Modular organization of cardiac energy metabolism: energy conversion, transfer and feedback regulation

Abstract

To meet high cellular demands, the energy metabolism of cardiac muscles is organized by precise and coordinated functioning of intracellular energetic units (ICEUs). ICEUs represent structural and functional modules integrating multiple fluxes at sites of ATP generation in mitochondria and ATP utilization by myofibrillar, sarcoplasmic reticulum and sarcolemma ion-pump ATPases. The role of ICEUs is to enhance the efficiency of vectorial intracellular energy transfer and fine tuning of oxidative ATP synthesis maintaining stable metabolite levels to adjust to intracellular energy needs through the dynamic system of compartmentalized phosphoryl transfer networks. One of the key elements in regulation of energy flux distribution and feedback communication is the selective permeability of mitochondrial outer membrane (MOM) which represents a bottleneck in adenine nucleotide and other energy metabolite transfer and microcompartmentalization. Based on the experimental and theoretical (mathematical modelling) arguments, we describe regulation of mitochondrial ATP synthesis within ICEUs allowing heart workload to be linearly correlated with oxygen consumption ensuring conditions of metabolic stability, signal communication and synchronization. Particular attention was paid to the structure-function relationship in the development of ICEU, and the role of mitochondria interaction with cytoskeletal proteins, like tubulin, in the regulation of MOM permeability in response to energy metabolic signals providing regulation of mitochondrial respiration. Emphasis was given to the importance of creatine metabolism for the cardiac energy homoeostasis.

Keywords: cardiac metabolism; creatine kinase; mitochondria; respiration regulation.

Figure 1

Mitochondria distribution in adult rat cardiomyocyte. (a) Individual mitochondria, visualized by green autofluorescence of flavoproteins, are localized at the A-band level of sarcomere. Immunofluorescent labelling of α-actinin (red colour) was used to mark sarcomeric Z-lines. (b) Fluorescence intensity plot shows peaks of mitochondrial flavoproteins intensities (dotted line) which correspond to regions of ‘zero’ intensity of α-actinin (solid line). Reproduced from Gonzalez-Granillo et al. (2012) with permission.

Figure 2

Intracellular energetic units (ICEUs). ICEUs include sites of ATP hydrolysis (myofibrillar ATPases, sarcoplasmic reticulum ATPase, ion-pump ATPase) which are relied to the mitochondrial ATP synthesis through the creatine kinase (CK)/PCr network. Metabolic or randle cycles include free fatty acids (FFA) and glucose (GLU) transport and degradation. FFA is taken up by a family of plasma membrane proteins (fatty acid transporter protein, FATP1, fatty acid translocase, CD36) and esterified to acyl-CoA via fatty acyl-CoA synthetase. The resulting acyl-CoA is then transported into mitochondria via carnitine palmitolyltransferase I (CPT and CPT II). Once inside, acyl-CoA becomes a substrate for the β-oxidation pathway (β-FAO), resulting in the production of one molecule of NADH, one molecule of FADH₂, and one molecule of Acetyl-CoA. Acetyl-CoA enters the TCA cycle, where it is further oxidized to CO₂ with the concomitant generation of three molecules of NADH, one molecule of FADH₂ and one molecule of ATP. GLU is taken up by glucose transporter-4 (GLUT4) and enters the glycolysis pathway, which converts glucose into two molecules of pyruvate (PYR), two net ATP and two NADH. NADH is transferred into mitochondria via the malate–aspartate shuttle (mal/asp shuttle). OAA, oxaloacetate. Pyruvate enters into the Krebs cycle and oxidative phosphorylation (OxPhosph) via Acetyl-CoA. NADH and FADH₂ issued from both metabolic pathways are oxidized in the respiratory chain. Mitochondrial creatine kinase (MtCK) catalyses the direct transphosphorylation of intramitochondrial ATP and cytosolic creatine (Cr) into ADP and phosphocreatine (PCr). ADP enters the matrix space to stimulate OxPhosph, while PCr is transferred via the cytosolic Cr/PCr shuttle to be used in the functional coupling between CK and ATPases (acto-myosin ATPase and ion pumps). Feedback regulation of mitochondrial ATP synthesis is performed by Cr/Pc, ADP, Pi ratios. Cell signaling via AMP kinase (AMPK) provides a parallel control of most of these processes, including substrate uptake via fatty acid and glucose transporters and flux via β-FAO and glycolysis. Reproduced from Saks et al. (2014) with permission.

Figure 3

Kinetic properties of mitochondrial creatine kinase (MtCK) in situ in permeabilized adult primary cardiomyocytes. (a) The experimental procedure used for the complete kinetic analysis of MtCK in permeabilized adult primary cardiomyocyte. The left scale and the blue trace indicate the oxygen concentration (nmolO₂ mL ⁻¹). The right scale and the red trace show the rate of oxygen uptake expressed in nmolO₂ min⁻¹ nmol⁻¹ cyt. aa3. The experiment was carried out in solution containing 5 mm glutamate/2 mm malate as respiratory substrates. First, the respiration is activated by addition of MgATP-inducing production of endogenous ADP in MgATPase reaction. Then, phosphoenolpyruvate–pyruvate kinase (PEP–PK) system is added to trap all extramitochondrial free ADP. This decreases the respiration rate, but not to initial level, due to structural organization of intracellular energetic unit (ICEU). Mitochondria are in privileged position to trap some of endogenous ADP. Addition of creatine activates MtCK reaction. The oxidative phosphorylation (OxPhosph) is stimulated mostly by intramitochondrial ADP, produced by MtCK, which is not accessible for PEP–PK. (b) Measurement of mitochondrial membrane potential (DΨ_m) using the TMRM flurescence was applied to show the control that Cr exerts on the respiration. TMRM is a positively charged lipophyle fluorescent probe that enters into negatively charged matrix when the inner membrane is energized. Incubation of permeabilized adult cardiomyocytes with TMRM gives the detectable level of its fluorescence outside the mitochondria. Respiratory substrates induce membrane polarization corresponding to state 2 of respiration according to Chance. The addition of ATP induced small change in mitochondrial membrane potential. The remove of ADP by PEP–PK system induces state 4 of respiration and complete membrane polarisation. Cr strongly increases respiration corresponding to state 3 and rapid decrease in DΨ_m due to the phosphorylation of ADP produced in activated MtCK reaction and transferred into the matrix due to MtCK/adenine nucleotide translocase (ANT) functional coupling. (c) Kinetic mechanism of MtCK reaction (bi-bi, random quasi-equilibrium type according to Cleland classification). Scheme shows the interconversion of productive ternary enzyme–substrate (CK.Cr.MgATP) and enzyme–product (CK.PCr.MgADP) complexes in the presence of MgATP²⁻, MgADP⁻, Cr and phosphocreatine (PCr). Every substrate and products is characterized by two constants of dissociation. (d) Measurement of ATP and PCr production during the stepwise addition of Cr (a) using ion pair HPLC/UPLC. Experiments were performed under conditions of activated (full circles) and inhibited (empty circles) complex I of the respiratory chain. The ATP level was stable during the experiment, while PCr production continuously increased. Adapted from Guzun et al. (2009) and Timohhina et al. (2009).

Figure 4

Confocal microscopy images of mitochondria [coimmunolabelled for voltage-depending anion channel (a, d)], α-actinin (b) and βII-tubulin (e) arrangement in adult cardiac muscle fibres. Images (c, f) show that both mitochondria and βII-tubulin are arranged regularly between Z-lines. Scale bar 2 µm. Reproduced from Varikmaa et al. (2014) with permission.

Figure 5

Role of mitochondrial outer membrane permeability and ADP signalling in the regulation of respiration of permeabilazed adult cardiomyocytes. (■) stepwise addition of MgATP activates respiration due to the production of endogenous extramitochondrial ADP. The apparent K_m(ATP) is equal to 158 ± 40.1 µm because of the restriction of ADP diffusion in situ. (●) stepwise addition of MgATP in the presence of 20 mm of creatine increases rapidly respiration rates due to the stimulation by endogenous extra- and intramitochondrial ADP decreasing the apparent K_m(ATP) to 24 ± 0.8 µm. (blue ▲) removal of extramitochondrial ADP by phosphoenolpyruvate –pyruvate kinase system mitochondrial creatine kinase increases significantly the apparent K_m(ATP) to 2 mm because of restricted diffusion. Reproduced from Guzun et al. (2009) with permission.

Figure 6

‘Mitochondrial Interactosome’ includes ATP synthasome formed by ATP synthase, adenine nucleotide translocase (ANT) and inorganic phosphate carrier (PiC), mitochondrial creatine kinase (MtCK) functionally coupled to ATP synthasome and voltage-dependent anion channel (VDAC) with regulatory proteins (βII-tubulin and other linker proteins). ATP regenerated by ATP synthase is transferred to MtCK due to its functional coupling with ATP syntasome. MtCK transfers the phosphate group from ATP to creatine. Produced PCr leaves mitochondria as a main energy flux due to high selective permeability of VDAC. Recycled ADP is returned to ATP syntasome via the functional coupling. Small signalling amounts of ADP can reach ATP synthase. Regenerated ATP is transferred directly to MtCK which recycles continuously the ADP maintaining production of the PCr energy flux. In this way, MtCK amplifies cytosolic ADP signal. Reproduced from Timohhina et al. (2009) with permission.

Figure 7

Development of intracellular energy units in cardiomyocytes isolated from rat hearts. (a–c) Immunofluorescent confocal micrograph of developmental distribution of mitochondria and βII-tubulin in 8-day-old cardiomyocytes coimmunolabelled for voltage-depending anion channel (VDAC) (a) and βII-tubulin (b). The lower panel (c) shows the colocalization of VDAC and βII-tubulin in the 8-day-old rat heart cells. Magnification bar: 2 µm. For immunolabelling mouse monoclonal antibody for βII-tubulin (Abcam, ab28036, Cambridge, UK) and the rabbit polyclonal serum for VDAC (kindly provided Dr. Catherine Brenner, Universite Paris-Sud, Paris, France) were used. Fluorescence images were acquired by Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a Plan-Apofluar 639/1.30 glycerol objective. (d) Relationship between content of mitochondrial creatine kinase (MtCK) and activation of the MtCK – phosphoryl transfer system during rat heart postnatal development. Ordinate – percentage of MtCK protein expression relative to adult – densitometric quantification of Western blot. Abscissa – stimulatory effect of creatine on the respiration rate of cardiomyocytes in the presence of 2 mm ATP and the trapping system for exogenous ADP (10 IU mL⁻¹ of the pyruvate kinase (PK) and 5 mm of the phosphoenolpyruvate (PEP). Under these conditions, changes in kinetics of respiration follow the kinetics of MtCK reaction. V_max(ADP) – theoretical maximal respiration rate in the presence of exogenous ADP (see Table 2).

Figure 8

Mechanisms of regulation of respiration controlled by MtCK. (a) Mathematically modelled oscillations of ADP concentrations in the core of myofibrils over cardiac cycle at workloads equivalent to 750 (black), 1500 (red) and 2250 (green) µmol ATP s⁻¹ kg⁻¹. (b) Graphical Michaelis–Menten representation of the dependence of mitochondrial respiration rate on the concentration of ADP. Coloured arrows on X-axes show ADP concentrations corresponding to increased workloads from panel (a). In isolated mitochondria (permeable mitochondrial outer membrane), ADP concentration corresponding to minimal workload falls in the region of maximal respiratory rate (saturated concentration) and does not allow any regulation. The apparent K_m for ADP in isolated mitochondria is 7.9 ± 1.6 lM. In permeabilized cardiomyocytes (restricted diffusion), the respiration rates become linearly dependent on ADP concentrations in agreement with heart workloads (Fig. 8a, c). The apparent K_m for ADP in permeabilized cardiomyocytes is 370.8 ± 30.6 µm. This linear dependence is amplified by creatine in the presence of activated MtCK. The apparent K_m for ADP in the presence of creatine decreases up to 50.2 ± 8.0 µm. (c) Linear increase in oxygen consumption rates as a function of increased relative workload. Reproduced from Saks et al. (2012) with permission.