Reduced Inotropic Reserve and Increased Susceptibility to Cardiac Ischemia/Reperfusion Injury in Phosphocreatine-Deficient Guanidinoacetate-
http://www.100md.com
《循环学杂志》
the Department of Cardiovascular Medicine (M.t.H., C.A.L., A.F., J.E.S., K.H., L.S., H.W., J.W., S.N.) and University Laboratory of Physiology (A.E.S., K.C.), University of Oxford, Oxford, England
Center for Molecular Neurobiology, Hamburg, Germany (D.I.).
Abstract
Background— The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase–knockout (GAMT–/–) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT–/– mice.
Methods and Results— GAMT–/– mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and ;-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT–/– mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT–/– hearts (101±8 mm Hg in WT versus 59±7 mm Hg in GAMT–/–; P<0.05). 31P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT–/– hearts utilized P-GA. During ischemia/reperfusion, GAMT–/– hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23±9 in WT and 51±5 mm Hg in GAMT–/– during reperfusion; P<0.05) and incomplete resynthesis of P-GA.
Conclusions— GAMT–/– mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT–/– mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.
Key Words: creatine kinase ; metabolism ; hemodynamics ; ischemia ; myocardium
Introduction
Despite decades of research,1,2 the true functional role of high-energy phosphate metabolism for cardiac function in normal and diseased states remains to be fully clarified. Creatine kinase (CK) catalyzes the transfer of the high-energy phosphate bond between ATP and phosphocreatine (PCr). Chemical inhibition of CK by iodoacetamide substantially reduces contractile reserve in normal hearts.3 CK function has also been shown to be compromised in postischemic4 and in failing myocardium,5,6 and this mechanism may contribute to the pathophysiology of heart failure.7
More recently, genetically manipulated mouse models have provided the opportunity to dissect the role of the CK/PCr system in greater detail. CK-knockout mice provided the first model of this kind, and M-CK–knockout,8 mito-CK–knockout, and double (M- and mito-CK)–knockout9 mice have been reported, the latter showing 3% of residual CK activity (in the form of BB-CK).10 However, thus far, results with regard to CK-knockout mice have been conflicting and await further clarification, with some groups showing significant functional alterations, adaptations, and left ventricular (LV) hypertrophy in CK double-knockout mice11–13 but others demonstrating relatively little functional, although energetic, impact.10,14 Importantly, CK-knockout mice show normal levels of myocardial creatine. An alternative intervention to impair the function of the CK/PCr system would be to create transgenic mouse models of altered total creatine, and thus PCr, content. Creatine is mainly produced in liver and kidney through 2 essential chemical reactions, the first catalyzed by arginine:glycine amidinotransferase, or AGAT (arginine+glycine/guanidinoacetate+ornithine), and the second step catalyzed by guanidinoacetate N-methyltransferase, or GAMT (guanidinoacetate/creatine).
We recently reported on a new model of altered energetics due to knockout of GAMT.15 Our initial report focused on the neurological and skeletal muscle phenotype of these mice but also demonstrated normal baseline perfused heart function (LV developed pressure [LVDP]) in GAMT-knockout (GAMT–/–) mice. GAMT–/– mice show substantial accumulation of the creatine precursor guanidinoacetate (GA) and its phosphorylated form, phospho-guanidinoacetate (P-GA). However, creatine levels in heart were 27% of those in WT mice.15 Although these animals were fed with a creatine-free diet, we now know that the source of the residual creatine is coprophagia by GAMT–/– mice when housed with heterozygous or WT littermate controls because the latter have creatine present in their feces.16 We have shown that cardiac creatine and PCr decline below levels of detectability if homozygous GAMT–/– mice are housed separately.17
Therefore, we now have a new mouse model, which shows undetectable myocardial (phospho)creatine levels and substantial accumulation of the precursor (P-)GA. Although P-GA participates in the CK reaction, the reaction velocity is 100 times slower compared with phosphocreatine.18 In the present study we took advantage of this new model of altered cardiac energetics. Using an array of sophisticated tools for murine cardiac phenotyping including MRI and MR spectroscopy (MRS), we tested the hypothesis that in vivo cardiac function at baseline and during inotropic stress, as well as susceptibility to ischemia/reperfusion injury, is compromised in GAMT–/– mice lacking creatine and PCr.
Methods
WT and GAMT–/– Mice
Heterozygous GAMT+/– mice, constructed as described before,15 were backcrossed for at least 8 generations onto a C57Bl/6 genetic background. Mice used for experiments were generated by intercrossing GAMT+/– mice and genotyped by polymerase chain reaction.15 From 6 weeks of age, mice were housed per genotype to prevent ingestion of creatine in GAMT–/– mice through coprophagia. Mice were kept in cages with 12-hour light/dark cycle and controlled temperature (20°C to 22°C) and fed creatine-free chow and water ad libitum. All studies were performed in 5- to 8-month-old mice and were conducted in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.
In Vivo MRI
High-resolution murine cine MRI was applied in vivo on GAMT–/– (n=13; 6 male, 7 female) and WT (n=14; 7 male, 7 female) mice as described previously19 to assess LV mass and volumes.
In Vivo Hemodynamics at Baseline and During Inotropic Stimulation
Measurements were made in closed-chest, spontaneously breathing mice (as described in Nemoto et al20). GAMT–/– and WT littermates (n=12; 5 males, 7 females in each group) were anesthetized with 2% isoflurane in oxygen on a homeothermic blanket. A 1.4F microtipped conductance cannula (SPR-839, Millar Instruments) was advanced retrogradely into the LV via the right carotid artery under echocardiographic guidance. The right jugular vein was cannulated with stretched polythene tubing for infusion of dobutamine. Preload conditions were altered by occlusion of the inferior vena cava. A small incision was made in the abdominal wall at the level of the xiphisternum, and a cotton swab was used to apply transient compression. Isoflurane was reduced to 1.25% to 1.5%, followed by equilibration until hemodynamic indices were stable for >15 minutes. Measurements were taken under steady state and inferior vena cava occlusion, at baseline and during infusion of low- and high-dose dobutamine (4 and 16 ng/g body wt per minute). Recordings were analyzed with the use of PVAN software version 3.0 (Millar Instruments). The cannula was calibrated to relative volume units with a cuvette system, and parallel conductance was measured by saline bolus method.20 No correction for the electrical field inhomogeneity () was made; therefore, the only occlusion parameter reported is preload recruitable stroke work (PRSW) because this is and chamber size independent. Systolic and diastolic blood pressures were measured invasively in separate groups of anesthetized mice (n=4 WT; n=5 GAMT–/–).
Conscious ECG
ECGs from conscious unrestrained mice (n=7 WT; n=9 GAMT–/–) were recorded with the use of the AnonyMOUSE ECG screening tool (Mouse Specifics Inc).21
Assessment of Myocyte Cross-Sectional Area by Histology
A papillary level short-axis slice of the LV (n=8 per group) was fixed in formaldehyde, dehydrated, and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined under an inverted microscope. Myocyte cross-sectional area was measured by computerized planimetry as previously described.22
Perfused Heart Experiments
Male mice were heparinized (5000 U/kg body wt) and anesthetized (pentobarbitone 140 mg/kg body wt IP). Hearts were excised, cannulated, and perfused in Langendorff mode at 80 mm Hg and 37°C with Krebs-Henseleit buffer gassed with 95% O2/5% CO2 (pH 7.4) containing the following (in mmol/L): 149 Na+, 5.9 K+, 1.2 Mg2+, 2.25 Ca2+, 1.2 SO42–, 126.2 Cl–, 0.5 EDTA2–, 25 HCO3–, 1.2 H2PO4–, 11 D-glucose, 4.5 pyruvate, and 0.5 lactate. Contractility was assessed with a fluid-filled intraventricular balloon connected to a pressure transducer (AD Instruments Ltd). The end-diastolic pressure (EDP) was set to 5 to 15 mm Hg.
31P-MRS
Five-minute 31P-nuclear MR spectra of perfused mouse hearts were acquired on a Bruker Avance 500 spectrometer equipped with a 11.7-T magnet and a 20-mm 1H-/31P-cross-cage resonator with a pulse-and-collect sequence (repetition time=100 ms, =29°, number of scans=3000) and corrected for partial saturation. Metabolites were quantified with respect to the signal intensity of a known amount of phenylphosphonic acid in the intraventricular balloon with the use of the AMARES algorithm and MRUI software. Baseline concentrations were calculated with the use of 20-minute spectra. Baseline [Pi] was too low for quantification, and therefore [Pi] is only shown from ischemia onward. Intracellular volume was assumed to be 0.5 mL/g wet wt. pHi was determined from the chemical shift between Pi and PCr with the use of pH=6.72–log(–5.72)/(3.17–). In the absence of PCr, phenylphosphonic acid (18 ppm) was used as a frequency reference. GAMT–/– mice show an additional resonance at –0.5 ppm, which previously has been assigned to P-GA15,16 as also described in a human patient with GAMT deficiency.23 In addition, this resonance has been shown to appear to the right of PCr in 31P-MR spectra of porcine coronary arteries perfused with GA.18 Finally, we detected GA in cardiac tissue from GAMT–/– mice with high-performance liquid chromatography (data not shown). Altogether this demonstrates that the resonance at –0.5 ppm originates from P-GA.
Isolated Heart Protocols
To test utilization of P-GA in the CK reaction during increased workload, hearts (n=5 per group) were perfused for 20 minutes with the ;-agonist isoproterenol (20 nmol/L) in the buffer. 31P-spectra from the last 10 minutes were used to quantify PCr/P-GA. Susceptibility to ischemia/reperfusion was tested in other hearts (n=5 WT, n=4 GAMT–/–) subjected to 10 minutes of total, global, and normothermic ischemia, followed by 30 minutes of reperfusion.
Biochemical and Molecular Measurements
Total creatine kinase and citrate synthase activities were determined by spectrophotometry as previously described.6 To measure relative quantities of atrial natriuretic factor (ANF) and ;-myosin heavy chain (MHC) mRNA, total RNA was extracted from frozen LV heart tissue (n=8 per group) with the use of the RNeasy Kit (Qiagen), involving treatment with proteinase K and DNase I and used in real time–polymerase chain reaction (Qiagen Quantitect SYBR Green RTPCR kit, Qiagen) with the use of previously described primers for ANF and ;-MHC24 and the Rotor-Gene system (Corbett Research Ltd). Results were normalized to the expression levels of the housekeeping gene MLN5125 and related to 100% in WT mice.
Statistical Analysis
Statistical significance was assessed by ANOVA or Student t test where appropriate. Data are mean±SD. Differences were considered significant at P<0.05.
Results
Characteristics of WT and GAMT–/– Mice
As shown in Table 1, GAMT–/– had a 28% lower body weight than WT mice, as previously described.15 Tibial length was 5% lower in GAMT–/– mice, suggesting a combination of growth retardation and lower body fat content in GAMT–/– mice. LV weight was 19% lower in GAMT–/– mice, and these mice had an increased LV weight/body weight ratio but a decreased LV weight/tibial length ratio. Thus, to clarify whether GAMT–/– hearts are hypertrophied, we determined myocyte cross-sectional areas, which were similar for WT and GAMT–/– mice. Furthermore, ANF and ;-MHC mRNA levels in LV tissue, which are well-known markers of cardiac hypertrophy, were not significantly different between GAMT–/– and WT mice (Table 1), suggesting that GAMT–/– mice do not develop significant LV hypertrophy.
Cardiac Function at Baseline and During Inotropic Stimulation
Heart rate in conscious mice was 707±50 bpm in WT and 672±68 bpm in GAMT–/– mice (P=NS), and there were no significant differences in any measurement of interval duration (data not shown). In vivo hemodynamics (Table 2) showed no significant differences for any indices of systolic (dP/dtmax, dP/dtmax/iP, PRSW) and diastolic (LV end-diastolic pressure [LVEDP], dP/dtmin, ) function, with the exception of a slightly lower LV systolic pressure in GAMT–/– mice. Systolic and diastolic blood pressure values were also similar. This suggests that baseline cardiac function was essentially normal in GAMT–/– mice. Correspondingly, in vivo MRI (Table 3) showed that hearts from GAMT–/– mice had normal LV ejection fractions (62±7% versus 64±7%) and LV volume indices as well as cardiac indices. LV volumes were somewhat smaller in GAMT–/– mice (data not shown), in keeping with smaller LV mass and body weight. In isolated heart experiments, baseline LVDP (137±30 and 143±18 mm Hg in WT and GAMT–/–, respectively) and heart rate (427±30 versus 408±58 bpm, respectively) were also similar in both groups.
Progressive ;-adrenergic inotropic stimulation with 4 and 16 ng/g body wt per minute dobutamine in vivo resulted in similar stepwise increases of both heart rate (Figure 1) and dP/dtmax (to 13 850±845 and 12 069±973 mm Hg/s in WT and GAMT–/–, respectively, at 16 ng). dP/dtmin did not increase significantly with dobutamine in either group. However, as shown in Figure 1, although there were similar increases in PRSW at low-dose dobutamine, at the higher dose of 16 ng/g body wt per minute dobutamine, PRSW rose to a maximum in WT but failed to increase any further in GAMT–/– mice. Because heart rate increased in a dose-dependent manner in both groups up to the maximum dose of 16 ng dobutamine, the reduced response on PRSW in the GAMT–/– hearts is unlikely to be caused by differences in dobutamine sensitivity because this would also be reflected in the heart rate response. Thus, the reduced maximum response of PRSW to dobutamine suggests that inotropic reserve was significantly impaired in GAMT–/– hearts.
Cardiac Energetics in WT and GAMT–/– Mice
Inotropic Stimulation
To test the usage of P-GA in the CK reaction, hearts were subjected to ;-adrenergic stimulation with 20 nmol/L isoproterenol. This led to similar changes of LVDP and heart rate, resulting in increases of the rate-pressure product to 140±37% and 145±23%. In WT hearts, this was accompanied by a 36±6% decrease of PCr. Interestingly, in GAMT–/– hearts, despite a 100-fold reduction in CK reaction velocity,18 P-GA levels decreased similarly by 38±12%, indicating that P-GA can serve as an energy storage compound, being utilized during increased energy demand.
Ischemia/Reperfusion Injury
Susceptibility of GAMT–/– hearts to ischemia/reperfusion injury was tested by subjecting isolated hearts to 10 minutes of total global ischemia and 30 minutes of reperfusion (Figures 3 and 4 ). With the onset of ischemia, contractile function rapidly deteriorated in both groups. Two of 4 GAMT–/– hearts but none of the WT hearts went into ischemic contracture just before the onset of reperfusion. This is also reflected in a small increase in mean LVEDP at the end of ischemia in this group. On reperfusion, LVEDP rapidly increased in all hearts but more in the GAMT–/– hearts, reaching 23±9 and 51±5 mm Hg in WT and GAMT–/– hearts, respectively, during the last 20 minutes of reperfusion (P<0.01; Figure 3). In accord with this, recovery of contractile function was significantly impaired in GAMT–/– compared with WT hearts (24±10% versus 53±18% of baseline; P<0.05). Both PCr and P-GA rapidly declined after the onset of ischemia in WT and GAMT–/– hearts, respectively (Figure 4A). During reperfusion, PCr showed almost complete recovery (83±13% of baseline), but P-GA showed only partial (36±7%) recovery (P<0.01 versus WT). ATP decreased more slowly during ischemia and showed little recovery during reperfusion in both groups (P=NS; Figure 4B). During ischemia, Pi accumulated similarly in both groups (Figure 4C). During reperfusion, Pi decreased rapidly in WT hearts but much more slowly in GAMT–/– hearts (P<0.05 versus WT; Figure 4C), indicating washout of Pi in the absence of resynthesis of P-GA. During ischemia, pHi dropped rapidly to 6.40±0.10 and 6.28±0.14 in WT and GAMT–/– hearts, respectively, followed by rapid recovery in both groups on reperfusion, but no differences were detected between groups (Figure 4D). Thus, GAMT–/– hearts showed markedly increased susceptibility to ischemia/reperfusion injury. P-GA could be utilized during acute ischemia but failed to compensate fully for the protective effects of PCr.
Discussion
Definition of the Model
We report on the cardiac phenotype of a new model of altered energetics due to knockout of the enzyme catalyzing the second and last essential step of creatine synthesis, GAMT. Other genetically manipulated models12,26,27 have shown LV hypertrophy as a uniform response to impaired cardiac energetics. In GAMT–/– mice, traditional indices of macroscopic hypertrophy, such as total heart weight, heart weight/body weight ratio, or tibial length/body weight ratio, are difficult to interpret because these mice show a change in body composition with a >50% reduction in fat mass,15 whereas we found tibial length to be only slightly reduced by 5%. Thus, to determine whether GAMT–/– mice develop LV hypertrophy, myocyte cross-sectional areas were determined. Although there was a trend for an increase (9%), this was not statistically significant (P=0.55). In addition, classic hypertrophy markers ANF and ;-MHC were not upregulated. Thus, unlike other models of altered energetics, GAMT–/– mice do not develop significant LV hypertrophy.
Because both GAMT–/– and WT animals were bred onto a homogeneous background and with the use of heterozygous parents, all mice used in this study have the same genetic background except for the GAMT gene. Therefore, all detected differences specifically are the result of deletion of the GAMT gene rather than due to differences in genetic background.
Cardiac Function and Energetics in GAMT–/– Mice
Our results indicate that GAMT–/– mice show normal cardiac performance under unstressed, "baseline" conditions. Systolic and diastolic function, measured in vivo with a conductance catheter or with MRI or in vitro in the perfused heart, were similar to those of WT mice. This clearly shows that (otherwise normal) hearts lacking PCr/creatine do not require >1% of CK flux for maintenance of baseline performance. Although we cannot completely rule out that unrecognized adaptations (eg, on the myofibrillar or Ca2+ regulatory level) have occurred in GAMT–/– mice, our results indicate that a high-capacity CK/PCr system does not play a crucial role in maintaining cardiac performance under low workload conditions. In contrast, our results clearly demonstrate the important role of the CK/PCr system under situations of myocardial stress either due to increased workload and thus energy demand or during demand/supply mismatch (ischemia).
Inotropic Stimulation
When hearts were stimulated inotropically in vivo with increasing doses of dobutamine to test contractile reserve, GAMT–/– mice initially increased PRSW, the best size- and preload-independent parameter of contractility,28 but failed to reach maximum levels of performance under high-dose dobutamine. Thus, in vivo contractile reserve was impaired in GAMT–/– mice, underscoring the importance of a high-capacity CK/PCr system for achieving high inotropic states. This finding is in agreement with previous studies in perfused hearts in which CK was inhibited by iodoacetamide3 or in vivo in hearts chronically fed ;-GP leading to 80% PCr depletion, in which maximum LV pressure during aortic occlusion was impaired.29 Our model could test this question more accurately, given that PCr depletion in GAMT–/– mice is much more severe than after ;-GP feeding, and the issue of potential nonspecific effects of a chemical CK inhibitor3 can be avoided. Interestingly, in CK double-knockout mice, inotropic reserve was also reported to be impaired.30 Thus, all available evidence strongly suggests that hearts lacking a high-capacity CK/PCr system are unable to attain high workload states.
To test the hypothesis that in GAMT–/– hearts P-GA is utilized via the CK reaction during increased workload, we studied perfused hearts with 31P-MRS during isoproterenol stimulation. We showed that, despite a CK reaction velocity that was 2 orders of magnitude lower, P-GA was utilized during high-workload conditions; whereas PCr levels decreased by 36% in WT mice, P-GA levels fell by a similar 38% in GAMT–/– mice. We therefore speculate that P-GA takes up the function of PCr as a source of high-energy phosphate bonds under inotropic stress conditions, thereby, at least in part, compensating for the lack of PCr. It should be noted that workloads in the isolated heart model both at baseline and under isoproterenol are generally lower than in the in vivo situation,31 and it would be interesting to study function and energetics in these mice in vivo. However, at the present time, techniques for in vivo cardiac 31P-MRS in the mouse26 are extremely challenging and unavailable to us and most other laboratories. Partial compensation by P-GA may also explain why, unlike in other models of altered energetics, LV hypertrophy does not develop in GAMT–/– mice.
Ischemia/Reperfusion
Ischemia/reperfusion is the most extreme situation of cardiac energy demand/supply mismatch, and we therefore tested susceptibility to this form of cardiac injury in GAMT–/– mice. Our data demonstrate that recovery of both systolic (LVDP) and diastolic (LVEDP) functions during reperfusion are substantially impaired in GAMT–/– mice, ie, these mice show increased susceptibility to ischemia/reperfusion injury. This is reflected by changes in high-energy phosphate metabolism. During ischemia, both PCr and P-GA rapidly declined to nearly undetectable levels. This showed that P-GA again provides a source of high-energy phosphate bonds during acute stress (ischemia). In parallel with reduced functional recovery, resynthesis of P-GA and reduction of Pi levels were significantly impaired during reperfusion. These findings clearly indicate that a high-capacity CK/PCr system protects the heart from ischemia/reperfusion injury. A reduction in the capacity of this system, through a combination of reduced energy storage and reduced CK flux, as is the case in GAMT–/– mice, has deleterious consequences for the ischemic myocardium. These findings are consistent with our previous work showing that susceptibility to ischemia/reperfusion is also impaired in CK double-knockout mice with 3% residual CK activity32 and that rats with 80% depletion of PCr stores cannot survive an acute coronary ligation.33
Why do GAMT–/– mice show increased susceptibility to ischemia/reperfusion; First, these hearts enter the ischemic period with 33% lower high-energy phosphate stores at baseline (in the form of P-GA versus PCr in WT), and this lack of energy reserve may account for the exacerbation of ischemic injury. Second, GA has been reported to inhibit Na+/K+-ATPase34; this may result in additional Na+ loading during ischemia in GAMT–/– hearts, which in turn may impair Ca2+ efflux via Na+-Ca2+ exchange or even result in Ca2+ influx via reversed Na+-Ca2+ exchange. Third, recovery of P-GA during reperfusion was significantly impaired in GAMT–/– mice. The discrepancy between unaltered breakdown of P-GA (compared with PCr) during ischemia and impaired resynthesis on reperfusion may be related to the different CK isoenzymes involved: PCr breakdown mainly occurs through the cytosolic CK isoenzymes MM and MB, and PCr synthesis mainly occurs via mitochondrial CK. The Vmax of mito-CK for Cr and PCr is substantially lower than that of cytosolic CK, and the reactivity of GA with MM-CK is 100 times lower than that of creatine.18 The reactivity of GA with mito-CK is unknown. It is tempting to speculate that incomplete resynthesis of GA may be due to the lower reaction velocity of mito-CK on reperfusion compared with P-GA breakdown during ischemia by MM-CK. In addition, differences in nonenzymatic degradation of P-GA and PCr could play a role during ischemia. It is also possible that more GA than creatine is lost from the cardiomyocyte during ischemia and/or early reperfusion. Although both compounds are electroneutral, GA is a smaller molecule than creatine, possibly leading to increased leakage across the sarcolemma.
Limitations and Future Directions
We did not use a time-specific GAMT-knockout model. Thus, in addition to the compensatory increase in P-GA, other adaptations to the lack of creatine and PCr may have occurred during fetal and adult development. This will be an interesting topic for future studies. However, unlike in other models of impaired energetics,12,26,27 potential adaptations did not lead to LV hypertrophy. The reasons for this discrepancy in hypertrophy development between different models of altered cardiac energetics clearly warrant further study. However, one explanation for a lack of hypertrophy in GAMT–/– mice may be that we used heterozygous parents to create GAMT–/– mice. Thus, during embryonic development and during the postnatal period, GAMT–/– mice were provided with a source of creatine by their mothers, and severe creatine depletion only develops in these mice after weaning. In contrast, CK double-knockout mice, for example, have a severely impaired CK system throughout their entire intrauterine, postneonatal, and adult life. It is conceivable that these differences in timing explain the difference in hypertrophy development.
In our in vivo LV conductance measurements, the difference in heart size between GAMT–/– and WT mice limited the number of parameters that were available for baseline comparison, eg, the slopes of the end-systolic pressure-volume relationship and of the dP/dtmax–end-diastolic volume relationship are both chamber size dependent.35,36 Little additional benefit would therefore be attained by measuring to calibrate these parameters; instead, we used PRSW, which is independent of heart size, insensitive to loading conditions, and independent of calibration factors such as and parallel conductance.28 As such, PRSW can be considered particularly reliable as a measure of contractile function. Under inotropic stimulation, dP/dtmax values were not significantly different between WT and GAMT–/– mice, but PRSW was. This underscores the limited validity of dP/dtmax as a parameter of contractility because it is highly preload dependent. Indeed, Kass et al31 have shown that a change of 1 mm Hg in EDP can result in an 18% change in dP/dtmax and that this load dependency increases under inotropic stimulation (eg, with dobutamine). As they also note, 1 mm Hg is close to the noise level for this type of pressure measurement.
GAMT–/– mice are a potentially interesting model to further clarify the role of the CK/PCr system for the development of heart failure. In the present study energetic abnormalities do not occur in isolation but in the context of changes in a variety of other cellular systems involved in contractile function and dysfunction. Thus, future work should test whether LV remodeling is aggravated in GAMT–/– mice in a chronic heart failure model, eg, after coronary ligation.
In summary, we have shown that GAMT–/– mice completely lacking myocardial PCr and creatine show a compensatory accumulation of P-GA, ie, these mice have severely reduced CK reaction velocity (1%) but partially maintained high-energy phosphate stores. These mice do not develop cardiac hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under these conditions. However, when stressed by inotropic stimulation or ischemia/reperfusion, GAMT–/– mice exhibit a markedly abnormal phenotype, demonstrating that hearts with a severely reduced capacity of the CK/PCr system show limited inotropic reserve and aggravated injury after acute stress.
Acknowledgments
This study was supported by the British Heart Foundation. Dr Watkins is a Fellow of the American Heart Association.
Footnotes
The first 2 authors have contributed equally to this work.
References
Pool PE, Spann JF, Buccino RA, Sonnenblick EH, Braunwald E. Myocardial high-energy phosphate stores in cardiac hypertrophy and heart failure. Circ Res. 1967; 21: 365–373.
Ingwall JS, Kramer MF, Fifer MA, Lorell BH, Shemin R, Grossman W, Allen PD. The creatine kinase system in normal and diseased human myocardium. N Engl J Med. 1985; 313: 1050–1054.
Tian R. Thermodynamic limitation for the sarcoplasmic reticulum Ca2+-ATPase contributes to impaired contractile reserve in hearts. Ann N Y Acad Sci. 1998; 853: 322–324.
Neubauer S, Hamman BL, Perry SB, Bittl JA, Ingwall JS. Velocity of the creatine kinase reaction decreases in postischemic myocardium: a 31P-NMR magnetization transfer study of the isolated ferret heart. Circ Res. 1988; 63: 1–15.
Nascimben L, Ingwall JS, Pauletto P, Friedrich J, Gwathmey JK, Saks V, Pessina AC, Allen PD. Creatine kinase system in failing and nonfailing human myocardium. Circulation. 1996; 94: 1894–1901.
Neubauer S, Horn M, Naumann A, Tian R, Hu K, Laser M, Friedrich J, Gaudron P, Schnackerz K, Ingwall JS, Ertl G. Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction. J Clin Invest. 1995; 95: 1092–1100.
Ingwall JS, Weiss RG. Is the failing heart energy starved; On using chemical energy to support cardiac function. Circ Res. 2004; 95: 135–145.
van Deursen J, Heerschap A, Oerlemans F, Ruitenbeek W, Jap P, ter Laak H, Wieringa B. Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity. Cell. 1993; 74: 621–631.
Steeghs K, Benders A, Oerlemans F, deHaan A, Heerschap A, Ruitenbeek W, Jost C, vanDeursen J, Perryman B, Pette D, Bruckwilder M, Koudijs J, Jap P, Veerkamp J, Wieringa B. Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies. Cell. 1997; 89: 93–103.
Saupe KW, Spindler M, Hopkins JC, Shen W, Ingwall JS. Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart: reaction kinetics of the creatine kinase isoenzymes in the intact heart. J Biol Chem. 2000; 275: 19742–19746.
Kaasik A, Veksler V, Boehm E, Novotova M, Minajeva A, Ventura-Clapier R. Energetic crosstalk between organelles: architectural integration of energy production and utilization. Circ Res. 2001; 89: 153–159.
Boehm E, Ventura-Clapier R, Mateo P, Lechene P, Veksler V. Glycolysis supports calcium uptake by the sarcoplasmic reticulum in skinned ventricular fibres of mice deficient in mitochondrial and cytosolic creatine kinase. J Mol Cell Cardiol. 2000; 32: 891–902.
Nahrendorf M, Spindler M, Hu K, Bauer L, Ritter O, Nordbeck P, Quaschning T, Hiller KH, Wallis J, Ertl G, Bauer WR, Neubauer S. Creatine kinase knockout mice show left ventricular hypertrophy and dilatation, but unaltered remodeling post-myocardial infarction. Cardiovasc Res. 2005: 65: 419–427.
Saupe KW, Spindler M, Tian R, Ingwall JS. Impaired cardiac energetics in mice lacking muscle-specific isoenzymes of creatine kinase. Circ Res. 1998; 82: 898–907.
Schmidt A, Marescau B, Boehm EA, Renema WK, Peco R, Das A, Steinfeld R, Chan S, Wallis J, Davidoff M, Ullrich K, Waldschutz R, Heerschap A, De Deyn PP, Neubauer S, Isbrandt D. Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency. Hum Mol Genet. 2004; 13: 905–921.
Kan HE, Renema WK, Isbrandt D, Heerschap A. Phosphorylated guanidinoacetate partly compensates for the lack of phosphocreatine in skeletal muscle of mice lacking guanidinoacetate methyltransferase. J Physiol. 2004; 560: 219–229.
Schneider JE, Tyler DJ, Ten Hove M, Sang AE, Cassidy PJ, Fischer A, Wallis J, Sebag-Montefiore LM, Watkins H, Isbrandt D, Clarke K, Neubauer S. In vivo cardiac 1H-MRS in the mouse. Magn Reson Med. 2004; 52: 1029–1035.
Boehm EA, Radda GK, Tomlin H, Clark JF. The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase. Biochim Biophys Acta. 1996; 1274: 119–128.
Schneider JE, Cassidy PJ, Lygate C, Tyler DJ, Wiesmann F, Grieve SM, Hulbert K, Clarke K, Neubauer S. Fast, high-resolution in vivo cine magnetic resonance imaging in normal and failing mouse hearts on a vertical 11.7 T system. J Magn Reson Imaging. 2003; 18: 691–701.
Nemoto S, DeFreitas G, Mann DL, Carabello BA. Effects of changes in left ventricular contractility on indexes of contractility in mice. Am J Physiol. 2002; 283: H2504–H2510.
Chu V, Otero JM, Lopez O, Morgan JP, Amende I, Hampton TG. Method for non-invasively recording electrocardiograms in conscious mice. BMC Physiol. 2001; 1: 6.
Shioi T, McMullen JR, Tarnavski O, Converso K, Sherwood MC, Manning WJ, Izumo S. Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation. 2003; 107: 1664–1670.
Schulze A, Bachert P, Schlemmer H, Harting I, Polster T, Salomons GS, Verhoeven NM, Jakobs C, Fowler B, Hoffmann GF, Mayatepek E. Lack of creatine in muscle and brain in an adult with GAMT deficiency. Ann Neurol. 2003; 53: 248–251.
Gaussin V, Tomlinson JE, Depre C, Engelhardt S, Antos CL, Takagi G, Hein L, Topper JN, Liggett SB, Olson EN, Lohse MJ, Vatner SF, Vatner DE. Common genomic response in different mouse models of beta-adrenergic-induced cardiomyopathy. Circulation. 2003; 108: 2926–2933.
Hamalainen HK, Tubman JC, Vikman S, Kyrola T, Ylikoski E, Warrington JA, Lahesmaa R. Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Anal Biochem. 2001; 299: 63–70.
Weiss RG, Chatham JC, Georgakopolous D, Charron MJ, Wallimann T, Kay L, Walzel B, Wang Y, Kass DA, Gerstenblith G, Chacko VP. An increase in the myocardial PCr/ATP ratio in GLUT4 null mice. FASEB J. 2002; 16: 613–615.
Spindler M, Saupe KW, Christe ME, Sweeney HL, Seidman CE, Seidman JG, Ingwall JS. Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy. J Clin Invest. 1998; 101: 1775–1783.
Yang B, Larson DF, Watson R. Age-related left ventricular function in the mouse: analysis based on in vivo pressure-volume relationships. Am J Physiol. 1999; 277: H1906–H1913.
Neubauer S, Hu K, Horn M, Remkes H, Hoffmann KD, Schmidt C, Schmidt TJ, Schnackerz K, Ertl G. Functional and energetic consequences of chronic myocardial creatine depletion by beta-guanidinopropionate in perfused hearts and in intact rats. J Mol Cell Cardiol. 1999; 31: 1845–1855.
Crozatier B, Badoual T, Boehm E, Ennezat PV, Guenoun T, Su J, Veksler V, Hittinger L, Ventura-Clapier R. Role of creatine kinase in cardiac excitation-contraction coupling: studies in creatine kinase-deficient mice. FASEB J. 2002; 16: 653–660.
Kass DA, Hare JM, Georgakopoulos D. Murine cardiac function: a cautionary tail [published correction appears in Circ Res. 1998;83:115]. Circ Res. 1998; 82: 519–522.
Spindler M, Meyer K, Stromer H, Leupold A, Boehm E, Wagner H, Neubauer S. Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia-reperfusion injury and impaired calcium homeostasis. Am J Physiol. 2004; 287: H1039–H1045.
Horn M, Remkes H, Stromer H, Dienesch C, Neubauer S. Chronic phosphocreatine depletion by the creatine analogue (beta)-guanidinopropionate is associated with increased mortality and loss of ATP in rats after myocardial infarction. Circulation. 2001; 104: 1844–1849.
Zugno AI, Stefanello FM, Streck EL, Calcagnotto T, Wannmacher CM, Wajner M, Wyse AT. Inhibition of Na+, K+-ATPase activity in rat striatum by guanidinoacetate. Int J Dev Neurosci. 2003; 21: 183–189.
Georgakopoulos D, Mitzner WA, Chen CH, Byrne BJ, Millar HD, Hare JM, Kass DA. In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol. 1998; 274: H1416–H1422.
Takaoka H, Esposito G, Mao L, Suga H, Rockman HA. Heart size-independent analysis of myocardial function in murine pressure overload hypertrophy. Am J Physiol. 2002; 282: H2190–H2207.(Michiel ten Hove, PhD; Cr)
Center for Molecular Neurobiology, Hamburg, Germany (D.I.).
Abstract
Background— The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase–knockout (GAMT–/–) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT–/– mice.
Methods and Results— GAMT–/– mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and ;-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT–/– mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT–/– hearts (101±8 mm Hg in WT versus 59±7 mm Hg in GAMT–/–; P<0.05). 31P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT–/– hearts utilized P-GA. During ischemia/reperfusion, GAMT–/– hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23±9 in WT and 51±5 mm Hg in GAMT–/– during reperfusion; P<0.05) and incomplete resynthesis of P-GA.
Conclusions— GAMT–/– mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT–/– mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.
Key Words: creatine kinase ; metabolism ; hemodynamics ; ischemia ; myocardium
Introduction
Despite decades of research,1,2 the true functional role of high-energy phosphate metabolism for cardiac function in normal and diseased states remains to be fully clarified. Creatine kinase (CK) catalyzes the transfer of the high-energy phosphate bond between ATP and phosphocreatine (PCr). Chemical inhibition of CK by iodoacetamide substantially reduces contractile reserve in normal hearts.3 CK function has also been shown to be compromised in postischemic4 and in failing myocardium,5,6 and this mechanism may contribute to the pathophysiology of heart failure.7
More recently, genetically manipulated mouse models have provided the opportunity to dissect the role of the CK/PCr system in greater detail. CK-knockout mice provided the first model of this kind, and M-CK–knockout,8 mito-CK–knockout, and double (M- and mito-CK)–knockout9 mice have been reported, the latter showing 3% of residual CK activity (in the form of BB-CK).10 However, thus far, results with regard to CK-knockout mice have been conflicting and await further clarification, with some groups showing significant functional alterations, adaptations, and left ventricular (LV) hypertrophy in CK double-knockout mice11–13 but others demonstrating relatively little functional, although energetic, impact.10,14 Importantly, CK-knockout mice show normal levels of myocardial creatine. An alternative intervention to impair the function of the CK/PCr system would be to create transgenic mouse models of altered total creatine, and thus PCr, content. Creatine is mainly produced in liver and kidney through 2 essential chemical reactions, the first catalyzed by arginine:glycine amidinotransferase, or AGAT (arginine+glycine/guanidinoacetate+ornithine), and the second step catalyzed by guanidinoacetate N-methyltransferase, or GAMT (guanidinoacetate/creatine).
We recently reported on a new model of altered energetics due to knockout of GAMT.15 Our initial report focused on the neurological and skeletal muscle phenotype of these mice but also demonstrated normal baseline perfused heart function (LV developed pressure [LVDP]) in GAMT-knockout (GAMT–/–) mice. GAMT–/– mice show substantial accumulation of the creatine precursor guanidinoacetate (GA) and its phosphorylated form, phospho-guanidinoacetate (P-GA). However, creatine levels in heart were 27% of those in WT mice.15 Although these animals were fed with a creatine-free diet, we now know that the source of the residual creatine is coprophagia by GAMT–/– mice when housed with heterozygous or WT littermate controls because the latter have creatine present in their feces.16 We have shown that cardiac creatine and PCr decline below levels of detectability if homozygous GAMT–/– mice are housed separately.17
Therefore, we now have a new mouse model, which shows undetectable myocardial (phospho)creatine levels and substantial accumulation of the precursor (P-)GA. Although P-GA participates in the CK reaction, the reaction velocity is 100 times slower compared with phosphocreatine.18 In the present study we took advantage of this new model of altered cardiac energetics. Using an array of sophisticated tools for murine cardiac phenotyping including MRI and MR spectroscopy (MRS), we tested the hypothesis that in vivo cardiac function at baseline and during inotropic stress, as well as susceptibility to ischemia/reperfusion injury, is compromised in GAMT–/– mice lacking creatine and PCr.
Methods
WT and GAMT–/– Mice
Heterozygous GAMT+/– mice, constructed as described before,15 were backcrossed for at least 8 generations onto a C57Bl/6 genetic background. Mice used for experiments were generated by intercrossing GAMT+/– mice and genotyped by polymerase chain reaction.15 From 6 weeks of age, mice were housed per genotype to prevent ingestion of creatine in GAMT–/– mice through coprophagia. Mice were kept in cages with 12-hour light/dark cycle and controlled temperature (20°C to 22°C) and fed creatine-free chow and water ad libitum. All studies were performed in 5- to 8-month-old mice and were conducted in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.
In Vivo MRI
High-resolution murine cine MRI was applied in vivo on GAMT–/– (n=13; 6 male, 7 female) and WT (n=14; 7 male, 7 female) mice as described previously19 to assess LV mass and volumes.
In Vivo Hemodynamics at Baseline and During Inotropic Stimulation
Measurements were made in closed-chest, spontaneously breathing mice (as described in Nemoto et al20). GAMT–/– and WT littermates (n=12; 5 males, 7 females in each group) were anesthetized with 2% isoflurane in oxygen on a homeothermic blanket. A 1.4F microtipped conductance cannula (SPR-839, Millar Instruments) was advanced retrogradely into the LV via the right carotid artery under echocardiographic guidance. The right jugular vein was cannulated with stretched polythene tubing for infusion of dobutamine. Preload conditions were altered by occlusion of the inferior vena cava. A small incision was made in the abdominal wall at the level of the xiphisternum, and a cotton swab was used to apply transient compression. Isoflurane was reduced to 1.25% to 1.5%, followed by equilibration until hemodynamic indices were stable for >15 minutes. Measurements were taken under steady state and inferior vena cava occlusion, at baseline and during infusion of low- and high-dose dobutamine (4 and 16 ng/g body wt per minute). Recordings were analyzed with the use of PVAN software version 3.0 (Millar Instruments). The cannula was calibrated to relative volume units with a cuvette system, and parallel conductance was measured by saline bolus method.20 No correction for the electrical field inhomogeneity () was made; therefore, the only occlusion parameter reported is preload recruitable stroke work (PRSW) because this is and chamber size independent. Systolic and diastolic blood pressures were measured invasively in separate groups of anesthetized mice (n=4 WT; n=5 GAMT–/–).
Conscious ECG
ECGs from conscious unrestrained mice (n=7 WT; n=9 GAMT–/–) were recorded with the use of the AnonyMOUSE ECG screening tool (Mouse Specifics Inc).21
Assessment of Myocyte Cross-Sectional Area by Histology
A papillary level short-axis slice of the LV (n=8 per group) was fixed in formaldehyde, dehydrated, and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined under an inverted microscope. Myocyte cross-sectional area was measured by computerized planimetry as previously described.22
Perfused Heart Experiments
Male mice were heparinized (5000 U/kg body wt) and anesthetized (pentobarbitone 140 mg/kg body wt IP). Hearts were excised, cannulated, and perfused in Langendorff mode at 80 mm Hg and 37°C with Krebs-Henseleit buffer gassed with 95% O2/5% CO2 (pH 7.4) containing the following (in mmol/L): 149 Na+, 5.9 K+, 1.2 Mg2+, 2.25 Ca2+, 1.2 SO42–, 126.2 Cl–, 0.5 EDTA2–, 25 HCO3–, 1.2 H2PO4–, 11 D-glucose, 4.5 pyruvate, and 0.5 lactate. Contractility was assessed with a fluid-filled intraventricular balloon connected to a pressure transducer (AD Instruments Ltd). The end-diastolic pressure (EDP) was set to 5 to 15 mm Hg.
31P-MRS
Five-minute 31P-nuclear MR spectra of perfused mouse hearts were acquired on a Bruker Avance 500 spectrometer equipped with a 11.7-T magnet and a 20-mm 1H-/31P-cross-cage resonator with a pulse-and-collect sequence (repetition time=100 ms, =29°, number of scans=3000) and corrected for partial saturation. Metabolites were quantified with respect to the signal intensity of a known amount of phenylphosphonic acid in the intraventricular balloon with the use of the AMARES algorithm and MRUI software. Baseline concentrations were calculated with the use of 20-minute spectra. Baseline [Pi] was too low for quantification, and therefore [Pi] is only shown from ischemia onward. Intracellular volume was assumed to be 0.5 mL/g wet wt. pHi was determined from the chemical shift between Pi and PCr with the use of pH=6.72–log(–5.72)/(3.17–). In the absence of PCr, phenylphosphonic acid (18 ppm) was used as a frequency reference. GAMT–/– mice show an additional resonance at –0.5 ppm, which previously has been assigned to P-GA15,16 as also described in a human patient with GAMT deficiency.23 In addition, this resonance has been shown to appear to the right of PCr in 31P-MR spectra of porcine coronary arteries perfused with GA.18 Finally, we detected GA in cardiac tissue from GAMT–/– mice with high-performance liquid chromatography (data not shown). Altogether this demonstrates that the resonance at –0.5 ppm originates from P-GA.
Isolated Heart Protocols
To test utilization of P-GA in the CK reaction during increased workload, hearts (n=5 per group) were perfused for 20 minutes with the ;-agonist isoproterenol (20 nmol/L) in the buffer. 31P-spectra from the last 10 minutes were used to quantify PCr/P-GA. Susceptibility to ischemia/reperfusion was tested in other hearts (n=5 WT, n=4 GAMT–/–) subjected to 10 minutes of total, global, and normothermic ischemia, followed by 30 minutes of reperfusion.
Biochemical and Molecular Measurements
Total creatine kinase and citrate synthase activities were determined by spectrophotometry as previously described.6 To measure relative quantities of atrial natriuretic factor (ANF) and ;-myosin heavy chain (MHC) mRNA, total RNA was extracted from frozen LV heart tissue (n=8 per group) with the use of the RNeasy Kit (Qiagen), involving treatment with proteinase K and DNase I and used in real time–polymerase chain reaction (Qiagen Quantitect SYBR Green RTPCR kit, Qiagen) with the use of previously described primers for ANF and ;-MHC24 and the Rotor-Gene system (Corbett Research Ltd). Results were normalized to the expression levels of the housekeeping gene MLN5125 and related to 100% in WT mice.
Statistical Analysis
Statistical significance was assessed by ANOVA or Student t test where appropriate. Data are mean±SD. Differences were considered significant at P<0.05.
Results
Characteristics of WT and GAMT–/– Mice
As shown in Table 1, GAMT–/– had a 28% lower body weight than WT mice, as previously described.15 Tibial length was 5% lower in GAMT–/– mice, suggesting a combination of growth retardation and lower body fat content in GAMT–/– mice. LV weight was 19% lower in GAMT–/– mice, and these mice had an increased LV weight/body weight ratio but a decreased LV weight/tibial length ratio. Thus, to clarify whether GAMT–/– hearts are hypertrophied, we determined myocyte cross-sectional areas, which were similar for WT and GAMT–/– mice. Furthermore, ANF and ;-MHC mRNA levels in LV tissue, which are well-known markers of cardiac hypertrophy, were not significantly different between GAMT–/– and WT mice (Table 1), suggesting that GAMT–/– mice do not develop significant LV hypertrophy.
Cardiac Function at Baseline and During Inotropic Stimulation
Heart rate in conscious mice was 707±50 bpm in WT and 672±68 bpm in GAMT–/– mice (P=NS), and there were no significant differences in any measurement of interval duration (data not shown). In vivo hemodynamics (Table 2) showed no significant differences for any indices of systolic (dP/dtmax, dP/dtmax/iP, PRSW) and diastolic (LV end-diastolic pressure [LVEDP], dP/dtmin, ) function, with the exception of a slightly lower LV systolic pressure in GAMT–/– mice. Systolic and diastolic blood pressure values were also similar. This suggests that baseline cardiac function was essentially normal in GAMT–/– mice. Correspondingly, in vivo MRI (Table 3) showed that hearts from GAMT–/– mice had normal LV ejection fractions (62±7% versus 64±7%) and LV volume indices as well as cardiac indices. LV volumes were somewhat smaller in GAMT–/– mice (data not shown), in keeping with smaller LV mass and body weight. In isolated heart experiments, baseline LVDP (137±30 and 143±18 mm Hg in WT and GAMT–/–, respectively) and heart rate (427±30 versus 408±58 bpm, respectively) were also similar in both groups.
Progressive ;-adrenergic inotropic stimulation with 4 and 16 ng/g body wt per minute dobutamine in vivo resulted in similar stepwise increases of both heart rate (Figure 1) and dP/dtmax (to 13 850±845 and 12 069±973 mm Hg/s in WT and GAMT–/–, respectively, at 16 ng). dP/dtmin did not increase significantly with dobutamine in either group. However, as shown in Figure 1, although there were similar increases in PRSW at low-dose dobutamine, at the higher dose of 16 ng/g body wt per minute dobutamine, PRSW rose to a maximum in WT but failed to increase any further in GAMT–/– mice. Because heart rate increased in a dose-dependent manner in both groups up to the maximum dose of 16 ng dobutamine, the reduced response on PRSW in the GAMT–/– hearts is unlikely to be caused by differences in dobutamine sensitivity because this would also be reflected in the heart rate response. Thus, the reduced maximum response of PRSW to dobutamine suggests that inotropic reserve was significantly impaired in GAMT–/– hearts.
Cardiac Energetics in WT and GAMT–/– Mice
Inotropic Stimulation
To test the usage of P-GA in the CK reaction, hearts were subjected to ;-adrenergic stimulation with 20 nmol/L isoproterenol. This led to similar changes of LVDP and heart rate, resulting in increases of the rate-pressure product to 140±37% and 145±23%. In WT hearts, this was accompanied by a 36±6% decrease of PCr. Interestingly, in GAMT–/– hearts, despite a 100-fold reduction in CK reaction velocity,18 P-GA levels decreased similarly by 38±12%, indicating that P-GA can serve as an energy storage compound, being utilized during increased energy demand.
Ischemia/Reperfusion Injury
Susceptibility of GAMT–/– hearts to ischemia/reperfusion injury was tested by subjecting isolated hearts to 10 minutes of total global ischemia and 30 minutes of reperfusion (Figures 3 and 4 ). With the onset of ischemia, contractile function rapidly deteriorated in both groups. Two of 4 GAMT–/– hearts but none of the WT hearts went into ischemic contracture just before the onset of reperfusion. This is also reflected in a small increase in mean LVEDP at the end of ischemia in this group. On reperfusion, LVEDP rapidly increased in all hearts but more in the GAMT–/– hearts, reaching 23±9 and 51±5 mm Hg in WT and GAMT–/– hearts, respectively, during the last 20 minutes of reperfusion (P<0.01; Figure 3). In accord with this, recovery of contractile function was significantly impaired in GAMT–/– compared with WT hearts (24±10% versus 53±18% of baseline; P<0.05). Both PCr and P-GA rapidly declined after the onset of ischemia in WT and GAMT–/– hearts, respectively (Figure 4A). During reperfusion, PCr showed almost complete recovery (83±13% of baseline), but P-GA showed only partial (36±7%) recovery (P<0.01 versus WT). ATP decreased more slowly during ischemia and showed little recovery during reperfusion in both groups (P=NS; Figure 4B). During ischemia, Pi accumulated similarly in both groups (Figure 4C). During reperfusion, Pi decreased rapidly in WT hearts but much more slowly in GAMT–/– hearts (P<0.05 versus WT; Figure 4C), indicating washout of Pi in the absence of resynthesis of P-GA. During ischemia, pHi dropped rapidly to 6.40±0.10 and 6.28±0.14 in WT and GAMT–/– hearts, respectively, followed by rapid recovery in both groups on reperfusion, but no differences were detected between groups (Figure 4D). Thus, GAMT–/– hearts showed markedly increased susceptibility to ischemia/reperfusion injury. P-GA could be utilized during acute ischemia but failed to compensate fully for the protective effects of PCr.
Discussion
Definition of the Model
We report on the cardiac phenotype of a new model of altered energetics due to knockout of the enzyme catalyzing the second and last essential step of creatine synthesis, GAMT. Other genetically manipulated models12,26,27 have shown LV hypertrophy as a uniform response to impaired cardiac energetics. In GAMT–/– mice, traditional indices of macroscopic hypertrophy, such as total heart weight, heart weight/body weight ratio, or tibial length/body weight ratio, are difficult to interpret because these mice show a change in body composition with a >50% reduction in fat mass,15 whereas we found tibial length to be only slightly reduced by 5%. Thus, to determine whether GAMT–/– mice develop LV hypertrophy, myocyte cross-sectional areas were determined. Although there was a trend for an increase (9%), this was not statistically significant (P=0.55). In addition, classic hypertrophy markers ANF and ;-MHC were not upregulated. Thus, unlike other models of altered energetics, GAMT–/– mice do not develop significant LV hypertrophy.
Because both GAMT–/– and WT animals were bred onto a homogeneous background and with the use of heterozygous parents, all mice used in this study have the same genetic background except for the GAMT gene. Therefore, all detected differences specifically are the result of deletion of the GAMT gene rather than due to differences in genetic background.
Cardiac Function and Energetics in GAMT–/– Mice
Our results indicate that GAMT–/– mice show normal cardiac performance under unstressed, "baseline" conditions. Systolic and diastolic function, measured in vivo with a conductance catheter or with MRI or in vitro in the perfused heart, were similar to those of WT mice. This clearly shows that (otherwise normal) hearts lacking PCr/creatine do not require >1% of CK flux for maintenance of baseline performance. Although we cannot completely rule out that unrecognized adaptations (eg, on the myofibrillar or Ca2+ regulatory level) have occurred in GAMT–/– mice, our results indicate that a high-capacity CK/PCr system does not play a crucial role in maintaining cardiac performance under low workload conditions. In contrast, our results clearly demonstrate the important role of the CK/PCr system under situations of myocardial stress either due to increased workload and thus energy demand or during demand/supply mismatch (ischemia).
Inotropic Stimulation
When hearts were stimulated inotropically in vivo with increasing doses of dobutamine to test contractile reserve, GAMT–/– mice initially increased PRSW, the best size- and preload-independent parameter of contractility,28 but failed to reach maximum levels of performance under high-dose dobutamine. Thus, in vivo contractile reserve was impaired in GAMT–/– mice, underscoring the importance of a high-capacity CK/PCr system for achieving high inotropic states. This finding is in agreement with previous studies in perfused hearts in which CK was inhibited by iodoacetamide3 or in vivo in hearts chronically fed ;-GP leading to 80% PCr depletion, in which maximum LV pressure during aortic occlusion was impaired.29 Our model could test this question more accurately, given that PCr depletion in GAMT–/– mice is much more severe than after ;-GP feeding, and the issue of potential nonspecific effects of a chemical CK inhibitor3 can be avoided. Interestingly, in CK double-knockout mice, inotropic reserve was also reported to be impaired.30 Thus, all available evidence strongly suggests that hearts lacking a high-capacity CK/PCr system are unable to attain high workload states.
To test the hypothesis that in GAMT–/– hearts P-GA is utilized via the CK reaction during increased workload, we studied perfused hearts with 31P-MRS during isoproterenol stimulation. We showed that, despite a CK reaction velocity that was 2 orders of magnitude lower, P-GA was utilized during high-workload conditions; whereas PCr levels decreased by 36% in WT mice, P-GA levels fell by a similar 38% in GAMT–/– mice. We therefore speculate that P-GA takes up the function of PCr as a source of high-energy phosphate bonds under inotropic stress conditions, thereby, at least in part, compensating for the lack of PCr. It should be noted that workloads in the isolated heart model both at baseline and under isoproterenol are generally lower than in the in vivo situation,31 and it would be interesting to study function and energetics in these mice in vivo. However, at the present time, techniques for in vivo cardiac 31P-MRS in the mouse26 are extremely challenging and unavailable to us and most other laboratories. Partial compensation by P-GA may also explain why, unlike in other models of altered energetics, LV hypertrophy does not develop in GAMT–/– mice.
Ischemia/Reperfusion
Ischemia/reperfusion is the most extreme situation of cardiac energy demand/supply mismatch, and we therefore tested susceptibility to this form of cardiac injury in GAMT–/– mice. Our data demonstrate that recovery of both systolic (LVDP) and diastolic (LVEDP) functions during reperfusion are substantially impaired in GAMT–/– mice, ie, these mice show increased susceptibility to ischemia/reperfusion injury. This is reflected by changes in high-energy phosphate metabolism. During ischemia, both PCr and P-GA rapidly declined to nearly undetectable levels. This showed that P-GA again provides a source of high-energy phosphate bonds during acute stress (ischemia). In parallel with reduced functional recovery, resynthesis of P-GA and reduction of Pi levels were significantly impaired during reperfusion. These findings clearly indicate that a high-capacity CK/PCr system protects the heart from ischemia/reperfusion injury. A reduction in the capacity of this system, through a combination of reduced energy storage and reduced CK flux, as is the case in GAMT–/– mice, has deleterious consequences for the ischemic myocardium. These findings are consistent with our previous work showing that susceptibility to ischemia/reperfusion is also impaired in CK double-knockout mice with 3% residual CK activity32 and that rats with 80% depletion of PCr stores cannot survive an acute coronary ligation.33
Why do GAMT–/– mice show increased susceptibility to ischemia/reperfusion; First, these hearts enter the ischemic period with 33% lower high-energy phosphate stores at baseline (in the form of P-GA versus PCr in WT), and this lack of energy reserve may account for the exacerbation of ischemic injury. Second, GA has been reported to inhibit Na+/K+-ATPase34; this may result in additional Na+ loading during ischemia in GAMT–/– hearts, which in turn may impair Ca2+ efflux via Na+-Ca2+ exchange or even result in Ca2+ influx via reversed Na+-Ca2+ exchange. Third, recovery of P-GA during reperfusion was significantly impaired in GAMT–/– mice. The discrepancy between unaltered breakdown of P-GA (compared with PCr) during ischemia and impaired resynthesis on reperfusion may be related to the different CK isoenzymes involved: PCr breakdown mainly occurs through the cytosolic CK isoenzymes MM and MB, and PCr synthesis mainly occurs via mitochondrial CK. The Vmax of mito-CK for Cr and PCr is substantially lower than that of cytosolic CK, and the reactivity of GA with MM-CK is 100 times lower than that of creatine.18 The reactivity of GA with mito-CK is unknown. It is tempting to speculate that incomplete resynthesis of GA may be due to the lower reaction velocity of mito-CK on reperfusion compared with P-GA breakdown during ischemia by MM-CK. In addition, differences in nonenzymatic degradation of P-GA and PCr could play a role during ischemia. It is also possible that more GA than creatine is lost from the cardiomyocyte during ischemia and/or early reperfusion. Although both compounds are electroneutral, GA is a smaller molecule than creatine, possibly leading to increased leakage across the sarcolemma.
Limitations and Future Directions
We did not use a time-specific GAMT-knockout model. Thus, in addition to the compensatory increase in P-GA, other adaptations to the lack of creatine and PCr may have occurred during fetal and adult development. This will be an interesting topic for future studies. However, unlike in other models of impaired energetics,12,26,27 potential adaptations did not lead to LV hypertrophy. The reasons for this discrepancy in hypertrophy development between different models of altered cardiac energetics clearly warrant further study. However, one explanation for a lack of hypertrophy in GAMT–/– mice may be that we used heterozygous parents to create GAMT–/– mice. Thus, during embryonic development and during the postnatal period, GAMT–/– mice were provided with a source of creatine by their mothers, and severe creatine depletion only develops in these mice after weaning. In contrast, CK double-knockout mice, for example, have a severely impaired CK system throughout their entire intrauterine, postneonatal, and adult life. It is conceivable that these differences in timing explain the difference in hypertrophy development.
In our in vivo LV conductance measurements, the difference in heart size between GAMT–/– and WT mice limited the number of parameters that were available for baseline comparison, eg, the slopes of the end-systolic pressure-volume relationship and of the dP/dtmax–end-diastolic volume relationship are both chamber size dependent.35,36 Little additional benefit would therefore be attained by measuring to calibrate these parameters; instead, we used PRSW, which is independent of heart size, insensitive to loading conditions, and independent of calibration factors such as and parallel conductance.28 As such, PRSW can be considered particularly reliable as a measure of contractile function. Under inotropic stimulation, dP/dtmax values were not significantly different between WT and GAMT–/– mice, but PRSW was. This underscores the limited validity of dP/dtmax as a parameter of contractility because it is highly preload dependent. Indeed, Kass et al31 have shown that a change of 1 mm Hg in EDP can result in an 18% change in dP/dtmax and that this load dependency increases under inotropic stimulation (eg, with dobutamine). As they also note, 1 mm Hg is close to the noise level for this type of pressure measurement.
GAMT–/– mice are a potentially interesting model to further clarify the role of the CK/PCr system for the development of heart failure. In the present study energetic abnormalities do not occur in isolation but in the context of changes in a variety of other cellular systems involved in contractile function and dysfunction. Thus, future work should test whether LV remodeling is aggravated in GAMT–/– mice in a chronic heart failure model, eg, after coronary ligation.
In summary, we have shown that GAMT–/– mice completely lacking myocardial PCr and creatine show a compensatory accumulation of P-GA, ie, these mice have severely reduced CK reaction velocity (1%) but partially maintained high-energy phosphate stores. These mice do not develop cardiac hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under these conditions. However, when stressed by inotropic stimulation or ischemia/reperfusion, GAMT–/– mice exhibit a markedly abnormal phenotype, demonstrating that hearts with a severely reduced capacity of the CK/PCr system show limited inotropic reserve and aggravated injury after acute stress.
Acknowledgments
This study was supported by the British Heart Foundation. Dr Watkins is a Fellow of the American Heart Association.
Footnotes
The first 2 authors have contributed equally to this work.
References
Pool PE, Spann JF, Buccino RA, Sonnenblick EH, Braunwald E. Myocardial high-energy phosphate stores in cardiac hypertrophy and heart failure. Circ Res. 1967; 21: 365–373.
Ingwall JS, Kramer MF, Fifer MA, Lorell BH, Shemin R, Grossman W, Allen PD. The creatine kinase system in normal and diseased human myocardium. N Engl J Med. 1985; 313: 1050–1054.
Tian R. Thermodynamic limitation for the sarcoplasmic reticulum Ca2+-ATPase contributes to impaired contractile reserve in hearts. Ann N Y Acad Sci. 1998; 853: 322–324.
Neubauer S, Hamman BL, Perry SB, Bittl JA, Ingwall JS. Velocity of the creatine kinase reaction decreases in postischemic myocardium: a 31P-NMR magnetization transfer study of the isolated ferret heart. Circ Res. 1988; 63: 1–15.
Nascimben L, Ingwall JS, Pauletto P, Friedrich J, Gwathmey JK, Saks V, Pessina AC, Allen PD. Creatine kinase system in failing and nonfailing human myocardium. Circulation. 1996; 94: 1894–1901.
Neubauer S, Horn M, Naumann A, Tian R, Hu K, Laser M, Friedrich J, Gaudron P, Schnackerz K, Ingwall JS, Ertl G. Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction. J Clin Invest. 1995; 95: 1092–1100.
Ingwall JS, Weiss RG. Is the failing heart energy starved; On using chemical energy to support cardiac function. Circ Res. 2004; 95: 135–145.
van Deursen J, Heerschap A, Oerlemans F, Ruitenbeek W, Jap P, ter Laak H, Wieringa B. Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity. Cell. 1993; 74: 621–631.
Steeghs K, Benders A, Oerlemans F, deHaan A, Heerschap A, Ruitenbeek W, Jost C, vanDeursen J, Perryman B, Pette D, Bruckwilder M, Koudijs J, Jap P, Veerkamp J, Wieringa B. Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies. Cell. 1997; 89: 93–103.
Saupe KW, Spindler M, Hopkins JC, Shen W, Ingwall JS. Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart: reaction kinetics of the creatine kinase isoenzymes in the intact heart. J Biol Chem. 2000; 275: 19742–19746.
Kaasik A, Veksler V, Boehm E, Novotova M, Minajeva A, Ventura-Clapier R. Energetic crosstalk between organelles: architectural integration of energy production and utilization. Circ Res. 2001; 89: 153–159.
Boehm E, Ventura-Clapier R, Mateo P, Lechene P, Veksler V. Glycolysis supports calcium uptake by the sarcoplasmic reticulum in skinned ventricular fibres of mice deficient in mitochondrial and cytosolic creatine kinase. J Mol Cell Cardiol. 2000; 32: 891–902.
Nahrendorf M, Spindler M, Hu K, Bauer L, Ritter O, Nordbeck P, Quaschning T, Hiller KH, Wallis J, Ertl G, Bauer WR, Neubauer S. Creatine kinase knockout mice show left ventricular hypertrophy and dilatation, but unaltered remodeling post-myocardial infarction. Cardiovasc Res. 2005: 65: 419–427.
Saupe KW, Spindler M, Tian R, Ingwall JS. Impaired cardiac energetics in mice lacking muscle-specific isoenzymes of creatine kinase. Circ Res. 1998; 82: 898–907.
Schmidt A, Marescau B, Boehm EA, Renema WK, Peco R, Das A, Steinfeld R, Chan S, Wallis J, Davidoff M, Ullrich K, Waldschutz R, Heerschap A, De Deyn PP, Neubauer S, Isbrandt D. Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency. Hum Mol Genet. 2004; 13: 905–921.
Kan HE, Renema WK, Isbrandt D, Heerschap A. Phosphorylated guanidinoacetate partly compensates for the lack of phosphocreatine in skeletal muscle of mice lacking guanidinoacetate methyltransferase. J Physiol. 2004; 560: 219–229.
Schneider JE, Tyler DJ, Ten Hove M, Sang AE, Cassidy PJ, Fischer A, Wallis J, Sebag-Montefiore LM, Watkins H, Isbrandt D, Clarke K, Neubauer S. In vivo cardiac 1H-MRS in the mouse. Magn Reson Med. 2004; 52: 1029–1035.
Boehm EA, Radda GK, Tomlin H, Clark JF. The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase. Biochim Biophys Acta. 1996; 1274: 119–128.
Schneider JE, Cassidy PJ, Lygate C, Tyler DJ, Wiesmann F, Grieve SM, Hulbert K, Clarke K, Neubauer S. Fast, high-resolution in vivo cine magnetic resonance imaging in normal and failing mouse hearts on a vertical 11.7 T system. J Magn Reson Imaging. 2003; 18: 691–701.
Nemoto S, DeFreitas G, Mann DL, Carabello BA. Effects of changes in left ventricular contractility on indexes of contractility in mice. Am J Physiol. 2002; 283: H2504–H2510.
Chu V, Otero JM, Lopez O, Morgan JP, Amende I, Hampton TG. Method for non-invasively recording electrocardiograms in conscious mice. BMC Physiol. 2001; 1: 6.
Shioi T, McMullen JR, Tarnavski O, Converso K, Sherwood MC, Manning WJ, Izumo S. Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation. 2003; 107: 1664–1670.
Schulze A, Bachert P, Schlemmer H, Harting I, Polster T, Salomons GS, Verhoeven NM, Jakobs C, Fowler B, Hoffmann GF, Mayatepek E. Lack of creatine in muscle and brain in an adult with GAMT deficiency. Ann Neurol. 2003; 53: 248–251.
Gaussin V, Tomlinson JE, Depre C, Engelhardt S, Antos CL, Takagi G, Hein L, Topper JN, Liggett SB, Olson EN, Lohse MJ, Vatner SF, Vatner DE. Common genomic response in different mouse models of beta-adrenergic-induced cardiomyopathy. Circulation. 2003; 108: 2926–2933.
Hamalainen HK, Tubman JC, Vikman S, Kyrola T, Ylikoski E, Warrington JA, Lahesmaa R. Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Anal Biochem. 2001; 299: 63–70.
Weiss RG, Chatham JC, Georgakopolous D, Charron MJ, Wallimann T, Kay L, Walzel B, Wang Y, Kass DA, Gerstenblith G, Chacko VP. An increase in the myocardial PCr/ATP ratio in GLUT4 null mice. FASEB J. 2002; 16: 613–615.
Spindler M, Saupe KW, Christe ME, Sweeney HL, Seidman CE, Seidman JG, Ingwall JS. Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy. J Clin Invest. 1998; 101: 1775–1783.
Yang B, Larson DF, Watson R. Age-related left ventricular function in the mouse: analysis based on in vivo pressure-volume relationships. Am J Physiol. 1999; 277: H1906–H1913.
Neubauer S, Hu K, Horn M, Remkes H, Hoffmann KD, Schmidt C, Schmidt TJ, Schnackerz K, Ertl G. Functional and energetic consequences of chronic myocardial creatine depletion by beta-guanidinopropionate in perfused hearts and in intact rats. J Mol Cell Cardiol. 1999; 31: 1845–1855.
Crozatier B, Badoual T, Boehm E, Ennezat PV, Guenoun T, Su J, Veksler V, Hittinger L, Ventura-Clapier R. Role of creatine kinase in cardiac excitation-contraction coupling: studies in creatine kinase-deficient mice. FASEB J. 2002; 16: 653–660.
Kass DA, Hare JM, Georgakopoulos D. Murine cardiac function: a cautionary tail [published correction appears in Circ Res. 1998;83:115]. Circ Res. 1998; 82: 519–522.
Spindler M, Meyer K, Stromer H, Leupold A, Boehm E, Wagner H, Neubauer S. Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia-reperfusion injury and impaired calcium homeostasis. Am J Physiol. 2004; 287: H1039–H1045.
Horn M, Remkes H, Stromer H, Dienesch C, Neubauer S. Chronic phosphocreatine depletion by the creatine analogue (beta)-guanidinopropionate is associated with increased mortality and loss of ATP in rats after myocardial infarction. Circulation. 2001; 104: 1844–1849.
Zugno AI, Stefanello FM, Streck EL, Calcagnotto T, Wannmacher CM, Wajner M, Wyse AT. Inhibition of Na+, K+-ATPase activity in rat striatum by guanidinoacetate. Int J Dev Neurosci. 2003; 21: 183–189.
Georgakopoulos D, Mitzner WA, Chen CH, Byrne BJ, Millar HD, Hare JM, Kass DA. In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol. 1998; 274: H1416–H1422.
Takaoka H, Esposito G, Mao L, Suga H, Rockman HA. Heart size-independent analysis of myocardial function in murine pressure overload hypertrophy. Am J Physiol. 2002; 282: H2190–H2207.(Michiel ten Hove, PhD; Cr)