Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes

Abstract

We measured mitochondrial NADH autofluorescence or Ca(2+) using Rhod-2, simultaneously with cell shortening in isolated guinea-pig ventricular myocytes. When both frequency and amplitude of twitch shortening (work intensity) were increased by raising stimulus frequency in incremental steps from 0.1 to 3.3 Hz, the steady level of NADH signal increased in a frequency-dependent manner. Mitochondrial Ca(2+) also increased with increasing work intensity. Applying Ru360, an inhibitor of mitochondrial Ca(2+) uniporter, largely attenuated the response of both NADH fluorescence and mitochondrial Ca(2+). The increase in mitochondrial Ca(2+) was slow with t(1/2)=~12 s and no obvious cyclic changes were observed in the NADH signal. When a step change from 0.1 to 3.3 Hz stimulation was applied, the NADH signal first decreased to 83% and then increased to 155% of the control level. Upon returning to 0.1 Hz, the NADH signal showed an overshoot before declining to the control level. The biphasic onset time course was well explained by the delayed Ca(2+) activation of the substrate dehydrogenation superimposed on the feedback control of the ATP synthesis, while the offset time course with a delayed deactivation of dehydrogenation. A computer simulation using an oxidative phosphorylation linked to the cardiac excitation contraction model well reconstructed the response of NADH. This model simulation predicts that the activation of substrate dehydrogenation provides ~23% of driving force of the ATP synthesis to meet the increased workload induced by the jump of stimulus from 0.1 to 3.3 Hz, and remaining ~77% is supplied by the feedback control.

Excitation-contraction coupling and mitochondrial energetics

“…Similarly, cytoplasmic high energy phosphate buffer systems (e.g., creatine kinase (CK) isoenzymes and the rapidly diffusable phosphocreatine, PCr) are present that can limit changes in bulk ATP while shuttling ADP to the mitochondrial adenine nucleotide transporter (ANT), effectively transferring the energetic signal from the site of ATP hydrolysis to the mitochondrion (reviewed in [164]). Hence, in agreement with recent experimental and computational evidence, Ca 2+ , ADP, and P i are likely to regulate respiration in a complementary way at sites both upstream and downstream of the respiratory chain, thus providing a balanced availability of ATP and reduced NADH [25,26,45,46,101,117,164] (see also further below in Bioenergetic consequences of mitochondrial Ca 2+ uptake).…”

“…These differences may be related to the fact that in prestretched cardiac trabeculae [26], work during contraction may be severalfold higher than in unstretched single myocytes. Thus, ADP-related oxidation of NADH was less pronounced in single cells [101,117] Fig. 9B; [101,117]).…”

“…9B; [101,117]), albeit with different relative amounts of the initial oxidation-and the secondary recovery phases, respectively. These differences may be related to the fact that in prestretched cardiac trabeculae [26], work during contraction may be severalfold higher than in unstretched single myocytes.…”

“…During CICR, bulk cytosolic Ca 2+ can be completely buffered with no effect of local EC coupling, as demonstrated in studies of "Ca 2+ spikes" [173,183]. Similarly, cytoplasmic high energy phosphate buffer systems (e.g., creatine kinase (CK) isoenzymes and the rapidly diffusable phosphocreatine, PCr) are present that can limit changes in bulk ATP while shuttling ADP to the mitochondrial adenine nucleotide transporter (ANT), effectively transferring the energetic signal from the site of ATP hydrolysis to the mitochondrion (reviewed in [164] [25,26,45,46,101,117,164] (see also further below in Bioenergetic consequences of mitochondrial Ca 2+ uptake). …”

“…In an earlier set of studies, White and Wittenberg [214,215] had observed that in response to 5 Hz stimulation, NADH was oxidized at 95% O 2 , whereas it was reduced at 20% O 2 . They concluded that if O 2 availability limits the rate of respiration (i. e., at 20% O 2 ), then the equilibrium of the NADH/NAD + redox potential is shifted towards NADH reduction by Ca 2+ -stimulated TCA cycle dehydrogenases, and away from respiratory chain-induced oxidation [214,215].The transient behavior of NADH determined experimentally [23,25,26,101,117,214,215] could be reproduced by computational modeling in different studies [45,46,101]. In our recent model that integrates the processes of EC coupling and mitochondrial bioenergetic responses of the cardiac myocyte [46], the transient changes of NADH and [Ca 2+ ] m in response to a change of workload (Fig.…”