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 (31)P 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.

Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases.

Steady-state activation of cardiac beta-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer-based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca(2+) channel current (I(Ca,L)) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. I(Ca,L) amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for approximately 5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 micromol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 micromol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 micromol/L), cAMP signals and I(Ca,L) declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to approximately 50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct beta-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.

31P NMR detection of subcellular creatine kinase fluxes in the perfused rat heart: contractility modifies energy transfer pathways.

The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by (31)P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, F(f) = PCr --> ATP, and reverse flux, F(r) = ATP --> PCr = 3.3 mm.s(-1)), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (F(f) = 1.7 x F(r)), and (iii) a mitochondrial CK displaced toward PCr synthesis (F(f) = 0.3 and F(r) = 2.6 mm.s(-1)). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed.

Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels.

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.