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Am J Physiol Heart Circ Physiol 294: H1459-H1466, 2008. First published January 11, 2008; doi:10.1152/ajpheart.00911.2007
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Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Ilka Pinz,1,* Sanford E. Ostroy,1,* Kirsten Hoyer,1 Hanna Osinska,2 Jeffrey Robbins,2 Jeffery D. Molkentin,2 and Joanne S. Ingwall1

1NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; and 2Division of Molecular Cardiovascular Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Submitted 6 August 2007 ; accepted in final form 4 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|{Delta}G~ATP|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |{Delta}G~ATP| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with β-adrenergic stimulation of TG hearts, during which, for similar changes in |{Delta}G~ATP|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.

adrenergic performance; ATP; 31P-NMR spectroscopy

CALCINEURIN (CLN) is a Ca2+-calmodulin-activated phosphatase that is activated by prolonged increases in intracellular Ca2+ concentration. Cardiac-specific overexpression of constitutively active CLN in mice causes a pronounced increase in cardiac size that rapidly progresses to pump failure and death (18, 33). Myocytes isolated from such mouse hearts are hypercontractile and have increased myofibrillar ATPase and sarcoplasmic reticulum Ca2+-ATPase activities (6). In contrast, contractile performance is decreased in isolated and in vivo hearts overexpressing CLN (24). This discrepancy has been accounted for by myocyte disarray and disruption of the functional syncytium caused by defects in connexin 43, a major component of gap junctions, the His bundle, and Purkinje fibers (6). More recently, it was shown that respiration rates and activities of cytochrome c oxidase (COX) subunits I and IV are lower in mitochondria isolated from CLN-overexpressing mouse hearts (23) and that gene expression for the mitochondrial enzyme citrate synthase (CS) and other enzymes involved in fatty acid and glucose metabolism is reduced in isolated heart cells (8). Recently, a direct association has been shown between CLN and certain mitochondrial proteins (superoxide dismutase, aconitase, and malate dehydrogenase) (30).

These reports suggest that decreased capacity for ATP synthesis (8, 23), as well as increased ATP utilization (6), may contribute to CLN-induced cardiac hypertrophy and failure. ATP content and the chemical driving force for ATP-utilizing reactions, however, have not been measured in CLN-overexpressing hearts. A major goal of this study was to investigate the interrelated issues of energetics, contractile performance, and hypertrophy and to provide further information on the role of CLN in proteins required for ATP synthesis in normal and failing hearts. We found that ATP concentration ([ATP]) was lower in TG hearts and that CLN inhibits glycolytic, mitochondrial, and creatine kinase (CK) enzyme activities in nontransgenic (NTG) and transgenic (TG) hearts. The results show that, under a variety of conditions, the free energy of ATP hydrolysis (|{Delta}G~ATP|) was not substantially different between TG and NTG hearts. In NTG hearts, increased systolic performance was accompanied by changes in |{Delta}G~ATP|; in TG hearts, the changes in |{Delta}G~ATP| occurred without an increase in systolic performance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Twenty-two hearts from 8- to 10-wk-old mice (B6C3F1) with cardiac-specific overexpression of the catalytic subunit of CLN (TG, line 37, 15 copies) and 19 NTG littermates were studied (18). Seven TG hearts were excluded from analysis: two failed to beat at the low pacing rate of 300 beats/min, two did not tolerate pacing rates of 600 beats/min, and three had sudden increases in coronary flow due to a rupture at the aortic root. Myocardial tissue from 1-wk-old TG mice (n = 8) and their NTG littermates (n = 4), 8-wk-old knockout mice and their littermates (n = 3 each), and NTG and TG mice that had been injected with cyclosporin A (CsA, n = 4 each, 20 mg·kg–1·day–1) starting at 7 wk of age for 9 days were also studied (15, 16). Experimental protocols were 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 mouse heart. Hearts of NTG and TG mice were isolated and perfused [Langendorff mode with a balloon in the left ventricle (LV)] with phosphate-free Krebs-Henseleit buffer containing 0.5 mM pyruvate, 2 mM CaCl2, 0.5 mM EGTA, and 10 mM glucose (7). Glucose was supplied at twice the normal serum concentration to drive glycolysis. Pyruvate was added at saturating amounts for pyruvate dehydrogenase (13) to provide a glycolysis-independent source of acetyl-CoA. Although the amount of pyruvate supplied may not be sufficient for fatty acid substitution when glycolysis is inhibited, experiments with wild-type mice showed similar [ATP], [PCr], and contractile performance in hearts perfused with glucose + pyruvate or a mixture of substrates including fatty acids (2). Systolic function was assessed as systolic pressure (SP), developed pressure [DevP = SP – end-diastolic pressure (EDP)], rate-pressure product (RPP = DevP x HR, where HR is heart rate), and wall stress [wall stress = SP x r2/h(2r + h), where r is wall radius and h is wall thickness] (17). Rate of tension development was assessed as +dP/dt. Diastolic function was assessed as EDP and rate of relaxation as –dP/dt. EDP was set to 8–10 mmHg at the lowest pacing rate and then allowed to vary.

Experimental protocols. TG hearts were initially paced at 300 beats/min for 30 min; then pacing was increased to 420 and, finally, 600 beats/min (each for 10 min). NTG hearts were initially not paced, since their spontaneous HR was 357 ± 8 beats/min; HR was increased by pacing to 420 and then 600 beats/min (each for 10 min). In a subset of these hearts, the inotropic agent dobutamine (300 nM) was added as an additional challenge to the last pacing step. Hearts were used for 31P-NMR spectroscopy experiments or myocardial O2 uptake (MVO2) measurements. At the end of each protocol, heart chambers were blotted dry, weighed, frozen, and stored at –80°C.

31P-NMR spectroscopy. 31P-NMR free induction decays were acquired at 161.8 MHz using an NMR spectrometer (Inova, Varian NMR Systems, Palo Alto, CA). Spectra acquisition and calculations have been described by Saupe et al. (22).

Biochemical measurements. MVO2 was measured using Clark-type microelectrodes (Microelectrodes, Bedford, NH) (22). Ventricular tissue was homogenized for measurements of protein, total creatine, and activities of CK, phosphofructokinase (PFK), lactate dehydrogenase (LDH), CS, and COX, as described previously (9). CK isozyme distribution was determined electrophoretically (21).

Electron microscopy. Mice were anesthetized with isoflurane; their hearts were fixed by perfusion with 3.5% glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.4), divided into small (1-mm3) fragments, and fixed in glutaraldehyde-cacodylate overnight at 4°C. They were postfixed on ice in cacodylate buffer and embedded in a Poly/Bed 812-resin mixture. Thin sections were counterstained with uranyl acetate and lead citrate and examined on an electron microscope (model 912, Zeiss).

Statistical analysis. Values are means ± SE. Data were tested using repeated-measures ANOVA; for enzyme activities, one-factor ANOVA was used. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Heart characteristics. In agreement with previous studies (18), TG hearts demonstrated a dilated hypertrophic phenotype. At 8–10 wk of age (all P < 0.05), heart weight was 106 ± 5 and 243 ± 18 mg, LV volume was 15.8 ± 0.9 and 29 ± 3 µl, and LV wall thickness was 1.3 ± 0.04 and 1.7 ± 0.1 mm in NTG and TG mice, respectively. Chamber weights were 5.1 ± 0.3 and 21 ± 3 mg (atria), 21 ± 2 and 56 ± 6 mg (right ventricle), and 85 ± 5 and 188 ± 1 mg (LV) in NTG and TG mice, respectively. Body weight was not different: 26 ± 1 and 25 ± 1 g in NTG and TG mice, respectively. Baseline coronary flow was also not different: 19 ± 1 and 16 ± 2 ml·min–1·g LV–1 in NTG and TG mice, respectively. At 1 wk of age, TG hearts were 24% larger than NTG hearts (22.4 ± 0.9 vs. 18 ± 2 mg, P < 0.05) compared with the 129% increase at 8–10 wk of age.

HR and pacing. Spontaneous HR was 78 ± 19 and 357 ± 8 beats/min for the isolated TG and NTG hearts, respectively. Pacing TG hearts was difficult and could be accomplished only by placement of the pacing electrodes on the LV, instead of the atria, and by use of a low initial pacing rate of 300 beats/min. Although TG hearts did not tolerate faster pacing rates initially, TG hearts beating at 300 beats/min for 30 min tolerated subsequent increases in pacing rates to 420 and then 600 beats/min. They did not tolerate pacing rates >600 beats/min, in contrast to NTG hearts, which could be paced at 900 beats/min. Moreover, in ~50% of the TG hearts paced at 600 beats/min, HR ceased at multiple 20- to 25-s intervals. These results are consistent with myocyte disarray and disruption of functional syncytium in TG hearts (6).

Isovolumic contractile performance. Figure 1 shows representative pressure traces obtained from NTG and TG hearts at three pacing rates and during inotropic challenge with dobutamine. Mean values for all indexes of isovolumic contractile performance are presented in Table 1. In NTG hearts, increasing pacing rate led to increases in all indexes of systolic performance: SP, DevP, RPP, and wall stress. +dP/dt and –dP/dt also increased. All indexes of systolic performance at each pacing rate were lower in TG than in NTG hearts. Also, in contrast to NTG hearts, in TG hearts, SP, DevP, and dP/dt did not significantly increase with increased HR. The slight increase in RPP was caused by the increase in HR, rather than by an increase in DevP. Dobutamine challenge led to the expected increase in isovolumic contractile performance for NTG, but not TG, hearts; on the contrary, all the parameters decreased (Table 1).


Figure 1
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  		Fig. 1. Representative left ventricular (LV) pressure traces of isovolumic contractile performance in nontransgenic (NTG) and transgenic (TG) hearts. NTG hearts were allowed to beat spontaneously (360 beats/min) and then paced at 420 and 600 beats/min. TG hearts were paced at 300, 420, and 600 beats/min. Also shown are representative traces from a different pair of NTG and TG hearts in which dobutamine (D) was added to hearts paced at 600 beats/min. Average values for these experiments are presented in Table 1.

 

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  		Table 1. Isovolumic contractile performance in NTG and TG hearts at different HR and with inotropic challenge

 
Energetics. We used 31P-NMR spectroscopy of perfused hearts to determine whether steady-state [ATP], |{Delta}G~ATP|, and the level of the energy reserve compound PCr were normal in TG hearts. Figure 2 shows representative spectra and the values for [PCr] and |{Delta}G~ATP| obtained for NTG and TG hearts at three pacing rates and during inotropic challenge with dobutamine. Table 2 presents the values for the energy metabolites. With increasing pacing rates and inotropic challenge in the NTG hearts, [PCr] decreased to maintain a constant [ATP], while Pi concentration ([Pi]) increased; calculated ADP and AMP concentrations ([ADP] and [AMP]) increased. A similar overall pattern was observed in the TG hearts; however, the absolute values for [PCr] and [ATP] were ~22% and ~15% lower in TG than NTG hearts, even at the lowest HR, whereas [ADP] and [AMP] were higher in TG than in NTG hearts. The [PCr] decrease in TG hearts was not due to a decrease of the total creatine concentration ([creatine]), since it was 16% higher in the TG hearts.


Figure 2
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  		Fig. 2. A: representative 31P-NMR spectra from NTG and TG hearts treated as described in Fig. 1 legend. Resonance areas represent amounts of phosphocreatine (PCr), ATP, and Pi in the tissue. Since the TG heart is approximately twice the size of the NTG heart, equal concentrations would require that the TG heart exhibit an area twice the size of the NTG heart. Areas are not 2 times greater; therefore, PCr and ATP concentrations ([PCr] and [ATP]) are lower in TG hearts. Average values are presented in Table 2. B: [PCr] as a function of heart rate (HR) and inotropic challenge with dobutamine in NTG (open bars) and TG (shaded bars) hearts. Average values are presented in Table 2. C and D: relationship between developed pressure (DevP) and free energy for ATP hydrolysis (|{Delta}G~ATP|) for NTG ({circ}) and TG (bullet) hearts. C: increasing HR and pacing at ~360, 420, and 600 beats/min in NTG and 300, 420, and 600 beats/min in TG hearts. Lower |{Delta}G~ATP| values are associated with higher HR. With increased HR, despite similar decreases in |{Delta}G~ATP| in both groups, DevP increased significantly only in NTG hearts (P < 0.05, 600 vs. 360 beats/min, by 1-factor ANOVA; see Table 1). DevP was significantly lower in TG hearts than in corresponding NTG hearts under all conditions (P < 0.05, by repeated-measures ANOVA). D: effect of dobutamine + pacing at 600 beats/min compared with HR of ~360 beats/min (NTG) and 300 beats/min (TG). Lower |{Delta}G~ATP| values are associated with added dobutamine. With addition of dobutamine, decreases in |{Delta}G~ATP| are similar in TG and NTG hearts, but the expected increase in DevP is observed only in NTG hearts (P < 0.05, 600 beats/min + D vs. 360 beats/min, by 1-factor ANOVA). {Delta}GATP = {Delta}Go + RT ln([ADP][Pi]/[ATP]), where Go is Gibbs free energy, R is universal gas constant, T is absolute temperature, and [ADP] and [Pi] are ADP and Pi concentrations. Values are means ± SE.

 

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  		Table 2. Concentrations of high- and low-energy phosphate metabolites, |{Delta}G~ATP|, and pH

 
Changes in [ATP], [ADP], and [Pi] can be described by a single value, |{Delta}G~ATP|, which, in the normal heart, varies between ~64 kJ/mol at low levels of work to 53 kJ/mol at high levels of work (10). The value of |{Delta}G~ATP| describes the energetic state of the heart and represents the chemical driving force for ATPase reactions in the cell. The threshold values for |{Delta}G~ATP| below which ATPase reactions fail rank in the following order: Ca2+-ATPase, Na+-K+-ATPase, and myosin ATPase (10). The relationship between DevP and |{Delta}G~ATP| in TG and NTG hearts is presented in Fig. 2 for the pacing and dobutamine protocols. For both protocols, the lower DevP generated by the TG hearts is illustrated by the position of the TG data compared with the NTG data. For the pacing protocol in NTG hearts, when the lowest HR was compared with HR at 600 beats/min, |{Delta}G~ATP| fell by 2.8 kJ/mol as DevP increased by 18 mmHg. The change in |{Delta}G~ATP| in TG hearts was similar (3.1 kJ/mol), but DevP did not change. The uncoupling between the change in energetic driving force and mechanical performance of the TG heart with increasing HR became even greater when hearts paced at 600 beats/min were challenged with dobutamine. When the lowest HR was compared with HR at 600 beats/min plus dobutamine challenge, |{Delta}G~ATP| decreased to a similar extent in NTG and TG hearts (~5 and ~6 kJ/mol, respectively), but although DevP increased (by 38 mmHg) in NTG hearts, it decreased (by 9 mmHg) in TG hearts. Similar results were obtained when other indexes of systolic performance were compared (Table 1). Thus, for equivalent physiological challenges under identical perfusion conditions, the changes in |{Delta}G~ATP| were the same or even higher in TG than in NTG hearts, but systolic performance did not increase in TG hearts. In fact, decreases in systolic performance were observed in TG hearts challenged with the β-adrenergic agent dobutamine.

Ultrastructure. Electron micrographs of LV myocytes were used to assess the integrity of sarcomeres and mitochondria in TG hearts (Fig. 3). NTG myocytes contain mostly relaxed regularly spaced sarcomeres, with all bands easily visible and separated by rows of mitochondria (Fig. 3A). In contrast, TG myocytes show many contracted sarcomeres, which are occasionally fragmented into numerous dense bodies (Fig. 3D, note the near absence of I bands). The intercalated disks in TG myocytes show changes in the distribution of electron-dense material in the area of desmosomes.


Figure 3
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  		Fig. 3. Electron micrographs of LV tissue from NTG and TG mice at 8 wk of age. Sarcomeres: in NTG hearts, sarcomeres are mostly relaxed and regularly spaced, with all bands easily seen, and are separated by rows of mitochondria; in TG hearts, sarcomeres are frequently contracted. Sarcomere length is denoted by double arrows in A and D; D is ~80% of A. Mitochondria: in NTG hearts, most of the mitochondria have regularly packed cristae and matrix with a moderate electron density (A–C); in TG hearts, mitochondria show a variety of sizes and shapes (D) and abnormal ultrastructure in a number of cells (E and F). In TG mitochondria, cristae show clear abnormalities (arrowheads in D), while outer mitochondrial membrane is intact. In other TG cells, mitochondria have abnormally distributed and/or a reduced number of cristae (E) or even damaged cristae (F), without obvious disruption of the mitochondrial outer membrane.

 
NTG mitochondria have an unremarkable morphology and are characterized by regularly packed cristae. In contrast, TG mitochondria show a variety of shapes and sizes, with striking changes in ultrastructure. In some mitochondria, the cristae are replaced by circular membranous profiles or myelin figures (Fig. 3D, arrowhead), whereas the outer membranes appear intact. In other mitochondria, cristae are abnormally distributed and/or reduced in number (Fig. 3E). Some display damaged cristae (Fig. 3F), without obvious disruption of the outer mitochondrial membrane. Mitochondrial density also appears to be lower in TG myocytes.

These results are consistent with a limited contractile function in TG hearts due to defects in myofibrillar structure and a diminished capacity for ATP synthesis due to altered mitochondrial ultrastructure.

O2 consumption. To evaluate mitochondrial function, MVO2 was measured in intact hearts at increasing pacing rates. In Fig. 4, MVO2 is shown as a function of wall stress. In NTG and TG hearts, similar slopes and overlapping values indicate similar mitochondrial adjustments to changes in energy demand as a function of systolic performance; however, the dynamic ranges of NTG and TG hearts are different. NTG hearts are able to increase wall stress and consume more O2 than any of the TG hearts, whereas wall stress and MVO2 were lower in some TG hearts than in any of the NTG hearts.


Figure 4
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  		Fig. 4. Relationship between myocardial O2 uptake (MVO2) and wall stress for NTG ({circ}) and TG (bullet) hearts during the pacing protocol. Lines are linear regressions obtained for all data points and are not different. In NTG hearts, MVO2 = 0.44 (wall stress) + 6.01 (r = 0.48, n = 6); in TG hearts, MVO2 = 0.42 (wall stress) + 5.3 (r = 0.75, n = 4).

 
Activities of key enzymes for ATP synthesis. To provide additional information on the capacity for ATP production and to evaluate the role of CLN in the expression of specific proteins important for ATP production, the activities of select enzymes involved in ATP production by mitochondria, glycolysis, and the CK system were measured (Table 3). Consistent with the abnormal mitochondrial ultrastructure, enzyme activities (Vmax) of CS in the tricarboxylic acid cycle and COX in the electron transport chain were lower in TG hearts (–30% and –17%, respectively). Increases in the capacity of other ATP synthesis pathways did not compensate for decreased capacity for ATP production by oxidative phosphorylation. Instead, decreases were observed in the capacities of the glycolytic enzymes PFK and LDH (–26% and –25%, respectively) and the phosphoryltransferase CK (–33%).


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  		Table 3. Creatine content and activities of key enzymes for ATP synthesis

 
For the CK system, normal ventricular myocardium contains four major CK isozymes: CK-MM, CK-MB, CK-BB, and the sarcomeric form of mitochondrial-CK (sMtCK). As shown in Table 3, hearts from 8- to 10-wk-old TG mice showed increases in CK-B-containing enzyme activity (+23%) and decreases in CK-M-containing activity (–26%) and an even larger decrease in sMtCK activity (–54%). These are characteristic changes associated with cardiac hypertrophy and failure (11, 12, 26). In contrast to a decrease of [creatine] in heart failure, however, [creatine] was increased (+16%) in these TG hearts.

Three strategies were used to investigate the role of CLN in the changes in enzyme activity: genetics, determination of age dependence, and pharmacology (CsA injection). The measured parameters of hearts in which the CLN gene had been deleted, in contrast to those of 8–10-wk-old TG hearts, were not significantly different from NTG littermates (8-wk-old knockout mice and their littermates; data not shown). The timing of CLN overexpression-directed inhibition of the enzyme activities was evaluated by measuring their age dependence. NTG and TG data from 1-wk-old mouse hearts were compared with data from 8- to 10-wk-old hearts (Table 3). The decreases in glycolytic enzyme activities in TG hearts were maximal by 1 wk of age and persisted to 8–10 wk of age. The decreases in CK, CK-M, sMtCK, and CS activity were significant at 1 wk of age but became much larger at 8–10 wk of age.

In contrast to the decreased enzyme activities in the CLN-overexpressed hearts, those in which CLN activity was normally expressed (NTG) or overexpressed (TG) and then inhibited with CsA at 7 wk of age (for 9 days) showed some increases in activity compared with the sham-injected animals (Table 3). In NTG and TG hearts, CsA injections increased PFK, LDH, total CK, CK-M, sMtCK, and COX activities, with the highest values in the NTG hearts. This is as expected, because the NTG hearts have less CLN and, therefore, would be more susceptible to an equal dose of CsA. Inhibition of CLN activity in TG hearts resulted in a return to normal NTG levels of activity for the glycolytic enzymes and most of the CK isozymes. Thus these data are consistent with a CLN-directed inhibition of ATP-producing pathways in NTG and TG hearts.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Decreased capacity for ATP synthesis. Decreased capacity for ATP synthesis via oxidative phosphorylation in the mitochondria of TG hearts was shown by decreases in CS activity in the tricarboxylic acid cycle, COX activity in the electron transport chain, and sMtCK activity in the inner membrane space; a decrease in the maximum MVO2; and a severely altered mitochondrial structure. Direct measurements of Vmax of ATP synthesis enzymes strongly support conclusions drawn from mitochondrial and gene expression studies of the same type in TG hearts (5, 7, 22), as well as recent data that showed a direct association between CLN and certain mitochondrial proteins (29). In contrast, an increase in mitochondrial capacity was observed in skeletal muscle of mice overexpressing constitutively active calmodulin kinase (CAMK) (34). CAMK is upstream of CLN, and an increase of CAMK or CLN would be expected to have directionally similar effects on mitochondrial biogenesis. The underlying mechanism for this unexpected difference between skeletal and cardiac muscles is unknown.

A metabolic hallmark of cardiac hypertrophy is increased glycolysis (1). Thus we expected to observe no change or an increase in the Vmax of glycolytic enzymes in TG hearts. Instead, PFK and LDH activities decreased, even in hearts of 1-wk-old TG mice. These measures of enzyme activities are consistent with other studies showing downregulation of the genes for uptake and utilization of glucose and fatty acids by CLN (8, 31). Analysis of creatine and purine pools suggests that, although cytosolic [AMP] was two- to fourfold higher in these hearts, any compensatory action of 5'-AMP-activated protein kinase to preserve or restore normal [ATP] (28) was likely offset by chronic activation of cytosolic AMP-specific 5'-nucleotidase (3, 4, 11). Our data suggest that CLN overexpression prevents compensatory increases in glucose and fatty acid utilization. However, no fatty acids were included in the perfusate, and experiments reported here do not directly determine relative substrate utilization.

Creatine, PCr, and [PCr]-to-[creatine] ratio. Our finding of a small, but significant, increase (+16%) in the total creatine pool in TG hearts was unexpected (Table 3). In cardiac hypertrophy with and without failure in human and animal models, [creatine] (and, hence, [PCr]) falls by as much as 60% (19, 29). On the basis of the chemistry of the CK reaction, we would expect that [PCr] would change in parallel with [creatine]. Instead of a 16% increase in [PCr] in TG hearts, however, we found a ~20% decrease. The ratio of [PCr] to free [creatine] ([PCr]/[creatine]) was 3.1 for NTG hearts and only 1.1 for TG hearts. The effects of low [PCr]/[creatine] on LV hypertrophy and contractile reserve have been demonstrated in hearts in which total [creatine] was increased nearly twofold due to overexpression of the creatine transporter (32). In that study, [PCr] did not increase in concert with the total creatine pool, and a lower [PCr]/[creatine] led to a phenotype remarkably similar to that observed in the present study for TG hearts and hearts caused to fail in other ways: LV contractile dysfunction, hypertrophy, and dilatation with increased cytosolic [ADP] but unchanged [Pi] at baseline and reduced contractile reserve on inotropic challenge. Decreased [PCr]/[creatine] is a common feature of the failing heart (14, 19, 20, 25). Thus decreased [PCr], as well as decreased Vmax for CK, contributes to a decreased capacity for ATP supply via the CK reaction in CLN hearts.

Energetic phenotype of hearts overexpressing CLN. In adult mouse hearts overexpressing CLN since late fetal development, a decreased capacity for ATP supply by major ATP-producing pathways could contribute to a ~15% decrease in [ATP]. The decrease in [ATP] in these TG hearts is similar to the loss of myocardial ATP observed in heart failure patients (5, 27) and hearts of animal models of severe heart failure due to a variety of etiologies (11, 12, 25, 29). To our knowledge, this is the first report showing that myocardial [ATP] is lower in a TG mouse model of hypertrophy and failure, suggesting that failure must be severe to detect a fall in [ATP] (25). Also, [PCr] and [PCr]/[creatine] were lower than in the normal heart, exacerbated by an unexpected increase in total [creatine]. Although [PCr] and [ATP] were lower in TG hearts, [Pi] did not accumulate, and, as a consequence, |{Delta}G~ATP| was maintained near normal. Near-normal |{Delta}G~ATP|, despite decreased [ATP] and [PCr], was also observed in the pacing-induced canine model of heart failure (25). During challenges to increase work, chemical energy was used but did not translate to systolic performance. We suggest that maintenance of normal [Pi] is an important compensatory property of chronic energy deprivation, allowing the severely failing heart to perform mechanical work, albeit at low levels.

Heart failure. The TG mouse hearts used in the present study were taken from the ~50% of the mice that survived to 11 wk of age (24), offering a rare opportunity to study the properties of the severe failing heart. Systolic performance was lower in CLN-overexpressing hearts than in littermate NTG hearts, even at low levels of work. Their inability to recruit contractile reserve became even more dramatic, when, at the highest pacing rate, the heart was also challenged with dobutamine. These severely failing hearts with compromised ATP synthesis capacity not only had no contractile reserve, but systolic performance decreased with β-adrenergic challenge.

In summary, tissue-specific overexpression of CLN in the mouse heart leads to severe heart failure, a decreased capacity to supply ATP, and a lower [ATP]. CLN-directed inhibition of Vmax for enzymes important for ATP synthesis is independent of the presence of hypertrophy in NTG and TG hearts. Our finding that overexpression of CLN led to an increase in [creatine] was unexpected. The lower capacity for ATP synthesis and [ATP] in CLN hearts raises the possibility that, in other models of heart failure, these changes may be due, at least in part, to increased CLN. We suggest that strategies directed to preserving a normal supply of ATP in severely failing myocardium may be an effective therapy.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-075619 and HL-52320.


   ACKNOWLEDGMENTS
 
We thank Linda Joy Johnson for assistance in manuscript preparation.

Present addresses: I. Pinz, Maine Medical Center Research Institute, Center for Molecular Medicine, Scarborough, ME 04074; K. Hoyer, CV Therapeutics, Palo Alto, CA 94304.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. S. Ingwall, NMR Laboratory for Physiological Chemistry, Brigham and Women's Hospital, 221 Longwood Ave., Rm. 247, Boston, MA 02115 (e-mail: jingwall{at}rics.bwh.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* I. Pinz and S. E. Ostroy contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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