Impaired Cardiac Energetics in Mice Lacking Muscle-Specific Isoenzymes of Creatine Kinase

From the NMR Laboratory for Physiological Chemistry, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Dr Kurt Saupe, NMR Laboratory for Physiological Chemistry, Room 247, 221 Longwood Ave, Boston, MA 02115. E-mail ksaupe{at}aol.com

	Abstract

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Abstract—Our purpose was to determine whether hearts from mice bioengineered to lack either the M isoform of creatine kinase (MCK^-/- mice) or both the M and mitochondrial isoforms (M/MtCK^-/- mice) have deficits in cardiac contractile function and energetics, which have previously been reported in skeletal muscle from these mice. The phenotype of hearts with deleted creatine kinase (CK) genes is of clinical interest, since heart failure is associated with decreased total CK activity and changes in the relative amounts of the CK isoforms in the heart. We measured isovolumic contractile performance in isolated perfused hearts from wild-type, MCK^-/-, and M/MtCK^-/- mice simultaneously with cardiac energetics (³¹P-nuclear magnetic resonance spectroscopy) at baseline, during increased cardiac work, and during recovery. Hearts from wild-type, MCK^-/-, and M/MtCK^-/- mice had comparable baseline function and responded to 10 minutes of increased heart rate and perfusate Ca²⁺ with similar increases in rate-pressure product (48±5%, 42±6%, and 51±6%, respectively). Despite a similar contractile response, M/MtCK^-/- hearts increased [ADP] by 95%, whereas wild-type and MCK^-/- hearts maintained [ADP] at baseline levels. The free energy released from ATP hydrolysis decreased by 3.6 kJ/mol in M/MtCK^-/- hearts during increased cardiac work but only slightly in wild-type (1.7 kJ/mol) and MCK^-/- (1.5 kJ/mol) hearts. In contrast to what has been reported in skeletal muscle, M/MtCK^-/- hearts were able to hydrolyze and resynthesize phosphocreatine. Taken together, our results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more "energetically costly" in terms of high-energy phosphate use, accumulation of ADP, and decreases in free energy released from ATP hydrolysis, but not in terms of myocardial oxygen consumption.

Key Words: transgenic mouse • bioenergetics • creatine kinase • heart failure

	Introduction

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The CK (EC 2.7.2.2) isoenzyme family consists of five isoenzymes. The isoenzymes are dimers formed from four distinct polypeptide chains, each encoded by separate genes. Two of these isoenzymes are located in the mitochondria (ubiquitous MtCK and sarcomeric (MtCK), and three are located in the cytoplasm (BBCK, MBCK, and MMCK). Each of these isoenzymes catalyzes the transfer of a phosphoryl group between PCr and ATP via the following reaction: PCr+ADP+H⁺ATP+creatine. By catalyzing this reaction, which has a large K_eq (1.66x10⁹ [mol/L]^-1), CK functions to maintain a high concentration of ATP and low concentrations of the products of ATP hydrolysis (ADP, P_i, and H⁺) in cells. This ensures that G_ATP will be adequate to maintain ion gradients and perform the mechanical work of the cell.¹

Recently, mice have been bioengineered to lack specific isoenzymes of CK.^{2 3} The impact of these gene deletions on contractile function and utilization of PCr has been defined for skeletal muscle.^{2 3 4 5} The first type of CK "knockout" mouse, referred to here as MCK^-/-, has a null mutation of the MCK gene. Loss of MCK from skeletal muscle causes a transient impairment in contractile function but no changes in the ability of the muscle to hydrolyze or resynthesize PCr.² A second type of CK knockout mouse, referred to as M/MtCK^-/-, lacks both the MCK and MtCK genes, leaving only BCK. Skeletal muscle of M/MtCK^-/- mice not only has a larger impairment of contractile function than MCK^-/- skeletal muscle but also is unable to hydrolyze PCr even though some BBCK is present.³ This surprising finding suggests that either the amount of BBCK remaining is inadequate to hydrolyze PCr, or that the BBCK and PCr are not localized in the same functional compartment.

The CK enzyme system in cardiac muscle differs from skeletal muscle in several important respects, making it difficult to predict how the heart will respond to loss of CK isoenzyme(s). Cardiac muscle has only 12% to 25% of the total CK activity of skeletal muscle, and the isoenzyme distribution in the heart is more diverse, with MCK and MtCK constituting 70% and 25% of total CK activity in the heart, respectively, as opposed to 95% and 5% in skeletal muscle.^{5 6} Cardiac muscle also has a much higher concentration of mitochondria than does skeletal muscle. Because of this, cardiac muscle has not only a larger capacity for aerobic ATP synthesis than skeletal muscle but also a shorter diffusion distance for movement of metabolites such as ATP between the mitochondria and cytosolic ATPases.

The physiological phenotype of hearts with deleted CK genes is of clinical interest, since heart failure of many different etiologies is associated with decreased total CK activity and large changes in the relative amounts of the CK isoforms in the myocardium.⁷ The net result of these changes in the CK enzyme system is a decreased capacity of the failing heart to synthesize ATP from PCr by as much as 70%.^{8 9} The causes of these impairments in the CK enzyme system and how they interact with the other changes in myocyte biochemistry that occur during heart failure are poorly understood.⁷ Thus, defining the cardiac biochemistry and physiology of hearts with deletions of specific CK isoenzymes is of interest.

The purpose of the present study was to define contractile function and energetic properties of mouse hearts lacking either one (MCK^-/-) or both (M/MtCK^-/-) of the major cardiac isoenzymes of CK. Specifically, we sought to determine whether hearts lacking CK isoenzyme(s) have the ability to hydrolyze PCr when challenged with increased work and whether these hearts are able to maintain [ADP] low and G_ATP high, especially when challenged with increased work. Since MtCK forms a functional compartment with porin and adenine nucleotide translocase in the inner mitochondrial membrane,¹⁰ another goal was to determine whether ATP synthesis via oxidative phosphorylation, assessed as MVO₂ over a range of cardiac workloads, would be altered in hearts of these mice. To accomplish these goals, we measured isovolumic contractile performance in isolated perfused hearts from wild-type, MCK^-/-, and M/MtCK^-/- mice. We measured contractile performance simultaneously with cardiac energetics using ³¹P-NMR spectroscopy at baseline, when HR and perfusate Ca²⁺ were increased to increase cardiac work, and during recovery.

	Materials and Methods

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Animals
MCK^-/- and M/MtCK^-/- mice were generated in the laboratory of Dr Bé Wieringa (University of Nijmegen, the Netherlands) by gene targeting, as previously reported.^{2 3} All mutant mice had a mixed inbred background (C57/BL6 and 129/Sv); wild-type mice were C57/BL6. Male and female mice 20 to 30 weeks of age were studied. There were no differences among the wild-type, MCK^-/-, and M/MtCK^-/- mice regarding hearts weights (134±5, 137±6, and 125±5 mg, respectively) or heart weight–to–body weight ratios (4.7±0.1, 4.4±0.1, and 4.7±0.1 mg/g, respectively). The genotype of each mouse was confirmed by measuring the isoenzymes of CK present in its heart using a Helena Cardio-Rep CK isoenzyme analyzer. The experimental protocol for the present study was approved by the Standing Committee on Animals of Harvard Medical Area and followed the recommendations of current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.

Isolated Perfused Heart Preparation
Hearts of wild-type and mutant mice were isolated and perfused in the Langendorff mode in a 10-mm glass NMR tube as described previously.¹¹ Briefly, the chest was opened, and the heart was rapidly excised and arrested in ice-cold buffer. Retrograde perfusion via the aorta was carried out at a constant coronary perfusion pressure of 75 mm Hg at 37°C. Right ventricular drainage was accomplished by incision of the pulmonary artery. The flow of thebesian veins was drained by a thin polyethylene tube (PE-10) pierced through the apex of the left ventricle. Coronary flow was measured by collecting coronary sinus effluent through the suction tube. Phosphate-free Krebs-Henseleit buffer containing (mmol/L) NaCl 118, KCl 5.3, CaCl₂ 2.0, MgSO₄ 1.2, EDTA 0.5, NaHCO₃ 25, and glucose 10 was prepared at the time of the experiment and equilibrated with 95% O₂/5% CO₂, yielding a pH of 7.4. Hearts were paced using monophasic square-wave pulses delivered from a Grass stimulator (model S 88) through salt-bridge pacing wires consisting of PE-160 tubing filled with 4 mol/L KCl in 2% agarose.

Measurement of Isovolumic Contractile Performance
A water-filled balloon custom-made from polyvinyl chloride film was inserted through the mitral valve into the left ventricle via an incision in the left atrium. The balloon was connected to a pressure transducer (Statham P23Db, Gould) for continuous recording of left ventricular pressure and HR. The size of the balloon was carefully matched to the size of the ventricle. The balloon was inflated to set left ventricular EDP between 6 and 8 mm Hg for all hearts, and the balloon volume was then held constant. Contractile performance data were collected on-line at a sampling rate of 200 Hz using a commercially available data acquisition system (MacLab ADInstruments). LVDevP (the difference between systolic pressure and EDP), the minimum and maximum values within a beat of the first derivative of left ventricular pressure (+dP/dt and -dP/dt), and RPP (product of LVDevP and HR) were calculated off-line.

Experimental Protocols
Studying the isovolumic contractile performance of mouse hearts presents several technical challenges. Our primary challenge was to find a technique for inducing a large rapid increase in cardiac work. We were limited in this regard by the fact that we could not increase HR above 600 bpm without diminishing our ability to accurately measure EDP because of the long fluid-filled pressure measuring line needed for experiments conducted in the magnet. Even if we had been able to increase HR to what occurs during maximal exercise in mice (900 bpm), it is unlikely that this would have caused large increases in RPP. Isolated mouse hearts have a "negative staircase"; increasing HR above 350 bpm causes decreases in developed pressure and positive and negative dP/dt.¹² To maintain developed pressure constant when we increased HR, perfusate Ca²⁺ was increased to 4.0 mmol/L (measured free Ca²⁺ 3.0 mmol/L). Pilot experiments defining Ca²⁺ dose-response curves showed that this concentration of Ca²⁺ maximizes developed pressure in mouse hearts as it does in rat hearts. Therefore to rapidly increase cardiac work, we increased perfusate Ca²⁺ from 2.0 mmol/L (free Ca²⁺, measured as 1.3 mmol/L) to 4.0 mmol/L and simultaneously increased HR from 420 to 600 bpm. Increasing perfusate [Ca²⁺] as a method of increasing contractile performance was preferred to pharmacological agents to avoid potential confounding influences of altered receptor systems in the bioengineered animals. In the present study, we refer to the work state created by perfusing hearts with 2.0 mmol/L Ca²⁺ and pacing at 420 bpm as baseline and that created by perfusing hearts with 4.0 mmol/L Ca²⁺ and pacing at 600 bpm as increased work. Two experimental protocols were used in the present study.

Protocol 1: Changes in Work State
In the first protocol, contractile performance and ³¹P-NMR spectroscopy measurements were made at baseline, during increased work, and during recovery in 15 wild-type, 8 MCK^-/-, and 8 M/MtCK^-/- hearts. Baseline measurements of contractile performance and ³¹P-NMR spectroscopy were made after a 20-minute stabilization period. After these baseline measurements (perfusate Ca²⁺, 2.0 mmol/L), the perfusate was switched to a buffer containing 4.0 mmol/L Ca²⁺. The transition to 4.0 mmol/L Ca²⁺ perfusate took 30 seconds as the high Ca²⁺ perfusate washed into the system. During this 30 seconds, HR was increased to 600 bpm. The increased workload condition was maintained for 10 minutes, and the functional and NMR measurements were initiated after 2 minutes. The perfusion was then switched back to baseline conditions, and hearts were allowed to recover for 4 minutes before the start of the recovery NMR measurement. At the end of the experiments, hearts were blotted, weighed, and stored at -80°C for later biochemical analysis.

Protocol 2: Measurement of MVO₂
In the second protocol, MVO₂ was measured over a range of cardiac workloads in 7 wild-type, 8 MCK^-/-, and 7 M/MtCK^-/- hearts. These hearts were perfused at a constant flow rate with the same buffer as the first group, except that 0.5 mmol/L pyruvate was included in the perfusate. Coronary flow was maintained constant to eliminate measurement error associated with this parameter, which is critical in the calculation of MVO₂. Also, the constant flow system allowed us to draw coronary venous effluent from the pulmonary artery at a constant rate, thereby maintaining a constant transit time from the coronary circulation to the O₂-measuring electrode. MVO₂ was measured at six workloads induced by stepwise increases in perfusate [Ca²⁺] (1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mmol/L) and during KCl-induced cardiac arrest (25 mmol/L KCl). To measure MVO₂, 1.5 mL/min of the perfusate leaving the pulmonary outflow tract was drawn across the face of an O₂ electrode in a 0.5-mL chamber. The O₂ tension in this coronary venous effluent as well as in the arterial perfusate was measured with an O₂ meter (model 860, Orion Research Inc). MVO₂ was calculated as follows and expressed as µmol O₂/min per gram dry weight: MVO₂=PO₂(A-V)x solubility of O₂/mm Hgxcoronary flow rate/dry heart weight.

Biochemical Assays
In a subset of mice, rapidly frozen heart and hindlimb skeletal muscle tissue was thawed and homogenized for measurement of maximal activities of CK and the major glycolytic enzymes PFK, GAPDH, LDH, and CS, a marker of mitochondrial mass. Cardiac and skeletal muscle (5 to 10 mg) was homogenized for 10 seconds at 4°C in potassium phosphate buffer containing 1 mmol/L EDTA and 1 mmol/L ß-mercaptoethanol, pH 7.4 (final concentration, 5 mg tissue/mL). Aliquots were removed for assays of protein by the method of Lowry et al¹³ (using bovine serum albumin as the standard) and for total creatine by a fluorometric assay.¹⁴ Triton X-100 was then added to the homogenate at a final concentration of 0.1%. The CK activity was measured in tissue homogenates at 37°C as previously described.¹⁵ The activities of PFK,¹⁶ LDH,¹⁷ GAPDH,¹⁸ and CS¹⁹ were also measured as previously described. Enzyme activities were measured (IU/mg protein) and converted (mmol · L^-1 · s^-1) using the measured concentrations of cardiac protein (0.145±0.006 versus 0.141±0.005 versus 0.146±0.004 mg protein/mg wet wt in wild-type, MCK^-/-, and M/MtCK^-/- hearts, respectively) and the assumption that the ratio of intracellular volume to cardiac mass of 0.48 µL/mg wet weight was the same for all hearts.²⁰ All values are expressed as mmol · L^-1 · s^-1 at 25°C with the exceptions of PFK and LDH (30°C) and CK (37°C).

³¹P-NMR Spectroscopy
³¹P-NMR spectra were obtained at 161.94 MHz using a GE-400 wide-bore Omega spectrometer. Hearts were placed in a 10-mm glass NMR tube and inserted into a custom-made ¹H/³¹P double-tuned probe situated in an 89-mm bore 9.4-T superconducting magnet. To improve homogeneity of the NMR-sensitive volume, the perfusate level was adjusted so that the heart was submerged in buffer. Spectra were collected without proton decoupling at a pulse width of 15 microseconds, pulse angle of 60°, recycle time of 2.14 seconds, and sweep width of 6000 Hz. Single spectra were collected during 8-minute periods and consisted of data averaged from 208 free induction decays.

Data Analysis
³¹P-NMR spectra were analyzed using 20-Hz exponential multiplication and zero and first-order phase corrections. The resonance areas corresponding to ATP, PCr, and P_i, which are proportional to the number of moles of the respective compound in the heart, were fitted to lorentzian function and calculated by a commercially available program (NMR1). By comparing the resonance areas of fully relaxed (recycle time, 10 seconds) and those of partially saturated (recycle time, 2.14 seconds) spectra, the corrections for partial saturation were calculated for ATP (1.0), PCr (1.2), and P_i (1.15).

Baseline values for [ATP] measured in a separate group of hearts using high-pressure liquid chromatography (W. Shen, unpublished data, 1997) were used to convert resonance area units into millimolar concentrations. Accordingly, for each wild-type heart, the average of the ß- and -ATP resonance areas at baseline was set at 9.6 mmol/L; for MCK^-/- hearts, 9.4 mmol/L; and for M/MtCK^-/- hearts, 9.3 mmol/L.

pH_i was determined by comparing the chemical shift of the P_i peak and relative position of PCr in each spectrum to values from a standard curve. Cytosolic free [ADP] was calculated using the equilibrium constant of the CK reaction and from values obtained by NMR spectroscopy. Total creatine values of 28.2±0.5, 32.1±0.7, and 30.5±0.6 mmol/L measured biochemically in a separate group of hearts using a standard assay (see above) were used in the calculation of ADP: [ADP]=([ATP][free creatine])/([PCr][H⁺]K_eq), where K_eq is 1.66x10⁹ (mol/L)^-1 for a [Mg²⁺] of 1.0 mmol/L.^{21 22}

The free energy stored in the high-energy phosphate bonds of ATP (G_ATP) is released by ATP hydrolysis.^{1 23} Although G_ATP is a negative value, the change in free energy state due to ATP hydrolysis is a positive value and is calculated as follows: G_ATP(kJ/mol)=G°+RT ln ([ADP][P_i]/[ATP]), where G° (-30.5 kJ/mol) is the value of G_ATP under standard conditions of molarity, temperature, pH, and [Mg²⁺],²³ R is the gas constant (8.3 J/mol K), and T is temperature (Kelvin).

All results were expressed as mean±SE. To test for differences among the three groups of hearts, one-way ANOVA was used. Statistical analyses were performed with the use of Statview (Brainpower), and a value of P0.05 was considered significant.

	Results

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Enzyme Activities in Cardiac and Skeletal Muscle
Total CK activity in cardiac muscle was 51.9±4.6 mmol · L^-1 · s^-1 in wild-type, 14.6±0.9 mmol · L^-1 · s^-1 in MCK^-/-, and 1.9±0.2 mmol · L^-1 · s^-1 in M/MtCK^-/- hearts. As noted by Steeghs et al,²⁴ hearts from M/MtCK^-/- mice contained no detectable ubiquitous MtCK and therefore contained only BBCK. Maximal activities of selected enzymes were measured to determine whether loss of CK isoenzymes causes changes in glycolytic and oxidative enzymes, as has been reported in fast-twitch skeletal muscle.^{2 4} In skeletal muscle, loss of CK isoenzyme(s) did not alter the maximal activities of PFK, GAPDH, or LDH, but consistent with a previous report, CS activity was increased in the skeletal muscle of MCK^-/-2 and also M/MtCK^-/- mice (Table 1). In cardiac muscle, there were no differences among the three groups in PFK, GAPDH, LDH, or CS activities.

Cardiac Function and Energetics Under Baseline Conditions
A representative tracing of left ventricular pressure, HR, and left ventricular dP/dt under baseline conditions from an M/MtCK^-/- heart is shown in the left panel of Figure 1. Mean values for all three groups are shown in Table 2. During baseline perfusion, there were no significant differences among the three groups of hearts with regard to LVDevP, RPP, and positive or negative dP/dt (Table 2). Isovolumic contractile performance was stable during the 16-minute baseline period, as can be seen in Figure 2, where RPP varied <5% in each group during this period. Coronary flow, an index of coronary vascular resistance under conditions of constant perfusion pressure, was also not different among the three groups (24.0±1.8, 21.2±1.1, and 20.3±1.2 mL · min^-1 · g^-1 in wild-type, MCK^-/-, and M/MtCK^-/- mice, respectively).

View larger version (26K): [in this window] [in a new window]   Figure 1. Representative tracing of left ventricular (LV) isovolumic contractile performance from a heart with the genes for MCK and MtCK knocked out (M/MtCK-/- mouse) at baseline (left), during increased cardiac work (middle), and during recovery (right). Balloon volume was set in each heart before the baseline period so that EDP was between 5 and 10 mm Hg. All hearts were paced at 420 bpm during baseline and recovery and at 600 bpm during increased cardiac work.

View larger version (21K): [in this window] [in a new window]   Figure 2. RPP at baseline, during increased cardiac work, and during recovery in isovolumic Langendorff-perfused hearts from wild-type, MCK-/-, and M/MtCK-/- mice. There were no differences in RPP among the three groups during any stage of the protocol.

In the left panels of Figure 3, representative ³¹P-NMR spectra during baseline perfusion are shown for wild-type, MCK^-/-, and M/MtCK^-/- hearts. Mean values for metabolite concentrations are shown in Figure 4. As the representative spectra demonstrate, [PCr] was significantly lower in M/MtCK^-/- hearts (8.2±0.3 mmol/L) than in wild-type hearts (10.7±0.5 mmol/L) or MCK^-/- hearts (11.5± 0.6 mmol/L) (Figure 4). There were no significant differences among the three groups in [ATP], [P_i], or pH during baseline (Figure 4). During baseline perfusion, [ADP], as calculated from the equilibrium equation for the CK reaction, was significantly higher in M/MtCK^-/- hearts (214±9 µmol/L) than in either wild-type hearts (149±11 µmol/L) or MCK^-/- hearts (150±12 µmol/L). The increase in [ADP] in M/MtCK^-/- hearts was not large enough to make G_ATP significantly different from the other two groups of hearts under baseline conditions (Figure 4).

View larger version (23K): [in this window] [in a new window]   Figure 3. Representative 31P-NMR spectra from hearts isolated from wild-type (top), MCK-/- (middle), and M/MtCK-/- (bottom) mice at baseline (left), during increased cardiac work (middle), and during recovery (right). At baseline, M/MtCK-/- hearts had a lower [PCr] than did wild-type or MCK-/- hearts but the same [ATP] and [Pi]. During increased cardiac work, M/MtCK-/- hearts used more PCr than did wild-type or MCK-/- hearts.

View larger version (25K): [in this window] [in a new window]   Figure 4. A, Cardiac energetics at baseline, during increased cardiac work, and during recovery. Baseline [PCr] was lower in M/MtCK-/- hearts and decreased more during increased cardiac work than in wild-type or MCK-/- hearts. The responses of [ATP] and [Pi] during the protocol were not different in the three groups of hearts. Both [ATP] and [Pi] were significantly below baseline levels during recovery in all three groups. [PCr] recovered to above baseline levels in wild-type and MCK-/- hearts but not in M/MtCK-/- hearts. B, [ADP] was significantly higher in M/MtCK-/- hearts at baseline than in wild-type or MCK-/- hearts. When cardiac work was increased, wild-type and MCK-/- hearts maintained [ADP] at baseline levels, whereas [ADP] was doubled in M/MtCK-/- hearts. During recovery, GATP was significantly above baseline, and [ADP] was significantly below baseline in all three groups. Data from baseline spectra are plotted at the end of the baseline period; data from spectra collected during increased work and recovery are plotted at the midpoint of the 8-minute period required to acquire the spectra. *P<0.05 vs wild-type and MCK-/- hearts.

Increased Cardiac Work
A representative example of the response of left ventricular pressure to increased perfusate [Ca²⁺] and HR is shown in the middle panel of Figure 1 for an M/MtCK^-/- heart. It can be seen that LVDevP is essentially maintained constant when HR and perfusate [Ca²⁺] are increased, resulting in an increase in RPP. The increases in RPP were not different among the three groups of hearts and averaged 48±5%, 42±6%, and 51±6% in the wild-type, MCK^-/-, and M/MtCK^-/- hearts, respectively (Table 2, Figure 2). During this increased RPP, there were no significant differences among the three groups of hearts for EDP, LVDevP, or positive or negative dP/dt (Table 2).

Increasing RPP caused [PCr] to decrease and [P_i] to increase in each group of hearts as can be seen in the middle panels of Figure 3. The decrease in [PCr] was greater in M/MtCK^-/- hearts (4.0±0.5 mmol/L) than in wild-type or MCK^-/- hearts (1.9±0.5 and 2.1±0.7 mmol/L) (Figures 3 and 4). The increases in [P_i] (4.0±0.5 mmol/L in wild-type hearts, 4.6±1.5 mmol/L in MCK^-/- hearts, and 5.5±0.8 mmol/L in M/MtCK^-/- hearts) were not different among groups. Increasing RPP caused [ATP] to decrease similarly in the three groups of hearts, by 2.1±0.3 mmol/L in wild-type, by 1.6±0.5 mmol/L in MCK^-/-, and by 1.8±0.5 mmol/L in M/MtCK^-/- hearts (Figure 4). pH decreased in each group of hearts, by 0.03±0.01 in wild-type, by 0.05±0.02 in MCK^-/-, and by 0.04±0.01 in M/MtCK^-/- hearts, in response to increased RPP. Free [ADP] nearly doubled (214±9 to 407±59 µmol/L) in M/MtCK^-/- hearts in response to increased RPP but remained at baseline levels in both wild-type and MCK^-/- hearts (Figure 4). During increased cardiac work, the free energy released from ATP hydrolysis (G_ATP) decreased by 1.7±0.3 kJ/mol in wild-type hearts and by 1.5±0.4 kJ/mol in MCK^-/- hearts, both significant decreases (Figure 5). In M/MtCK^-/- hearts, there was a significantly larger decrease in G_ATP (3.6±0.4 kJ/mol) in response to increased cardiac work.

View larger version (22K): [in this window] [in a new window]   Figure 5. Relationship between MVO2 and RPP in wild-type, MCK-/-, and M/MtCK-/- hearts. Each point is a different perfusate [Ca2+] with KCl-induced cardiac arrest (25 mmol/L) the leftmost point. Hearts were paced at 420 bpm. There was no difference in the relationship between MVO2 and RPP among the three groups. Note the lack of a further increase in RPP as perfusate Ca2+ was increased from 4.0 to 5.0 mmol/L. Regression equations are as follows: MVO2=0.00012RPP+2.30 (wild-type hearts), MVO2=0.00013RPP+1.61 (MCK-/- hearts), and MVO2=0.00013RPP+1.44 (M/MtCK-/- hearts).

Recovery From Increased Cardiac Work
When perfusate [Ca²⁺] was returned to 2.0 mmol/L and HR was restored to 420 bpm, RPP returned to 78±2%, 80±4%, and 83±3% of baseline in wild-type, MCK^-/-, and M/MtCK^-/- hearts, respectively, after 10 minutes (not different among groups) (Figure 1, Table 1). There were no significant differences in EDP, LVDevP, or positive or negative dP/dt among the three groups after 10 minutes of recovery (Table 2). In all three groups, RPP remained significantly below baseline during the recovery period.

The amount of PCr resynthesized during recovery was not different among the three groups of hearts (5.5±0.5 mmol/L in wild-type, 6.8±0.8 mmol/L in MCK^-/-, and 5.3±0.7 mmol/L in M/MtCK^-/- hearts). In wild-type and MCK^-/- hearts (but not M/MtCK^-/- hearts), [PCr] was significantly higher during recovery than at baseline. In all three groups of hearts, [ATP] and [P_i] remained significantly below baseline during recovery. Total cardiac phosphate ([P_i]+[PCr]+3x[ATP]) decreased by only 11% to 14% from baseline to recovery in the three groups of hearts (not different among groups). After 10 minutes of recovery, [ADP] was significantly below baseline in each group of hearts but remained 2-fold higher in M/MtCK^-/- (151±20 µmol/L) than in wild-type (71±6 µmol/L), or MCK^-/- (67±9 µmol/L) hearts (Figure 4). During the 10 minutes of recovery, G_ATP increased to levels significantly greater than baseline in each group of hearts but remained lower in M/MtCK^-/- hearts (54.1±0.4 kJ/mol) than in wild-type or MCK^-/- hearts (56.5±0.3 and 56.4±0.8 kJ/mol, respectively) (Figure 5).

Myocardial Oxygen Consumption
Since cardiac ATP is primarily synthesized by oxidative phosphorylation, we tested whether MVO₂ over a large range of cardiac workloads would be altered by loss of CK isoenzyme(s). In this separate group of hearts, MVO₂ was measured at six different workloads induced by perfusate [Ca²⁺] of 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mmol/L and during KCl-induced cardiac arrest (Figure 5). There was a linear relationship between RPP and MVO₂ for each group of hearts, and there were no significant differences among groups for the relationship between RPP and MVO₂. Similarly, during KCl arrest, MVO₂ was not significantly different among the three groups of hearts.

	Discussion

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The purpose of the present study was to determine how hearts lacking the major isoenzyme(s) of CK respond to increased contractile work, both in terms of cardiac energetics and contractile performance. The question of how loss of specific CK isoenzymes affects cardiac physiology is clinically important, since heart failure of many different etiologies is associated with decreased total CK activity as well as changes in the relative amounts of the CK isoforms.⁷ Although recent data have described several interesting characteristics of skeletal muscle from mice lacking the adult CK isoenzymes, little is known regarding the cardiac energetics and ventricular function of these mice.^{2 3}

The major findings of the present study were as follows: First, deletion of MCK (72% decrease in total CK activity) did not cause changes in cardiac energetics or left ventricular contractile function. Second, deletion of MCK in combination with loss of MtCK (96% decrease in total CK activity) rendered hearts incapable of maintaining [ADP] and G_ATP at baseline levels when challenged with the same increase in work as MCK^-/- and wild-type hearts. Third, in contrast to skeletal muscle, where PCr is metabolically inert, hearts from M/MtCK^-/- mice not only used PCr during increased work but used more PCr than did wild-type or MCK^-/- hearts. Fourth, deletion of MCK and, more interestingly, deletion of both MCK and MtCK did not alter the relationship between cardiac work (as assessed by RPP) and MVO₂ over the range studied. Taken together, these results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more energetically costly in terms of high-energy phosphate use, accumulation of ADP, and decreases in G_ATP, but not in terms of MVO₂.

Energetic Responses to Increased HR and Perfusate Ca²⁺
Hearts from wild-type and MCK^-/- mice had indistinguishable contractile and energetic responses to increased cardiac work. In contrast, hearts from M/MtCK^-/- mice differed from both wild-type and MCK^-/- hearts regarding their energetic response to increased cardiac work. The first difference was that in response to the same increase in isovolumic contractile work, [ADP] nearly doubled in M/MtCK^-/- hearts, whereas it remained unchanged in wild-type and MCK^-/- hearts. This inability to maintain low concentrations of ADP during a time of increased ATP hydrolysis is consistent with the view that one of the primary roles of CK in the heart is to maintain low concentrations of the hydrolysis products of ATP.²⁵ A second major difference in the M/MtCK^-/- hearts was that G_ATP decreased by twice as much during increased work as it did in wild-type and MCK^-/- hearts. The observation that hearts that chronically have only 4% of normal CK activity demonstrate a large fall in G_ATP during increased work is consistent with the observation of Tian and Ingwall,²⁶ who reported that rat hearts with CK acutely inhibited respond to increased work with a large decrease in G_ATP.

The cause of the work-induced changes in [ADP] and G_ATP in the M/MtCK^-/- hearts was not simply loss of MCK, since MCK^-/- hearts behaved like hearts from wild-type mice. Instead, the large work-induced changes in [ADP] and G_ATP are likely caused by (1) loss of MtCK, (2) loss of MtCK in concert with loss of MCK, or (3) a decrease in the total CK activity below some critical threshold necessary to maintain "normal" energetics at this level of cardiac work. The first possibility is unlikely, since loss of MtCK alone, at least in skeletal muscle, does not impair baseline energetics or the ability to hydrolyze PCr during ischemia.^{3 24} The recent finding that MCK and BCK are functionally equivalent in skeletal muscle would support the idea that changes in energetics in M/MtCK^-/- hearts are due to a low total CK.²⁷

Why do M/MtCK^-/- hearts hydrolyze more PCr during the same increase in cardiac work than either wild-type or MCK^-/- hearts? Since CK is the only known enzyme capable of hydrolyzing PCr, our observation that the hearts with the lowest total CK activity (M/MtCK^-/- hearts) hydrolyzed the most PCr in response to the same increase in cardiac work is unexpected. Because [PCr] reflects the balance between synthesis and hydrolysis, this result demonstrates that during increased cardiac work, the rate of synthesis of PCr is slower than its rate of hydrolysis in all three groups of hearts but that this difference is largest in M/MtCK^-/- hearts. This is consistent with the view that MtCK, localized in an environment of high [ATP] in the mitochondrial matrix, primarily functions to synthesize PCr from ATP, whereas BBCK is localized in an environment where PCr hydrolysis is favored. This is not to say that BBCK is incapable of synthesizing PCr, since during the recovery from increased work, [PCr] in M/MtCK^-/- hearts increased to baseline values. The increased PCr hydrolysis in M/MtCK^-/- hearts may be necessitated by an impaired ability of these hearts to rapidly increase oxidative ATP synthesis secondary to loss of both MCK and MtCK. Whatever the cause, this increased reliance on PCr as a source of high-energy phosphates demonstrates that increasing cardiac work is more energetically costly in hearts with low CK activity. It should be emphasized that the small size of the mouse hearts limits the temporal resolution of our ³¹P-NMR spectroscopy measurements. Our measure of energetics during increased work is the average of what occurs between 2 and 10 minutes after the workload was increased.

Neither of the two types of CK "knockout" mice demonstrated any signs of adaptations in other ATP-producing pathways to compensate for chronic loss of CK. Adaptation to deletion of genes for CK isoenzymes has been shown to occur in fast but not slow twitch skeletal muscle from M/MtCK^-/- and MCK^-/- mice, in which mitochondrial enzymes levels are increased compared with levels in wild-type mice.³ Although values for V_max, the maximal activity of an enzyme under conditions of saturating substrates, were not different among the three groups of hearts studied here, values for V_max do not report the in vivo reaction rates. Therefore, we cannot rule out the possibility that there are differences in the in vivo rates for ATP-synthesizing reactions among the three groups of hearts.

In studies of M/MtCK^-/- mice, Steeghs et al³ demonstrated that the [PCr] in skeletal muscle does not change either during ischemia or high-frequency electrical stimulation in contrast to wild-type and MCK^-/- skeletal muscle. This observation has led to the conclusion that PCr is metabolically inert in the skeletal muscle of M/MtCK^-/- mice, even though some BBCK is present in the muscle. In stark contrast to skeletal muscle, in the present study we demonstrate that hearts from M/MtCK^-/- mice are not only capable of hydrolyzing PCr but that [PCr] decreased twice as much in response to the same increase in cardiac work as it does in wild-type or MCK^-/- hearts. Possible explanations for this major qualitative difference between skeletal and cardiac muscle from M/MtCK^-/- animals include the following: (1) Cardiac muscle has more total CK activity than does skeletal muscle, allowing the heart to hydrolyze PCr. (2) In skeletal muscle, BBCK may be localized in such a way that substrates are not available to it. The first possibility is unlikely, since we report that there is only modestly more CK in cardiac (1.9 mmol · L^-1 · s^-1) than in skeletal muscle (1.0 mmol · L^-1 · s^-1). The fact that cardiac muscle can use large amounts of PCr with a total CK activity of only 1.9 mmol · L^-1 · s^-1 suggests that it is not a low total CK activity that causes PCr to be metabolically inert in skeletal muscle. Therefore, we favor the second possibility, that BBCK in skeletal muscle from M/MtCK^-/- mice is localized in such a way that no net hydrolysis of PCr occurs during ischemia or increased work. This would mean either that BBCK and its substrates are not in the same compartment or that the concentrations of these substrates in the vicinity of BBCK were not changing in a way that leads to net hydrolysis of PCr.

Contractile Responses to Increased HR and Perfusate Ca²⁺
We found, as have others,¹² that mouse hearts perform less work in the isolated perfused preparation than they do under in vivo conditions, where they receive a high degree of sympathetic drive as well as a larger variety of metabolic substrates. For that reason, RPP during the baseline condition studied here is only 50% of what one would expect in a resting mouse in vivo. Accordingly, our condition of increased cardiac work corresponds to the RPP expected for a resting mouse, not an exercising mouse. It is difficult to determine what constitutes a high workload condition for a mouse heart, since little data exist on the in vivo cardiovascular responses to maximal exercise in mice. There is reason to believe that the increase in cardiac work in mice during maximal exercise is less than the 4- to 5-fold increase observed in healthy humans, since HR increases only by 50% to 75% during maximal exercise in mice as opposed to 300% in young healthy humans. The high-energy requirement of the "resting" (HR, 600 bpm; mean arterial pressure, 90 mm Hg) mouse heart may suggest that it has a lower cardiac energy reserve than is found in larger species.

We found no differences among the three types of hearts regarding the contractile response to increased HR and Ca²⁺ over the range studied. The fact that MCK^-/- hearts had a normal contractile response was not surprising, since (1) in rat hearts, total CK activity must be inhibited to <5% of normal before contractile reserve is diminished,²⁶ and (2) MCK^-/- hearts had enough remaining CK (28% of normal total activity) to have a normal energetic response. In contrast, our observation that M/MtCK^-/- hearts had a normal contractile response to increased HR and perfusate Ca²⁺ was surprising, since these hearts had only 4% of normal CK and dramatically impaired energetics.²⁶ The ability of M/MtCK^-/- hearts to increase contractile functions to the same degree as wild-type hearts could be due to a species difference in the dependence on CK. It could also be related to the fact that acute chemical inhibition of CK affects all isoforms, whereas in knockout mice, certain isoforms are completely eliminated but the BB isoform is unaltered. Whatever the reason, our data clearly demonstrate that in this model, BBCK alone is adequate to allow a 50% increase in contractile performance.

In a previous study by Tian et al,²⁸ a correlation between EDP and [ADP] was observed when [ADP] was increased without altering [P_i], [ATP], or pH. In the present study, in which work-induced increases in [ADP] occurred in the M/MtCK^-/- hearts simultaneous with changes in [ATP], [P_i], and pH, increases in [ADP] were not associated with increases in EDP. We speculate that the lack of a correlation between [ADP] and EDP in the present study is either because the correlation described by Tian et al applies only under narrowly defined circumstances or because the M/MtCK^-/- hearts have adapted to a state of chronically higher [ADP].

In skeletal muscle from MCK^-/- and M/MtCK^-/- animals, contractile performance is transiently impaired when resting muscle is electrically stimulated at high frequency.^{2 3} The fact that we found no evidence of impaired contractile function in hearts from MCK^-/- and M/MtCK^-/- mice during our protocol is likely due to differences in our protocols and to biochemical differences between cardiac and skeletal muscle. Our protocol, where RPP increases 40% over the course of 60 seconds, should not lead to the type of energy supply/demand mismatch that occurs when resting skeletal muscle is maximally electrically stimulated, a condition that induces a many-fold increase in ATP consumption in a few seconds. In our protocol, cardiac contractile performance could not be accurately measured until 4 minutes after the start of the increased cardiac work. Since impaired contractile performance lasts for <60 seconds in skeletal muscle from CK knockout mice, it is likely that even if the contractile phenotypes seen in skeletal muscle had been present in the heart, we would not have seen them.² A comparison of skeletal and cardiac muscle biochemistry indicates that cardiac muscle has a higher concentration of mitochondria than does skeletal muscle, as indicated by the higher concentration of CS.³ This means that cardiac muscle has both a larger capacity to synthesize ATP oxidatively and a shorter diffusion distance between mitochondria and cytoplasmic ATPases than does skeletal muscle. Cardiac muscle may therefore be able to increase ATP synthesis very rapidly and hence have little, if any, need for a high-energy phosphate reserve to overcome the ATP supply/demand mismatch when RPP is only modestly increased. If this is the case, then hearts from larger animals with lower resting cardiac workloads may respond differently to loss of CK activity than do mouse hearts. Finally, cardiac muscle may tolerate disruption of the "CK shuttle" better than skeletal muscle because of the shorter distances between ATP synthesis (mitochondria) and cytoplasmic ATPases.

Myocardial Oxygen Consumption
We measured MVO₂ to determine whether loss of MtCK, which is closely linked to adenine nucleotide translocase, would alter oxidative metabolism. We observed that MVO₂ was not different among the three groups of hearts over a large range of cardiac workloads, demonstrating that loss of MtCK does not necessarily disrupt the oxidative synthesis of ATP. In skeletal muscle, Steeghs et al²⁴ observed that mice lacking MtCK has no deficits in oxidative metabolism in vitro.²⁴ In the present study, we report a similar finding at the level of the whole heart. A second purpose of measuring MVO₂ was to determine whether the higher [ADP] in M/MtCK^-/- hearts would lead to an elevated MVO₂. If so, this would suggest that MVO₂ is limited by the supply of ADP in intact mouse hearts as shown for isolated mitochondrial preparations.⁵ The ability of increased [ADP] to stimulate respiration in isolated mitochondria from MCK^-/- ventricular muscle has been demonstrated by Veksler et al,⁵ who showed that [ADP] stimulation of respiration was not different in MCK^-/- ventricular muscle compared with wild-type ventricular muscle if creatine was present. Our observation that the elevated [ADP] in M/MtCK^-/- hearts did not cause increased MVO₂ suggests that [ADP] is not the dominate determinate of MVO₂ in the intact mouse heart.

Limitations
One puzzling observation in the present study is that [ATP] decreased 1.6 to 2.1 mmol/L during increased work in each of the three groups of hearts, about twice the decrease observed in a similar protocol in rats.²⁶ Since this decrease was the same in wild-type, MCK^-/-, and M/MtCK^-/- hearts, it is not caused by the loss of CK isoenzyme(s) nor is it responsible for the differences in energetics among groups during increased cardiac work. A decrease in [ATP] during increased cardiac work is unlikely to occur in vivo. The cause of this decrease in [ATP] is likely related to a loss of purine nucleosides and bases during increased work, since [ATP] is not restored during recovery, even though PCr is resynthesized to above baseline levels. One potential explanation for these decreases in [ATP] might be that the supply of oxygen to the hearts is inadequate and that during increased RPP hearts become mildly hypoxic. Evidence against this comes from our observation that oxygen extraction, which is 58% at baseline, increases only to 70% during increased work (data not shown).

Our calculation of free [ADP] is based on the assumptions that (1) the CK reaction is near equilibrium, (2) the concentration of total creatine (the sum of free creatine and PCr) is constant during the protocol, and (3) the K_eq for the CK reaction is close to 1.66x10⁹ (at pH 7.0 and 1.0 mmol/L Mg²⁺), a value measured in vitro. Could our calculated values for ADP and G_ATP in the M/MtCK^-/- hearts be wrong because of violation of these assumptions? Although creatine can be lost acutely from hearts,²⁹ our observation that there is enough total creatine during recovery to allow PCr to be restored to above baseline levels strongly suggests that total creatine is not significantly decreased during our protocol. Less certain is the value for K_eq of the individual CK isoenzymes under in vivo conditions. It is possible that if M/MtCK^-/- hearts (which contain only BBCK) have a different K_eq, our baseline differences in ADP would not exist. We calculate that this would require K_eq at baseline to be 2.19x10⁹ in M/MtCK^-/- hearts as opposed to the value of 1.66x10⁹ used in the present study for all hearts. Even if this large difference in K_eq between wild-type and M/MtCK^-/- hearts does exist, to explain the doubling of [ADP] during increased work would require K_eq to increase further to 3.86x10⁹ in M/MtCK^-/- hearts. Therefore, we believe that it is unlikely that the differences in ADP and G_ATP between wild-type and M/MtCK^-/- hearts are due to differences in K_eq for the CK reaction.

Summary
The present study demonstrates that loss of MCK (72% decrease in total CK activity) in otherwise normal mouse hearts does not cause observable changes in cardiac energetics or left ventricular contractile function. In contrast, a combined loss of MtCK and MCK (96% decrease in CK activity) impaired cardiac energetics when hearts were challenged with increased work. Hearts lacking both MtCK and MCK hydrolyzed large amounts of PCr when challenged with increased work, in direct contrast to data showing that PCr is "metabolically inert" in the skeletal muscles of these mice. This difference in PCr metabolism between cardiac and skeletal muscle could be caused by enzyme and substrate being in separate functional compartments in skeletal, but not cardiac, muscle. Taken together, these results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more "energetically costly" in terms of high-energy phosphate use, accumulation of ADP, and decreases in G_ATP but not in terms of MVO₂.

	Selected Abbreviations and Acronyms

 

|GATP| = free energy released from ATP hydrolysis BCK = B isoform of CK CK = creatine kinase CS = citrate synthase EDP = end-diastolic pressure HR = heart rate Keq = equilibrium constant LVDevP = left ventricular developed pressure M/MtCK-/- = lacking M and mitochondrial isoforms of CK MCK = M isoform of CK MCK-/- = lacking M isoform of CK MtCK = mitochondrial isoform of CK MVO2 = myocardial oxygen consumption NMR = nuclear magnetic resonance PCr = phosphocreatine PFK = phosphofructokinase RPP = rate-pressure product

	Acknowledgments

 
This study was supported by National Research Service Award HL-09259 (Dr Saupe), a Deutsche Forschungsgemeinschaft research fellowship (Dr Spindler), and National Institutes of Health SCOR grant HL-52350 (Dr Ingwall). We would like to thank the laboratories of Dr Bé Wieringa and Nelson Goldberg for providing the founder mice used in this study and to Jonathan Rose for his technical assistance.

	Footnotes

 
This manuscript was sent to Ketty Schwartz, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received December 18, 1997; accepted February 12, 1998.

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