This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brandes, R. Articles by Bers, D. M. Search for Related Content PubMed PubMed Citation Articles by Brandes, R. Articles by Bers, D. M.

(Circulation Research. 1997;80:82-87.)
© 1997 American Heart Association, Inc.

Articles

Intracellular Ca²⁺ Increases the Mitochondrial NADH Concentration During Elevated Work in Intact Cardiac Muscle

Rolf Brandes, Donald M. Bers

the Department of Physiology, Loyola University Chicago, School of Medicine, Maywood, Ill. E-mail rbrande@luc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is not clear how mitochondrial energy production is regulated in intact tissue when energy consumption suddenly changes. Whereas mitochondrial [NADH] ([NADH]_m) may regulate cellular respiration rate and energetic state, it is not clear how [NADH]_m itself is controlled during increased work in vivo. We have varied work and [Ca²⁺] in intact cardiac muscle while assessing [NADH]_m using fluorescence spectroscopy. When increased work was accompanied by increasing average [Ca²⁺]_c (by increasing [Ca²⁺]_o or pacing frequency), [NADH]_m initially fell and subsequently recovered to a new steady state level. Upon reduction of work, [NADH]_m overshot and then returned to control levels. In contrast, when work was increased without increasing average [Ca²⁺]_c (by increasing sarcomere length), [NADH]_m fell similarly, but no recovery or overshoot was observed. This Ca²⁺-dependent recovery and overshoot may be attributed to Ca²⁺-dependent stimulation of mitochondrial dehydrogenases. We conclude that the immediate initial increase in respiration rate upon elevation of work is not activated by increased [NADH]_m (since [NADH]_m rapidly fell) or by [Ca²⁺]_c (since work could also be increased at constant [Ca²⁺]_c). However, during sustained high work, a Ca²⁺-dependent mechanism causes slow recovery of [NADH]_m toward control values. This demonstrates a Ca²⁺-dependent feed-forward control mechanism of cellular energetics in cardiac muscle during increased work.

Key Words: heart • force • ATP hydrolysis • oxidative phosphorylation • dehydrogenase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The regulation of oxidative phosphorylation has been the subject of extensive research.^{1 2} By use of isolated mitochondria, external conditions can easily be manipulated to study the effects of various parameters on the oxidative phosphorylation rate.¹ A disadvantage with this approach, however, is that it is not possible to accurately simulate in vivo conditions. In particular, during dynamic changes of the ATP consumption rate (eg, during increasing work), intracellular conditions are not at steady state, and these time-dependent changes may be critical for energetic regulation. To circumvent this problem, we have used intact cardiac muscle to evaluate the dynamic effects of changing work on the mitochondrial energy state during conditions of nonlimiting O₂ supply.

Although various parameters have been proposed to control flux through the respiratory chain, matching ATP production to energy demand, no general model has been agreed on.^{1 2} Nevertheless, most investigators believe that [O₂] is not limiting in vivo,¹ and in this case, most current theories and experimental results indicate that respiration rate (and therefore ATP production rate) depends on three cytosolic parameters (see Fig 1), [ADP], [ATP], and [P_i]³ (or combinations such as [ATP]/[ADP][P_i]⁴ or [ATP]/[ADP]⁵ ), and on [NADH]_m^{2 3 6} (or [NADH]_m/[NAD⁺]_m⁴ ). In particular, during conditions of nonlimiting [ADP], it has been suggested that [NADH]_m may exert strong control of the oxidative phosphorylation rate and cellular energetic state.^{2 3 7}

View larger version (36K): [in this window] [in a new window]   Figure 1. Increased [Ca2+]c causes increased work and ATP hydrolysis rate (by myofilament and transport ATPases). Consequently, increased ATP hydrolysis rate may cause changes in cytosolic phosphates (eg, increased [ADP] or [Pi]), and increased [Ca2+]c may cause increased [Ca2+]m. This suggests the following possible regulatory mechanisms: (1) Direct control of NADH production: the changes in cytosolic phosphates and/or increased [Ca2+] may cause increased mitochondrial NADH production rate by stimulating pyruvate dehydrogenase or tricarboxylic acid cycle enzymes. (2) Direct control of NADH consumption and oxidative phosphorylation: the changes in cytosolic phosphates and/or increased [Ca2+] may also cause increased mitochondrial NADH consumption rate by stimulating oxidative phosphorylation (eg, kinetic substrate control by ADP or by Ca2+ activation of the ATP synthase). (3) Indirect control of oxidative phosphorylation: if the NADH production rate is increased more than the NADH consumption rate, [NADH]m may increase. Such an increase of [NADH]m could, in turn, cause stimulation of oxidative phosphorylation. Acetyl-CoA indicates acetyl coenzyme A.

During increased work, either increased [ADP]/[ATP], [NAD⁺]_m/[NADH]_m, or [Ca²⁺]_m may cause increased NADH production rate⁸ and, consequently, [NADH]_m (provided that the [NADH]_m consumption rate is not correspondingly increased; see Fig 1). [Ca²⁺]_m has been implicated for two reasons. First, increases in [Ca²⁺]_c often accompany increased energy consumption (eg, during muscle contraction, hormone secretion, or neuronal activity), and increased [Ca²⁺]_c has been shown to increase [Ca²⁺]_m.⁹ Second, there is clear evidence from isolated mitochondria and isolated enzymes that Ca²⁺ can stimulate three key mitochondrial enzymes: pyruvate dehydrogenase, NAD-linked isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase.^{8 10 11} Thus, as outlined in Fig 1, when work is associated with increased [Ca²⁺]_c and, subsequently, [Ca²⁺]_m, NADH production may be stimulated.

We have previously shown that with nonlimiting O₂ supply in intact heart muscle, increased work caused decreased [NADH]_m and that prolonged stimulation caused a partial recovery of [NADH]_m.¹² However, we had no evidence regarding the mechanism of this recovery. We hypothesized that the recovery may have been caused by a work-dependent increase of [ADP]/ATP], [NAD⁺]_m/[NADH]_m, or [Ca²⁺]_m. Furthermore, during increased work in vivo, there is currently no direct evidence demonstrating a Ca²⁺-dependent stimulation of [NADH]_m or the role of Ca²⁺ in regulation of the oxidative phosphorylation rate. The main goal of the present study was therefore to explicitly test, for the first time, whether the recovery of [NADH]_m during sustained increases in work depended on Ca²⁺. This was examined by increasing work by Ca²⁺-dependent and Ca²⁺-independent means.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Trabeculae Preparation and Solutions
Thin multicellular trabeculae (200-µm diameter) were isolated from rat right ventricle as described previously.¹² Briefly, brown male LBN-F1 rats (370 to 510 g) were deeply anesthetized and anticoagulated by injecting 65 mg pentobarbital and 1000 U heparin intraperitoneally. The hearts were excised and then perfused at 28°C using the Langendorff method before removing the trabeculae. The perfusion solution contained (mmol/L) NaCl 108, KCl 21, MgCl₂ 1.2, CaCl₂ 0.5, NaHCO₃ 24, glucose 4, and sodium pyruvate 10, along with insulin (20 U/L), and was equilibrated with a 95% O₂/5% CO₂ gas mixture to produce pH 7.40 (at 24°C).

After dissection, the muscle was mounted in a muscle chamber, paced at 0.5 Hz, and superfused at 15 mL/min with the solution described above, except that 6 mmol/L KCl, instead of 21 mmol/L, was used and the temperature was 24°C. This solution was used for all experiments except for the [CaCl₂], which depended on protocol (see below). Muscle force was measured during isometric contractions, and sarcomere length was varied by adjusting muscle stretch. Sarcomere length was assessed from laser light (Uniphase 102-4) diffraction patterns, created by transmitting the light through the muscle.¹³

[NADH]_m Measurements
[NADH]_m was assessed using methods described previously.¹² Briefly, the trabeculae were excited by light at 350 nm, and fluorescence was detected at 385 and 456 nm. The fluorescence signal at 456 nm predominantly arises from mitochondrial NADH^{14 15} and motion artifact, whereas the reference signal at 385 nm mainly reflects motion artifact.¹² We used a previously developed method to eliminate the motion artifact from the fluorescence signal at 456 nm by dividing it with a modified reference signal.¹² [NADH]_m was calibrated to obtain the NADH ratio (NADH/NAD⁺) by using sodium cyanide to maximally reduce NAD⁺ and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to maximally oxidize it; see Brandes and Bers¹² for further technical details.

Assessment of ATP Hydrolysis Rate and Data Analysis
Time-averaged force was used as an index of work-related ATP hydrolysis rate.¹² This indirect and simplified measure assumes that the ATP hydrolysis rate is related to active force during the whole (isometric) contraction cycle.¹⁶ A 0.2-Hz low-pass filter was applied to the force signal to obtain average force by using software digital filtering (MicroCal Software Inc). The signal-to-noise ratio of the fluorescence ratio was similarly improved by digital low-pass filtering. Results were reported as mean±SE. Statistical analysis was performed using Student's t test (paired where applicable), and differences were considered significant at P<.05.

Experimental Protocols
To separate effects of Ca²⁺-dependent and Ca²⁺-independent mechanisms regulating [NADH]_m during altered work, three different types of protocol were used. In the first, various standard perfusion solutions with increasing [Ca²⁺]_o (0.3, 0.6, and 2.0 mmol/L) were used at constant pacing frequency (1 Hz) and maximum sarcomere length. This is expected to increase the intracellular Ca²⁺ transient amplitude and time-averaged [Ca²⁺]_c, as well as time-averaged force (work). In the second protocol, the pacing frequency was varied from 0.25 to 3 Hz at constant [Ca²⁺]_o (2 mmol/L) and maximum sarcomere length. This is also expected to increase the time-averaged [Ca²⁺]_c and work, although the individual Ca²⁺ transients may be less affected.¹⁷ In the third protocol, sarcomere length (and consequently myofilament overlap) was varied from a length (at peak of contraction) of 1.7 µm to maximum (2.1 to 2.2 µm) at constant pacing frequency (1 Hz) and [Ca²⁺]_o (2 mmol/L). This is expected to increase work without significantly altering the Ca²⁺ transient amplitudes.¹³

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺-Dependent Changes of Work
Increasing [Ca²⁺]_o is a direct way to increase both [Ca²⁺]_c transient amplitudes and work (energy consumption rate)* in cardiac muscle.^{18 19 20} Fig 2A indeed shows that when [Ca²⁺]_o was increased from 0.3 mmol/L to either 0.6 or 2 mmol/L at a constant stimulation frequency (1 Hz), the amplitude of the muscle force transients increased substantially. Because work was varied in different ways in the present study, time-averaged force (middle trace) was calculated and used as an index of muscle work or energy consumption rate (ATP hydrolysis rate).¹² Direct determination of ATP hydrolysis or O₂ consumption rates are not practical because of the small muscle size and kinetic resolution required. Furthermore, because the contractions are nearly isometric, the time-varying elastance model²¹ is not appropriate for estimating O₂ consumption. Both Cooper¹⁶ and Hasenfuss et al²² have shown that there is a linear relationship between energy consumption and the force-time integral, making time-averaged force a reasonable index of ATP hydrolysis rate and work. Furthermore, increased [Ca²⁺]_o, pacing frequency, and sarcomere length²³ are all known to increase the O₂ consumption rate, and this was reflected here as increased time-averaged force (see below for frequency and sarcomere length protocols).

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of transiently increased [Ca2+]o, pacing frequency, and sarcomere length (SL) on isometric force (top traces, in mN/mm2), work, assessed by calculating time-averaged force (middle traces, in mN/mm2) and [NADH]m (bottom traces, expressed as NADH/NAD+12 ). A, [Ca2+]o was raised from a control value of 0.3 mmol/L to either 0.6 or 2 mmol/L, causing increased time-averaged force (work) and [Ca2+]c. B, Pacing frequency was raised from a control value of 0.25 Hz to either 0.5, 1, or 2 Hz, causing increased work and time-averaged [Ca2+]c. C, SL was increased from a slack length of 1.7 µm up to that producing maximum force (2.1 to 2.2 µm). In contrast to panels A and B, increasing SL is expected to increase work without increasing [Ca2+]c significantly.

Fig 2A demonstrates that the increase in time-averaged force, or work, caused [NADH]_m to rapidly decrease to MIN. However, during sustained work elevation, [NADH]_m partially recovered to a new steady state level (SS) slightly below the original control value, creating an apparent undershoot. When [Ca²⁺]_o was reduced back to 0.3 mmol/L, there was a transient overshoot of [NADH]_m, causing it to reach MAX before it eventually returned to the original control level. These dynamic changes in [NADH]_m were more pronounced when increasing [Ca²⁺]_o from 0.3 to 2 mmol/L rather than 0.6.

Increased pacing frequency is a second direct way to increase both time-averaged [Ca²⁺]_c and work. Fig 2B shows that in contrast to increased [Ca²⁺]_o, increased pacing frequency did not dramatically alter the amplitudes of individual contractions.¹⁷ The slight negative force-frequency relationship typically observed in rat ventricle was also observed here and may be associated with a small reduction in individual Ca²⁺ transients.¹⁷ However, our large increase in the number of Ca²⁺ transients per unit time (twofold to eightfold) more than offset this effect, such that time-averaged [Ca²⁺]_c and force would be considerably elevated, as shown in Fig 2B. Fig 2B further demonstrates an undershoot and overshoot of [NADH]_m qualitatively similar to that seen for altered [Ca²⁺]_o (Fig 2A). Again, larger increments in work (average force) produced larger initial decreases in [NADH]_m (MIN) and larger overshoots (MAX) upon the abrupt reduction in work and ATP consumption rate.

The rapid initial decrease in [NADH]_m, upon a sudden increase in work using either protocol, can be explained by a rapid increase of NADH consumption via an increased oxidative phosphorylation rate, which is not matched by a corresponding stimulation of the NADH production rate (via the tricarboxylic acid cycle). That is, decreased [NADH]_m is a consequence of stimulation of oxidative phosphorylation by a parameter other than NADH. Furthermore, since [NADH]_m is lowered under these conditions, the initial increase in the oxidative phosphorylation rate cannot be controlled by increased [NADH]_m (or redox potential).

The recovery of [NADH]_m from the initial nadir, however, could reflect an additional mechanism that causes stimulation of NADH production, thereby opposing the initial decline of [NADH]_m. If this mechanism was also slow to deactivate, that would explain the overshoot as well (ie, maintained stimulation of NADH production after consumption had declined). We hypothesize that the [NADH]_m undershoot and overshoot are a consequence of slowly increasing and decreasing [Ca²⁺]_m, which in turn would be related to the time-averaged value of [Ca²⁺]_c.^{8 9} To test whether the initial decline and recovery of [NADH]_m are Ca²⁺ dependent, work alone was next altered by a protocol that does not alter time-averaged [Ca²⁺]_c.

Ca²⁺-Independent Changes of Work
To increase work without changing time-averaged [Ca²⁺]_c, the sarcomere length was abruptly increased, because this has previously been shown to result in unaltered [Ca²⁺]_c.^{13 24} Fig 2C shows the effects of increasing work by increasing sarcomere length from a slack control length to various lengths, up to that generating maximum force, using constant frequency (1 Hz) and [Ca²⁺]_o (2 mmol/L). At each length, the force transient amplitudes remained constant, suggesting that the Ca²⁺-transient amplitudes were also constant. The immediate response to the stretch is similar to that obtained by Allen and Kurihara,²⁴ who reported that force quickly increased but that [Ca²⁺]_c did not change significantly after a quick stretch from slack to optimum (maximum force). However, in contrast to our results, they also observed slowly increasing force and [Ca²⁺]_c after the initial changes. This discrepancy may be explained by their use of a slack force that was only 5% of maximum compared with our shortest length, which produced 30% of maximum force. Furthermore, the time constant for the slow increase was 4 minutes, whereas our measurement time, at each length, was only 1 minute.

Fig 2C demonstrates that the fall of [NADH]_m was similar to that observed initially when increased work was associated with increased time-averaged [Ca²⁺]_c (Fig 2A and 2B). However, in this case, in which time-averaged [Ca²⁺]_c did not increase, there was neither recovery of [NADH]_m during sustained work nor overshoot of [NADH]_m on return to the initial sarcomere length. In Fig 2C, MIN is equal to SS and therefore corresponds to the MIN values obtained in Fig 2A and 2B. This result supports the hypothesis that the partial recovery of [NADH]_m, when work was elevated by increased frequency or [Ca²⁺]_o, is Ca²⁺ dependent. Furthermore, this result also indicates that feedback by [ADP]/[ATP] or by [NAD⁺]_m/[NADH]_m was not responsible for the recovery or overshoot of [NADH]_m observed in Fig 2A and 2B.⁸

Work-Dependent Initial Changes of [NADH]_m
Fig 3A demonstrates that the initial decrease of [NADH]_m (MIN) as a function of work was qualitatively similar for all three protocols used to vary work. Thus, both the Ca²⁺-dependent protocols (changing [Ca²⁺]_o or frequency) and the Ca²⁺-independent protocol (changing sarcomere length) caused [NADH]_m to initially fall with increasing work. The initial decline in [NADH]_m, resulting from increased rate of oxidative phosphorylation, was therefore independent of [Ca²⁺]_c but depended on work level. Consequently, we conclude that the oxidative phosphorylation rate is initially not uniquely controlled by [NADH]_m³ or [Ca²⁺]_c.²

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of increased work (time-averaged force) on [NADH]m, with or without a simultaneous increase in time-averaged [Ca2+]c. Each data point was pooled from five experiments, similar to those shown in Fig 2. A, Comparison of the initial fall of [NADH]m (MIN) following increased work by increasing [Ca2+]o (x), pacing frequency (), or sarcomere length (SL, ). For all three protocols, both time-averaged force and [NADH]m were normalized to the same experimental control (Ctrl) condition ([Ca2+]o, 2 mmol/L; frequency, 1 Hz; and maximal SL, producing developed force, 48±5 mN/mm2). B, Comparison, using the frequency protocol, of initial fall of [NADH]m (MIN), steady state level after NADH recovery (SS), and overshoot (MAX) upon reducing work back to Ctrl value (0.25 Hz). Both work and [NADH]m were normalized to Ctrl values. The data points for the initial fall of [NADH]m (MIN) are taken from Fig 3A. C, Calculated recovery of [NADH]m [(SS/MIN)-1] as a function of Ca2+-dependent increases in work by the [Ca2+]o protocol (x) and frequency protocol () as well as Ca2+-independent increases in work by the SL protocol ().

The Ca²⁺-independent mechanism of [NADH]_m downregulation during the initial stages of increased work is opposed by the Ca²⁺-dependent mechanism, which caused [NADH]_m to recover (and overshoot) when using the frequency or [Ca²⁺]_o protocol (Fig 2A and 2B). Based on Fig 3A, the Ca²⁺-independent mechanism appears to be faster than the Ca²⁺-dependent recovery mechanism, because the slopes of the frequency and [Ca²⁺]_o-dependent data were not shallower than the slope of the sarcomere length–dependent data. This is consistent with the results of Fig 2A and 2B and with the notion that increased [Ca²⁺]_m and activation of dehydrogenases occurs with some time lag after abrupt changes in time-averaged [Ca²⁺]_c.^{8 9}

Work- and Ca²⁺-Dependent Changes of [NADH]_m During Sustained Work
Fig 3B shows pooled data from experiments in which frequency was altered (as in Fig 2B). It is clear that the Ca²⁺-dependent recovery mechanism limits the decline of [NADH]_m (SS) versus the initial Ca²⁺-independent fall (MIN). Indeed, at higher work, SS fell only to a level that was within 12% of the control value. MAX during the overshoot also increases monotonically with work (measured immediately before the sudden reduction back to control). Pooled data using the protocol in which [Ca²⁺]_o was altered produced qualitatively similar results.

Fig 3C shows the relationship between the amount of [NADH]_m recovery and work for the three protocols. The amount of [NADH]_m recovery increases as a function of work when using increasing [Ca²⁺]_o or frequency, whereas increasing sarcomere length shows no recovery of [NADH]_m. This once again demonstrates the strict Ca²⁺ dependence of the [NADH]_m recovery during sustained elevated work.

Kinetics of Ca²⁺-Dependent NADH Recovery
Our results also provide some kinetic information about the Ca²⁺-dependent activation of NADH production. When work was altered by increasing the pacing frequency from 0.25 to 1 Hz, [NADH]_m recovered with 30 seconds (see Fig 2B). This is similar to the rate of Ca²⁺ uptake by mitochondria in intact ventricular myocytes when the sarcoplasmic reticulum Ca²⁺ content was discharged into the cytosol (41 seconds)²⁵ and the rate of increased [Ca²⁺]_m in intact rat ventricular myocytes in response to increased contraction frequency from 0 to 4 Hz (40 seconds).⁹ The overshoot declined with 35 seconds, similar to the kinetics of [Ca²⁺]_m decline or Ca²⁺ efflux from the mitochondria in intact cells, 21 seconds⁹ and 45 seconds, respectively.²⁶ Thus, the kinetics of the Ca²⁺-dependent stimulation of NADH production observed here in intact cardiac muscle likely reflect the kinetics of mitochondrial Ca²⁺ transport and the time needed for activation or deactivation of the mitochondrial dehydrogenases.

Work and Temperature Regimens
As we have discussed previously,¹² a limitation of the results obtained here may be a result of using a nonphysiological temperature (24°C) and low work load. However, Fig 3A demonstrated that the initial decrease of [NADH]_m was related to work over the whole range of work levels studied here, from 25% to 160% of control, approaching the level in vivo.¹² This suggests that increasing work further may lower the initial [NADH]_m even more.

To evaluate the effects of using a physiological temperature and an even larger range of work levels, the frequency protocol was repeated at 37°C. Fig 4 shows that for the whole range of work levels (>13-fold increase) the results were qualitatively similar to those in Fig 2B (at 24°C with a 6-fold increase in work levels). This suggests that the phenomena described here are not unique to studies at 24°C but also occur at physiological temperatures and pacing frequencies.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of transiently increased pacing frequency at 37°C on isometric force (top trace, in mN/mm2), work, assessed by calculating time-averaged force (middle trace, in mN/mm2), and [NADH]m (bottom trace, not quantified in this experiment). Pacing frequency was raised from a control value of 0.25 Hz to 1, 2, 3, 4, 5, and 6 Hz, causing increased work and time-averaged [Ca2+]c.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Upon a sudden increase in work and ATP consumption rate, during either increasing [Ca²⁺]_c or constant [Ca²⁺]_c, there was a rapid decrease in [NADH]_m. This implies that the initial increase in the oxidative phosphorylation rate is Ca²⁺ independent (eg, insignificant Ca²⁺ control of the ATP synthase² ) and also that it is not controlled by increased [NADH]_m. Furthermore, because there was initially a net decrease in [NADH]_m, the NADH production rate (via the tricarboxylic acid cycle) is not adequately stimulated to match the respiratory demand. Initial control of the oxidative phosphorylation rate may therefore be controlled by alterations in cytosolic phosphates (eg, [ADP], [P_i], [ATP]/[ADP][P_i], or [ATP]/[ADP]) rather than [NADH]_m or [Ca²⁺]_c.

When the increase in work was caused by increased time-averaged [Ca²⁺]_c, [NADH]_m slowly recovered. This is most likely a result of slowly increasing [Ca²⁺]_m, which may stimulate mitochondrial dehydrogenases and, consequently, the flux of reducing equivalents through the tricarboxylic acid cycle to raise [NADH]_m. The increased [NADH]_m may subsequently further stimulate oxidative phosphorylation to better match the rate of ATP production to its consumption. In this scenario, the initially decreasing [NADH]_m may correlate with increasing [ADP] and [P_i] (decreasing phosphorylation potential), whereas during [NADH]_m recovery, [ADP] and [P_i] may decrease (increasing phosphorylation potential). Such large changes in [P_i] and [ADP] would occur only transiently and might not be observed in a typical (³¹P NMR) experiment, in which steady state phosphate levels are measured during increased work loads in vivo.^{3 27}

However, using time-resolved ³¹P NMR, a transient increase in [ADP] was observed in intact perfused hearts within 30 seconds of increased work load by the addition of isoprenaline.²⁸ In a pacing protocol, similar to that used in the present study, Elliott et al²⁹ also demonstrated that [P_i] followed a time dependence that was the mirror image of the time dependence of [NADH]_m observed here. Although increased [NADH]_m and phosphorylation potential could occur without an increase in O₂ consumption rate (or oxidative phosphorylation rate),⁷ Elliott et al showed that the O₂ consumption rate also slowly increased after increased work (pacing frequency). These observations are consistent with the results obtained here and argues for shared control of the oxidative phosphorylation rate by [NADH]_m (via [Ca²⁺]_c) and phosphates. Specifically, the recovery of [NADH]_m may switch some of the control of oxidative phosphorylation rate away from cytosolic phosphate control over to [NADH]_m control.³

It has previously been suggested that oxidative phosphorylation may be exclusively controlled by either ADP (if [ADP] but not [NADH]_m is rate limiting) or by NADH (if [NADH]_m but not [ADP] is rate limiting).³ Therefore, at low but increasing work loads, [NADH]_m may be constant while [ADP] increases and thereby increases the oxidative phosphorylation rate. Conversely, at higher increasing work loads, [ADP] may be constant while [NADH]_m increases and thereby increases the oxidative phosphorylation rate. The progressive decline of [NADH]_m with work observed in Fig 3A (and Fig 4), however, suggests that there was no such shift from an ADP- to an NADH-limited domain within the work regimen studied here.

The changes in cytosolic phosphates, driven by changes in [NADH]_m, may also have important consequences for regulation of the cytosolic phosphorylation potential. Initially, with increased work, the phosphorylation potential may fall, but this may be opposed by rising [NADH]_m, causing the phosphorylation potential to recover.⁷ This recovery may be necessary for maintaining the cytosolic energetic state needed for processes that hydrolyze ATP (eg, sarcoplasmic reticulum Ca²⁺ uptake).

Summarizing Model
Our results are most consistent with the following control mechanisms (see Fig 1): Increased cytosolic [Ca²⁺] causes increased work and ATP hydrolysis rate (by myofilament and transport ATPases). Increased ATP hydrolysis rate, in turn, may cause increased [ADP] (and/or P_i), which could stimulate the oxidative phosphorylation rate (more than the NADH production rate) and thereby a fall in [NADH]_m. On a slower time scale, cytosolic Ca²⁺ may enter the mitochondria and activate pyruvate dehydrogenase, NAD-linked isocitrate dehydrogenase, or 2-oxoglutarate (-ketoglutarate) dehydrogenase. (Note that in the present study 10 mmol/L pyruvate was used, and this may limit the amount of Ca²⁺-dependent activation of pyruvate dehydrogenase.) Activation of the dehydrogenases is expected to increase the NADH production rate and consequently cause [NADH]_m to recover. Increased [NADH]_m may further increase the oxidative phosphorylation rate and/or cause [ADP] (and the cytosolic phosphorylation potential) to return toward control levels.

Thus, we provide the first clear experimental evidence in intact cardiac muscle to support the idea that [Ca²⁺]_c is an important dynamic control signal for stimulating the reduction of mitochondrial NAD⁺ to NADH, thereby actively adjusting the cellular energy state during conditions of increased ATP consumption rate. Since an increase in time-averaged [Ca²⁺]_c is a physiological stimulus causing the immediate increase of myofilament ATP consumption rates, the parallel but time-delayed Ca²⁺-dependent activation of mitochondrial dehydrogenases constitutes part of a feed-forward control of cellular energetic state.

	Selected Abbreviations and Acronyms

 

= exponential time constant [Ca2+]c = cytosolic [Ca2+] [Ca2+]m = mitochondrial [Ca2+] MAX = maximum value of [NADH]m after decreased work MIN = minimum value of [NADH]m during increased work [NADH]m, [NAD+]m = mitochondrial [NADH] and [NAD+] NMR = nuclear magnetic resonance SS = steady state [NADH]m during increased work

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid from the American Heart Association of Metropolitan Chicago (Dr Brandes) and by National Institute of Health grants HL-30077 and HL-52478 (Dr Bers).

	Footnotes

 
*Although the energy consumption rate is strictly related to work/time (power), the term "work" will be used here rather than work/time.

Received July 23, 1996; accepted October 15, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Brand MD, Murphy MP. Control of electron flux through the respiratory chain in mitochondria and cells. Biol Rev Camb Philos Soc. 1987;62:141-193. Review.[Medline] [Order article via Infotrieve]

2. Harris DA, Das AM. Control of mitochondrial ATP synthesis in the heart. Biochem J. 1991;280:561-573. Review.

3. From AH, Zimmer SD, Michurski SP, Mohanakrishnan P, Ulstad VK, Thoma WJ, Ugurbil K. Regulation of the oxidative phosphorylation rate in the intact cell. Biochemistry. 1990;29:3731-3743.[Medline] [Order article via Infotrieve]

4. Erecinska M, Wilson DF. Regulation of cellular energy metabolism. J Membr Biol. 1982;70:1-14. Review.[Medline] [Order article via Infotrieve]

5. Heldt HW, Klingenberg M. Differences between the reactivity of endogenous and exogenous adenine nucleotides in mitochondria as studied at low temperature. Eur J Biochem. 1968;4:1-8.[Medline] [Order article via Infotrieve]

6. Koretsky AP, Katz LA, Balaban RS. Determination of pyridine nucleotide fluorescence from the perfused heart using an internal standard. Am J Physiol. 1987;253:H856-H862.[Abstract/Free Full Text]

7. Scholz TD, Laughlin MR, Balaban RS, Kupriyanov VV, Heineman FW. Effect of substrate on mitochondrial NADH, cytosolic redox state, and phosphorylated compounds in isolated hearts. Am J Physiol. 1995;268:H82-H91.[Abstract/Free Full Text]

8. Crompton M. The role of Ca²⁺ in the function and dysfunction of heart mitochondria. In: Langer GA, ed. Calcium and the Heart. New York, NY: Raven Press Publishers; 1990:167-199.

9. Miyata H, Silverman HS, Sollott SJ, Lakatta EG, Stern MD, Hansford RG. Measurement of mitochondrial free Ca²⁺ concentration in living single rat cardiac myocytes. Am J Physiol. 1991;261:H1123-H1134.[Abstract/Free Full Text]

10. McCormack JG, Halestrap AP, Denton RM. Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev. 1990;70:391-425. Review.[Free Full Text]

11. Hansford RG. Dehydrogenase activation by Ca²⁺ in cells and tissues. J Bioenerg Biomembr. 1991;23:823-854. Review.[Medline] [Order article via Infotrieve]

12. Brandes R, Bers DM. Increased work in cardiac trabeculae causes decreased mitochondrial NADH fluorescence followed by slow recovery. Biophys J. 1996;71:1024-1035.[Medline] [Order article via Infotrieve]

13. Backx PH, Ter Keurs HE. Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt. Am J Physiol. 1993;264:H1098-H1110.[Abstract/Free Full Text]

14. Eng J, Lynch RM, Balaban RS. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys J. 1989;55:621-630.[Medline] [Order article via Infotrieve]

15. Nuutinen EM. Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart. Basic Res Cardiol. 1984;79:49-58.[Medline] [Order article via Infotrieve]

16. Cooper G IV. Myocardial energetics during isometric twitch contractions of cat papillary muscle. Am J Physiol. 1979;236:H244-H253.

17. Frampton JE, Harrison SM, Boyett MR, Orchard CH. Ca²⁺ and Na⁺ in rat myocytes showing different force-frequency relationships. Am J Physiol. 1991;261:C739-C750.[Abstract/Free Full Text]

18. Spurgeon HA, Stern MD, Baartz G, Raffaeli S, Hansford RG, Talo A, Lakatta EG, Capogrossi MC. Simultaneous measurement of Ca²⁺, contraction, and potential in cardiac myocytes. Am J Physiol. 1990;258:H574-H586.[Abstract/Free Full Text]

19. Frampton JE, Orchard CH, Boyett MR. Diastolic, systolic and sarcoplasmic reticulum [Ca²⁺] during inotropic interventions in isolated rat myocytes. J Physiol (Lond). 1991;437:351-375.[Abstract/Free Full Text]

20. Chapman JB, Gibbs CL, Gibson WR. Heat and fluorescence changes in cardiac muscle: effects of substrate and calcium. J Mol Cell Cardiol. 1976;8:545-558.[Medline] [Order article via Infotrieve]

21. Yasumura Y, Nozawa T, Futaki S, Tanaka N, Suga H. Time-invariant oxygen cost of mechanical energy in dog left ventricle: consistency and inconsistency of time-varying elastance model with myocardial energetics. Circ Res. 1989;64:764-778.[Abstract/Free Full Text]

22. Hasenfuss G, Mulieri LA, Blanchard EM, Holubarsch C, Leavitt BJ, Ittleman F, Alpert NR. Energetics of isometric force development in control and volume-overload human myocardium: comparison with animal species. Circ Res. 1991;68:836-846.[Abstract/Free Full Text]

23. Kentish JC, Stienen GJ. Differential effects of length on maximum force production and myofibrillar ATPase activity in rat skinned cardiac muscle. J Physiol (Lond). 1994;475:175-184.[Abstract/Free Full Text]

24. Allen DG, Kurihara S. The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle. J Physiol (Lond). 1982;327:79-94.[Abstract/Free Full Text]

25. Bassani RA, Bassani JW, Bers DM. Mitochondrial and sarcolemmal Ca²⁺ transport reduce [Ca²⁺]_i during caffeine contractures in rabbit cardiac myocytes. J Physiol (Lond). 1992;453:591-608.[Abstract/Free Full Text]

26. Bassani JW, Bassani RA, Bers DM. Ca²⁺ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes. J Physiol (Lond). 1993;460:603-621.[Abstract/Free Full Text]

27. Katz LA, Koretsky AP, Balaban RS. Respiratory control in the glucose perfused heart: a 31P NMR and NADH fluorescence study. FEBS Lett. 1987;221:270-276.[Medline] [Order article via Infotrieve]

28. Unitt JF, McCormack JG, Reid D, MacLachlan LK, England PJ. Direct evidence for a role of intramitochondrial Ca²⁺ in the regulation of oxidative phosphorylation in the stimulated rat heart: studies using 31P n.m.r. and ruthenium red. Biochem J. 1989;262:293-301.[Medline] [Order article via Infotrieve]

29. Elliott AC, Smith GL, Allen DG. The metabolic consequences of an increase in the frequency of stimulation in isolated ferret hearts. J Physiol (Lond). 1994;474:147-159.[Abstract/Free Full Text]

This article has been cited by other articles:

				M.-H. T. Nguyen, S. J. Dudycha, and M. S. Jafri Effect of Ca2+ on cardiac mitochondrial energy production is modulated by Na+ and H+ dynamics Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2004 - C2020. [Abstract] [Full Text] [PDF]

				L. Zhou, M. E. Cabrera, H. Huang, C. L. Yuan, D. K. Monika, N. Sharma, F. Bian, and W. C. Stanley Parallel activation of mitochondrial oxidative metabolism with increased cardiac energy expenditure is not dependent on fatty acid oxidation in pigs J. Physiol., March 15, 2007; 579(3): 811 - 821. [Abstract] [Full Text] [PDF]

				J. Talbot, J. N. Barrett, E. F. Barrett, and G. David Stimulation-induced changes in NADH fluorescence and mitochondrial membrane potential in lizard motor nerve terminals J. Physiol., March 15, 2007; 579(3): 783 - 798. [Abstract] [Full Text] [PDF]

				J. Satrustegui, B. Pardo, and A. del Arco Mitochondrial Transporters as Novel Targets for Intracellular Calcium Signaling Physiol Rev, January 1, 2007; 87(1): 29 - 67. [Abstract] [Full Text] [PDF]

				M. Sedova, E. N. Dedkova, and L. A. Blatter Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes Am J Physiol Cell Physiol, November 1, 2006; 291(5): C840 - C850. [Abstract] [Full Text] [PDF]

				C. J. Bell, N. A. Bright, G. A. Rutter, and E. J. Griffiths ATP Regulation in Adult Rat Cardiomyocytes: TIME-RESOLVED DECODING OF RAPID MITOCHONDRIAL CALCIUM SPIKING IMAGED WITH TARGETED PHOTOPROTEINS J. Biol. Chem., September 22, 2006; 281(38): 28058 - 28067. [Abstract] [Full Text] [PDF]

				B. Korzeniewski Oxygen consumption and metabolite concentrations during transitions between different work intensities in heart Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1466 - H1474. [Abstract] [Full Text] [PDF]

				C. Maack, S. Cortassa, M. A. Aon, A. N. Ganesan, T. Liu, and B. O'Rourke Elevated Cytosolic Na+ Decreases Mitochondrial Ca2+ Uptake During Excitation-Contraction Coupling and Impairs Energetic Adaptation in Cardiac Myocytes Circ. Res., July 21, 2006; 99(2): 172 - 182. [Abstract] [Full Text] [PDF]

				G. Cherednichenko, A. V. Zima, W. Feng, S. Schaefer, L. A. Blatter, and I. N. Pessah NADH Oxidase Activity of Rat Cardiac Sarcoplasmic Reticulum Regulates Calcium-Induced Calcium Release Circ. Res., March 5, 2004; 94(4): 478 - 486. [Abstract] [Full Text] [PDF]

				M. L Riess, A. K.S Camara, L. G Kevin, J. An, and D. F Stowe Reduced reactive O2 species formation and preserved mitochondrial NADH and [Ca2+] levels during short-term 17 {degrees}C ischemia in intact hearts Cardiovasc Res, February 15, 2004; 61(3): 580 - 590. [Abstract] [Full Text] [PDF]

				R. Ferrari Healthy versus sick myocytes: metabolism, structure and function Eur. Heart J. Suppl., November 1, 2002; 4(suppl_G): G1 - G12. [Abstract] [PDF]

				G. Hajnoczky, G. Csordas, M. Madesh, and P. Pacher The machinery of local Ca2+ signalling between sarco-endoplasmic reticulum and mitochondria J. Physiol., November 15, 2000; 529(1): 69 - 81. [Abstract] [Full Text] [PDF]

				R. C. Scaduto Jr. and L. W. Grotyohann 2,3-Butanedione monoxime unmasks Ca2+-induced NADH formation and inhibits electron transport in rat hearts Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1839 - H1848. [Abstract] [Full Text] [PDF]

				G. Szalai, G. Csordas, B. M. Hantash, A. P. Thomas, and G. Hajnoczky Calcium Signal Transmission between Ryanodine Receptors and Mitochondria J. Biol. Chem., May 12, 2000; 275(20): 15305 - 15313. [Abstract] [Full Text] [PDF]

				M. Vendelin, O. Kongas, and V. Saks Regulation of mitochondrial respiration in heart cells analyzed by reaction-diffusion model of energy transfer Am J Physiol Cell Physiol, April 1, 2000; 278(4): C747 - C764. [Abstract] [Full Text] [PDF]

				J. H. G. M. van Beek, M. H. van Wijhe, M. H. J. Eijgelshoven, and J. B. Hak Dynamic adaptation of cardiac oxidative phosphorylation is not mediated by simple feedback control Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1375 - H1384. [Abstract] [Full Text] [PDF]

				C. Gibbs Respiratory control in normal and hypertrophic hearts Cardiovasc Res, June 1, 1999; 42(3): 567 - 570. [Full Text] [PDF]

				S. Pepe, N. Tsuchiya, E. G. Lakatta, and R. G. Hansford PUFA and aging modulate cardiac mitochondrial membrane lipid composition and Ca2+ activation of PDH Am J Physiol Heart Circ Physiol, January 1, 1999; 276(1): H149 - H158. [Abstract] [Full Text] [PDF]

				R. Brandes, L. S. Maier, and D. M. Bers Regulation of Mitochondrial [NADH] by Cytosolic [Ca2+] and Work in Trabeculae From Hypertrophic and Normal Rat Hearts Circ. Res., June 15, 1998; 82(11): 1189 - 1198. [Abstract] [Full Text] [PDF]

				L. S. Maier, R. Brandes, B. Pieske, and D. M. Bers Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium Am J Physiol Heart Circ Physiol, April 1, 1998; 274(4): H1361 - H1370. [Abstract] [Full Text] [PDF]

				Z. Zhou, M. A Matlib, and D. M Bers Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes J. Physiol., March 1, 1998; 507(2): 379 - 403. [Abstract] [Full Text] [PDF]

				C.J. Zuurbier and J.H.G.M. van Beek Mitochondrial Response to Heart Rate Steps in Isolated Rabbit Heart Is Slowed After Myocardial Stunning Circ. Res., July 19, 1997; 81(1): 69 - 75. [Abstract] [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brandes, R. Articles by Bers, D. M. Search for Related Content PubMed PubMed Citation Articles by Brandes, R. Articles by Bers, D. M.