hearts. The cardiac performance was changed either through variation of external calcium in the presence or absence of isoprenaline or through variation of LV balloon inflation. The recordings were analyzed by mathematical models composed on the basis of different energy transfer pathway configurations. According to our results, the total CK unidirectional flux was relatively stable when the cardiac performance was changed by increasing the calcium concentration or variation of LV balloon volume. The stability of total CK unidirectional flux is lost at extreme energy demand levels leading to a rise in inorganic phosphate, a drop of ATP and phosphocreatine, a drop of total CK unidirectional flux, and to a bypass of CK shuttle by direct ATP transfer. Our results provide experimental evidence for the existence of two pathways of energy transfer, direct ATP transfer, and PCr transfer through the CK shuttle, whose contribution may vary depending on the metabolic status of the heart.