Excitation-contraction coupling and mitochondrial energetics

Abstract

Cardiac excitation-contraction (EC) coupling consumes vast amounts of cellular energy, most of which is produced in mitochondria by oxidative phosphorylation. In order to adapt the constantly varying workload of the heart to energy supply, tight coupling mechanisms are essential to maintain cellular pools of ATP, phosphocreatine and NADH. To our current knowledge, the most important regulators of oxidative phosphorylation are ADP, Pi, and Ca2+. However, the kinetics of mitochondrial Ca2+-uptake during EC coupling are currently a matter of intense debate. Recent experimental findings suggest the existence of a mitochondrial Ca2+ microdomain in cardiac myocytes, justified by the close proximity of mitochondria to the sites of cellular Ca2+ release, i. e., the ryanodine receptors of the sarcoplasmic reticulum. Such a Ca2+ microdomain could explain seemingly controversial results on mitochondrial Ca2+ uptake kinetics in isolated mitochondria versus whole cardiac myocytes. Another important consideration is that rapid mitochondrial Ca2+ uptake facilitated by microdomains may shape cytosolic Ca2+ signals in cardiac myocytes and have an impact on energy supply and demand matching. Defects in EC coupling in chronic heart failure may adversely affect mitochondrial Ca2+ uptake and energetics, initiating a vicious cycle of contractile dysfunction and energy depletion. Future therapeutic approaches in the treatment of heart failure could be aimed at interrupting this vicious cycle.

Fig. 1

Overview on processes of excitation-contraction coupling and mitochondrial energetics. SR sarcoplasmic reticulum; SERCA SR Ca²⁺ ATPase; Mito mitochondria; TCA tricarboxylic acid cycle; RC respiratory chain; Δψ_m mitochondrial membrane potential; NCE mitochondrial Na⁺/Ca²⁺-exchanger; NHE mitochondrial Na⁺/H⁺ -exchanger; NKA sarcolemmal Na⁺/K⁺-ATPase; NCX sarcolemmal Na⁺/Ca²⁺-exchanger; RyR2 ryanodine receptor type 2; mCU mitochondrial Ca²⁺-uniporter; I_Na and I_Ca, currents of voltage-gated Na⁺- or Ca²⁺-channels, respectively

Fig. 2

Longitudinal (A) or scanning (B) electron micrographs of cardiac myocytes. M mitochondria; SR sarcoplasmic reticulum. With permission from Territo et al. [194] and Yoshikane et al. [220], respectively

Fig. 3

(A) Enzymatic reactions of the tricarboxylic acid (TCA) cycle, and their regulation by Ca²⁺. DH dehydrogenase. (B) Stimulation of pyruvate-dehydrogenase phosphatase (PDH-PPase), NAD-isocitrate dehydrogeNase (NAD-ICDH) and α-ketoglutarate dehydrogenase (α-KGDH) enzymatic activity by Ca²⁺ in extracts of mitochondria. Activity in the presence of EGTA was ∼ 10–20% of maximal velocity (V_max), respectively. Reproduced from Denton & McCormack [54] with permission

Fig. 4

Processes of oxidative phosphorylation at the mitochondrial respiratory chain. I–V complexes I–V of the respiratory chain. Complex V F₁/F₀-ATPase; c cytochrome c. Electrons (e⁻) donated by NADH to complex I (and by succinate to complex II; not shown) elicit sequential redox reactions at complexes I–IV, promoting H⁺-translocation from the matrix to the intermembrane space across the inner mitochondrial membrane (IMM). This creates a proton gradient (ΔpH), which together with the electrochemical gradient (Δψ_m) constitutes the driving force for protons to flow back into the matrix space via F₁/F₀-ATPase, promoting ATP-production from ADP. At complex III (and I; not shown), the superoxide anion radical is produced already under physiological conditions. It is dismutated to hydrogen peroxide (H₂O₂) by Mn²⁺-dependent superoxide-dismutase (Mn-SOD), which is eliminated to H₂O by glutathione-peroxidase (GPX). While Ca²⁺ activates (+) TCA-cycle dehydrogenases (but also complex V of the respiratory chain; not shown), ADP regulates respiration by increasing complex V activity (+)

Fig. 5

[Ca²⁺]_c and [Ca²⁺]_m during a single contraction in the presence and absence of norepinephrine (NE): myocyte loaded with indo-1/salt (A); myocyte loaded with indo-1/AM and quenched with Mn²⁺ (B). A and B each show results from a single cell, stimulated at steady-state 0.2 Hz in presence and absence of 1 µM NE. It is seen that there is no response of [Ca²⁺]_m to a single electrical stimulation. (C) Kinetics of rise and fall of [Ca²⁺]_m in response to changed pacing rate. Top: [Ca²⁺]_m for a single myocyte, superfused with medium containing 1 µM NE; bottom: mechanical response of cell to electrical stimulation. At 1^st arrow, stimulation frequency was increased from 0 to 4 Hz; at 2^nd arrow, cell begins to contract in synchrony with 4 Hz stimulation; at 3^rd arrow, pacing was discontinued. (D) Relation between [Ca²⁺]_m and mean [Ca²⁺]_c during Na⁺-replacement experiments with slow cytosolic and mitochondrial Ca²⁺ uptake. Reproduced with permission from Miyata et al. [137]

Fig. 6

(A-D) Representative experiment determining [Ca²⁺]_c and [Ca²⁺]_m in the same cell. [Ca²⁺]_c transients were elicited at 1 Hz in the absence and presence of isoproterenol (Iso, 1–100 nmol/L; (A), (C)). The [Ca²⁺] transients in (B) and (D) were recorded at the indicated time points of the protocol (arrows in (A), (C)). (E) and (F) Cytosolic (E) and mitochondrial Ca²⁺-transients (F) in the absence and presence of Ru360 (10 nmol/L)

Fig. 7

Full activation of mitochondrial Ca²⁺ uptake during RyR-mediated Ca²⁺ release. (A) Ca²⁺- and caffeine induced [Ca²⁺]_m and Δψ_m responses were measured in permeabilized myotubes using rhod-2 and TMRM, respectively. Uncoupler (1 µM CCCP and 2.5 µg/ml oligomycin) was added as indicated. (B) The rate of mitochondrial Ca²⁺ uptake was measured at varying [Ca²⁺] obtained by the addition of CaCl₂ in adherent rhod-2 loaded permeabilized cells. The added CaCl₂ concentration values are indicated with arrows below the x axis, and the effective [Ca²⁺]_c were calculated. To prevent Ca²⁺-induced Ca²⁺-release via RyR2, the cells were preincubated with thapsigargin to inhibit the SR Ca²⁺ ATPase and hence to deplete the SR prior to Ca²⁺ addition. Reproduced from Szalai et al. [191] with permission

Fig. 8

Computational modeling of [Ca²⁺]_m, assuming a microdomain (MD) in which mitochondria are exposed to [Ca²⁺] pulses of 10–20 µmol/L for 50 ms (D; representative pulse taken from t = 215 s in A). Starting from steady-state conditions with [Ca²⁺]_MD oscillating from 0.1 to 10 µmol/L at 1 Hz, an increase of amplitude (A, from 10 to 20 µmol/L), frequency (B, from 1 to 2 Hz) or both (C) of [Ca²⁺]_MD transients were simulated. Reproduced from Maack et al. [117] with permission

Fig. 9

Time-dependent behavior of [Ca²⁺]_m and NADH after changes in workload. (A) Experimental data showing the responses of NADH (upper panel) and [Ca²⁺]_m (lower panel) to an increase in stimulation frequency from 0.5 to 2 Hz in a rat cardiac trabecula. (B) NADH (upper panel) and [Ca²⁺]_m (lower panel) in response to an increase of stimulation frequency from 0 to 4 Hz in the presence of isoproterenol (100 nM). (C) Computational modeling of NADH and [Ca²⁺]_m by integrating the kinetics of EC coupling, mitochondrial Ca²⁺ transporters and energetics in response to a change in stimulation frequency from 0.25 Hz to 2 Hz (for parameters, see [46]). I, undershoot; II, recovery, and III, overshoot of NADH. Reproduced from Brandes and Bers (A; [26]), Maack et al. (B; [117]) and Cortassa et al. (C; [46]) with permission

Fig. 10

Effect of changing the actomyosin- (AM-) ATPase and mCU on the computational model (described by Cortassa et al. [46]; compare Fig. 9C) on energetic behavior after changes in workload. (A) The behavior of NADH with the maximal rate of ATP hydrolysis by the AM-ATPase being decreased to onehalf (solid trace; V_max/AM = 3.6·10⁻³ mM ms⁻¹) of the control value (shaded trace). (B) The profile of average ATP_i for changes in workload under control conditions (shaded trace) or with decreased AM-ATPase activity (solid trace). (C) The NADH profile with the maximal rate of the mCU decreased to 1/10^th (solid trace; V_max/mCU = 2.75·10⁻³ mM ms⁻¹) of the control value (shaded trace). (D) The profile of [Ca²⁺]_m accumulation for the low mCU condition described in (C) (solid trace) as compared to the control (shaded trace). Reproduced from Cortassa et al. [46] with permission

Fig. 11

Stimulation of α-ketoglutarate oxidation by Ca²⁺ in the absence and presence of Na⁺ (15 mM) and the mCU-inhibitor ruthenium red (RR). Reproduced from Denton et al. [55] with permission

Fig. 12

[Ca²⁺]_c (A), [Ca²⁺]_m (B) and reduced NADH (C) in guinea-pig cardiac myocytes paced at 3 Hz and exposure to 10 and 100 nM isoproterenol, with either 5 or 15 mM [Na⁺] in the pipet. Reproduced from Maack et al. [117] with permission

Fig. 13

Hypothetical vicious cycle (and potential targets for therapeutic interventions) of defects in EC coupling and energy depletion in chronic heart failure. PCr phosphocreatine; PLB phospholamban; SERCA SR Ca²⁺-ATPase