Energy status of the rapidly paced canine myocardium in congestive heart failure C. MONTGOMERY, N. HAMILTON, AND C. D. IANUZZO Departments of Biology and Physical Education, Faculty of Pure and Applied Science, Center for Health Studies, York University, Toronto, Ontario M3J lP3, Canada MONTGOMERY,C.,N.HAMILTON, AND C.D. IANUZZO.Energy status of the rapidly paced canine myocardium in congestive heart failure. J. Appl. Physiol. 73(6): 2363-2367, 1992.-Rapid ventricular pacing (RVP) is used as an experimental model of congestive heart failure (CHF). The purpose of this study was to determine the energy status of the dog myocardium after the development of CHF via chronic RVP. The myocardium had a significantly lower (P < 0.05) energy charge (EC) during CHF (0.63 k 0.01) than in sham-operated controls (0.82 & 0.02). This was due to significant differences in concentrations in ATP (-48%), ADP (29%), and AMP (275%) in the RVP group. However, the total adenine nucleotide pool was not different between groups. Myocardial lactate concentration was also similar. Glycogen was significantly lower (P < 0.05) by 20% at peak CHF. The adenine nucleotides were similar among the different myocardial layers (endo-, mid-, and epicardium). The administration of enalapril (an inhibitor of angiotension-converting enzyme) to decrease vascular resistance had no effect on the myocardial energy status of CHF dogs. These findings suggest that the lower EC in CHF animals is not the result of subendocardial ischemia. Also, lower EC is not associated with endogenous glycogen depletion or increased lactate concentration. The energy status of the myocardium in RVP-induced CHF is unlike that seen in ischemia-induced heart failure. This suggests that CHF in RVP is not vascular in origin. adenine charge
nucleotides;
glycogen;
lactate;
cardiac
muscle energy
processes of contraction and calcium regulation. The capacities of these pathways are correlated with resting heart rate (6). Recently, we evaluated the response of these three systems to the stress of chronic RVP (23). Dogs judged to be in CHF by clinical, hemodynamic, and radiographic evaluation (1, 20, 22) showed some cellular myocardial changes. The energy-transforming pathways (i.e., the tricarboxylic acid cycle, ,&oxidation, and glycolysis) changed only slightly, whereas the energy-utilizing systems [i.e., myofibrillar adenosinetriphosphatase (ATPase) and sarcoplasmic reticulum ATPase] had reduced capacities (23). These changes suggest that the strategy of the myocardium was to balance energy supply with energy demand by decreasing the energy-utilizing processes of contraction and calcium flux (23). To evaluate the efficacy of these altered energy-converting and -utilizing systems to maintain a normal energy status in this paced dog model of CHF, selected metabolite concentrations were determined in failing and control canine myocardia. The results show that normal adenine nucleotide molar concentrations were not maintained in the failing myocardium, whereas the total adenine nucleotide (TAN) concentration was unchanged. Myocardial lactate was the same in both groups, whereas glycogen stores were moderately reduced in RVP. METHODS
DESPITE CONSIDERABLE PROGRESSin the prevention and treatment of congestive heart failure (CHF), the pathogenic mechanism(s) of this disease remains elusive. If this problem is to be solved, the adaptive strategies employed by heart muscle in the face of physiological stress must be understood. Continuous rapid myocardial ventricular pacing (RVP) in dogs produces a clinical, hemodynamic, and neuroendocrine profile symptomatic of CHF (1, 20, 22, 24, 29). After 4.8 t 1.9 wk of RVP, dogs have increased cardiac size, left ventricular filling pressure, and plasma norepinephrine and decreased cardiac index, mean arterial pressure, and tissue norepinephrine (1). Chronic pacing has also been shown to result in a sixfold increase in myocardial 0, uptake (29). This model provides a unique opportunity to study CHF induced by chronic tachycardia. Three major subcellular systems confer physiological expression to the myocardium. They are the energy-supplying pathways of metabolism and the energy-requiring 0161-7567192
$2.00 Copyright
Mongrel dogs weighing 18-25 kg were housed at the University of Guelph Animal Care Facility for 7 days, where they were screened for health disorders. They were then sent to St. Michael’s Hospital (Toronto) and taught to lie quietly in the right decubitus position to permit hemodynamic and echocardiographic assessment in the conscious state. Throughout the study, dogs were allowed food (Purina Dog Chow 5006) and water ad libiturn. As paced dogs approached CHF, their appetites waned and they were given Derby meat mix, which they consumed more readily. Dogs were anesthetized for pacemaker implantation with thiopental sodium [20 t 2 (SD) mg/kg] and maintained with thiopental sodium (6.5 t 3 mg kg-’ h-l) and morphine sulfate (253 t 16 pg. kg-l . h-l). By use of fluoroscopic visualization, a unipolar pacemaker lead (Medtronic silicone/polyethylene) was placed in the right ventricular apex via the right external jugular vein. The pulse generator (Medtronix Spectrax) was then placed in a subcutaneous pocket anterior to the first rib. The ani-
0 1992 the American
l
Physiological
Society
l
2363
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 14, 2019.
2364
ENERGY
STATUS
AT CONGESTIVE
HEART
FAILURE
mals were allowed to recover from surgery for 1 wk be- 1). The cRVP group had significantly lower ATP (-48%) fore cardiac pacing was initiated. Hemodynamic assess- and higher ADP (29%) and AMP (275%) concentrations. ment during normal sinus rhythm in the conscious state This resulted in a significant reduction in the energy was performed, and the pacing was begun at 250 imcharge (EC) in cRVP compared with the sham group. pulses/min asynchronously. There was no significant change in the TAN concentraThe RVP dogs were divided into two groups. Some tion. animals were treated with enalapril (eRVP), an inhibitor Myocardial lactate concentration was similar between of angiotensin-converting enzyme (ACE), to decrease groups (sham = 3.90 t 0.66 vs. cRVP = 3.77 t 0.48 pmoll vascular resistance to blood flow. eRVP animals received g), whereas glycogen was 20% less in the cRVP (38 t 4 enalapril (10 mg/day po) beginning 1 wk after the onset pmol/g) than in the sham group (48 t 2 pmol/g, P < of pacing. The other paced dogs received a placebo 0.05). (pRVP). Sham animals underwent surgical implantation Table 2 contains the values obtained in the different of the pacemaker but were not paced for the period of transmural layers of the myocardium in each of the three time before they were killed. experimental groups. Within the sham group, there were Severe heart failure was defined as a 225% increase in no significant differences in the metabolites and subheart size accompanied by pulmonary edema and/or a strates in different layers of the myocardium. Within the ->lO% increase in body weight (1). This occurred 4-6 wk eRVP group, ADP and AMP levels were higher in the after the onset of pacing. At this time, dogs were anesthemid- than in the epicardium. This resulted in a statistitized with thiopental sodium (20 mg/kg) and ventilated cally significant difference in EC in the epicardium comwith room air to maintain normal arterial PO, values at pared with other layers of the eRVP myocardium. Howthe time of tissue sampling. A thoracotomy was per- ever, the full-transection EC was not significantly differformed. A full transmural section of the left ventricular ent from any myocardial layer in these animals. free wall midway between the apex and the base of the Furthermore, the EC was still significantly lower in the heart was obtained using a dermatological tissue corer eRVP than in the sham epicardium but not different cooled in liquid nitrogen. The sample was immediately from the pRVP epicardium and was still within the range removed from the corer, clamped with liquid nitrogenwhere tissue viability is at risk (2). Thus it appears that cooled tongs, submersed in liquid nitrogen, and divided the within-group differences in the eRVP animals are of into endo-, mid-, and epicardial portions. A second sam- little biological significance. There were no significant ple was taken immediately in a similar fashion but was differences among layers for the pRVP group. As in the not separated into layers. The time lapse from sampling full-transection data, there were no significant differthe heart to immersion in liquid nitrogen was -10 s. ences between eRVP and pRVP animals, with the single Metabolites and high-energy phosphates were ex- exception of higher AMP in the midmyocardium in the tracted by placing a lo- to 30-mg sample of frozen tissue eRVP group. in 0.42 M perchloric acid at -2°C. This preparation was homogenized, neutralized with KOH, and centrifuged at DISCUSSION 6,500 g (19, 27). Adenine nucleotides were measured in the extract either by high-pressure liquid chromatograChronic RVP. In this study we have observed signifiphy or by fluorometry. High-pressure liquid chromatogcant alterations in myocardial energy status as a result of raphy separation was done isocratically in a mobile phase RVP. Previous studies indicate that chronic tachycardia consisting of NH,H2P0,, tetrabutylammonium hydroresults in changes in myocardial metabolism (9, 13, 23). gen sulfate, and acetonitrile at pH 6.5 on a Waters Cl8 These studies suggested that the dog myocardium is unRadial-Pak column, as described previously (27). Fluoroable to completely satisfy the increased energy demand metric detection of ATP was done by coupling the reducof chronic pacing and that the resulting imbalance betion of NADP with the glucose-6-phosphate dehydrogetween high-energy phosphate production and utilization nase reaction at pH 8.1 (19). Lactate in the extract was may be due to a redistribution of blood flow between the measured fluorometrically by coupling the reduction of different layers of the myocardium. It is also possible NAD with the lactate dehydrogenase reaction at pH 10 in that the metabolic capacity of the myocardium, which is the presence of hydrazine (19). Glycogen was measured correlated with resting heart rate (6), is encroached on by digesting the tissue sample in 30% KOH and deterby RVP. mining glucose concentration spectrophotometrically at The perfused heart has been shown to function well at 620 nm with the anthrone reagent (25, 26). reduced absolute levels of ATP (11). Therefore the absoExperimental groups were compared using a two-way lute tissue concentrations of the different adenine nuanalysis of variance with Scheffb’s post hoc test at the cleotides provide limited insight into the metabolic sta0.05 confidence level. Data are presented as means t SE. tus of the myocardium when examined independently. For this reason, the EC was calculated for each myocardial preparation. EC is a traditional measure of the enerRESULTS getic status of the cell and is simply a measure of the Table 1 summarizes the metabolite concentrations for molar ratio of the TAN (2). EC has been related to metafull transection samples of sham and RVP myocardia. bolic regulation, function, and viability of cells (2). EC eRVP animals did not differ from pRVP animals in any was normal in sham myocardia (0.82) compared with the of these indexes. Therefore these two groups were comsignificantly lower value in RVP animals (0.62). EC of bined (cRVP) and compared with the sham group (Fig. 0.62 is approaching the value at which the viability of the Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 14, 2019.
ENERGY
STATUS
AT
CONGESTIVE
1. Metabolic parameters from transverse sections of the
TABLE Group
n
WW
WPI
Sham pRVP eRVP
4 4 6
4.951029 2.43&0.18* 2.74&0.12*
1.50~0.04 1.83k0.12 2.00~0.10
Values are means adenine nucleotides;
[TANI
0.4640.13 l-32&0.24* 1.24-t-0.04*
6,90+0.32 5.58kO.46 5.97-to.21
-t SE in pmol/g; n, no. of dogs. pRVP, placebo EC, energy charge; La, lactate; Gly, glycogen.
rapidly paced * Significantly
6 5 4 3 2 1 0I
a z 3 E '
s-[ATP]
c-[ATP]
s-[ADP]
2365
FAILURE
left ventricular
[AMP1
tissue may be threatened (2). Hence, it appears that, at peak heart failure, RVP results in severe metabolic perturbations in the dog myocardium. Although the ATPproducing pathways must be working at high levels, the adenine nucleotide concentrations are not maintained in normal proportions. ATP declines while both ADP and AMP rise at the RVP failure state. The 47% decrease in ATP seen here is considerably greater than the 16% drop reported by Coleman et al. (9). This difference appears to be related to differences in experimental protocol. In the study by Coleman et al., dogs were paced at 280 beats/ min for an average of only 19 days before tissue analysis. Furthermore those animals showed increased cardiac outputs, whereas the dogs in our study showed a significant decrease in cardiac index (1). Also in the study of Coleman et al., samples were taken while the heart rate was 150 beats/min. In this study, sampling was accomplished while pacing was maintained at 250 beats/min. Despite the significant changes that occur in the concentrations of the three adenine nucleotides in the RVP group, TAN is not different between the sham and paced groups. This is in contrast to studies of acute ischemia that show that TAN decreases as the ischemic period increases (10,12). In heart muscle, the major pathway of A
HEART
free wall EC
La1
0.82kO.02 0.6OkO,O2* 0.63+0.01*
group; eRVP, enalapril-treated different from sham, P
nucleotides;
glycogen;
lactate;
cardiac
muscle energy
processes of contraction and calcium regulation. The capacities of these pathways are correlated with resting heart rate (6). Recently, we evaluated the response of these three systems to the stress of chronic RVP (23). Dogs judged to be in CHF by clinical, hemodynamic, and radiographic evaluation (1, 20, 22) showed some cellular myocardial changes. The energy-transforming pathways (i.e., the tricarboxylic acid cycle, ,&oxidation, and glycolysis) changed only slightly, whereas the energy-utilizing systems [i.e., myofibrillar adenosinetriphosphatase (ATPase) and sarcoplasmic reticulum ATPase] had reduced capacities (23). These changes suggest that the strategy of the myocardium was to balance energy supply with energy demand by decreasing the energy-utilizing processes of contraction and calcium flux (23). To evaluate the efficacy of these altered energy-converting and -utilizing systems to maintain a normal energy status in this paced dog model of CHF, selected metabolite concentrations were determined in failing and control canine myocardia. The results show that normal adenine nucleotide molar concentrations were not maintained in the failing myocardium, whereas the total adenine nucleotide (TAN) concentration was unchanged. Myocardial lactate was the same in both groups, whereas glycogen stores were moderately reduced in RVP. METHODS
DESPITE CONSIDERABLE PROGRESSin the prevention and treatment of congestive heart failure (CHF), the pathogenic mechanism(s) of this disease remains elusive. If this problem is to be solved, the adaptive strategies employed by heart muscle in the face of physiological stress must be understood. Continuous rapid myocardial ventricular pacing (RVP) in dogs produces a clinical, hemodynamic, and neuroendocrine profile symptomatic of CHF (1, 20, 22, 24, 29). After 4.8 t 1.9 wk of RVP, dogs have increased cardiac size, left ventricular filling pressure, and plasma norepinephrine and decreased cardiac index, mean arterial pressure, and tissue norepinephrine (1). Chronic pacing has also been shown to result in a sixfold increase in myocardial 0, uptake (29). This model provides a unique opportunity to study CHF induced by chronic tachycardia. Three major subcellular systems confer physiological expression to the myocardium. They are the energy-supplying pathways of metabolism and the energy-requiring 0161-7567192
$2.00 Copyright
Mongrel dogs weighing 18-25 kg were housed at the University of Guelph Animal Care Facility for 7 days, where they were screened for health disorders. They were then sent to St. Michael’s Hospital (Toronto) and taught to lie quietly in the right decubitus position to permit hemodynamic and echocardiographic assessment in the conscious state. Throughout the study, dogs were allowed food (Purina Dog Chow 5006) and water ad libiturn. As paced dogs approached CHF, their appetites waned and they were given Derby meat mix, which they consumed more readily. Dogs were anesthetized for pacemaker implantation with thiopental sodium [20 t 2 (SD) mg/kg] and maintained with thiopental sodium (6.5 t 3 mg kg-’ h-l) and morphine sulfate (253 t 16 pg. kg-l . h-l). By use of fluoroscopic visualization, a unipolar pacemaker lead (Medtronic silicone/polyethylene) was placed in the right ventricular apex via the right external jugular vein. The pulse generator (Medtronix Spectrax) was then placed in a subcutaneous pocket anterior to the first rib. The ani-
0 1992 the American
l
Physiological
Society
l
2363
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 14, 2019.
2364
ENERGY
STATUS
AT CONGESTIVE
HEART
FAILURE
mals were allowed to recover from surgery for 1 wk be- 1). The cRVP group had significantly lower ATP (-48%) fore cardiac pacing was initiated. Hemodynamic assess- and higher ADP (29%) and AMP (275%) concentrations. ment during normal sinus rhythm in the conscious state This resulted in a significant reduction in the energy was performed, and the pacing was begun at 250 imcharge (EC) in cRVP compared with the sham group. pulses/min asynchronously. There was no significant change in the TAN concentraThe RVP dogs were divided into two groups. Some tion. animals were treated with enalapril (eRVP), an inhibitor Myocardial lactate concentration was similar between of angiotensin-converting enzyme (ACE), to decrease groups (sham = 3.90 t 0.66 vs. cRVP = 3.77 t 0.48 pmoll vascular resistance to blood flow. eRVP animals received g), whereas glycogen was 20% less in the cRVP (38 t 4 enalapril (10 mg/day po) beginning 1 wk after the onset pmol/g) than in the sham group (48 t 2 pmol/g, P < of pacing. The other paced dogs received a placebo 0.05). (pRVP). Sham animals underwent surgical implantation Table 2 contains the values obtained in the different of the pacemaker but were not paced for the period of transmural layers of the myocardium in each of the three time before they were killed. experimental groups. Within the sham group, there were Severe heart failure was defined as a 225% increase in no significant differences in the metabolites and subheart size accompanied by pulmonary edema and/or a strates in different layers of the myocardium. Within the ->lO% increase in body weight (1). This occurred 4-6 wk eRVP group, ADP and AMP levels were higher in the after the onset of pacing. At this time, dogs were anesthemid- than in the epicardium. This resulted in a statistitized with thiopental sodium (20 mg/kg) and ventilated cally significant difference in EC in the epicardium comwith room air to maintain normal arterial PO, values at pared with other layers of the eRVP myocardium. Howthe time of tissue sampling. A thoracotomy was per- ever, the full-transection EC was not significantly differformed. A full transmural section of the left ventricular ent from any myocardial layer in these animals. free wall midway between the apex and the base of the Furthermore, the EC was still significantly lower in the heart was obtained using a dermatological tissue corer eRVP than in the sham epicardium but not different cooled in liquid nitrogen. The sample was immediately from the pRVP epicardium and was still within the range removed from the corer, clamped with liquid nitrogenwhere tissue viability is at risk (2). Thus it appears that cooled tongs, submersed in liquid nitrogen, and divided the within-group differences in the eRVP animals are of into endo-, mid-, and epicardial portions. A second sam- little biological significance. There were no significant ple was taken immediately in a similar fashion but was differences among layers for the pRVP group. As in the not separated into layers. The time lapse from sampling full-transection data, there were no significant differthe heart to immersion in liquid nitrogen was -10 s. ences between eRVP and pRVP animals, with the single Metabolites and high-energy phosphates were ex- exception of higher AMP in the midmyocardium in the tracted by placing a lo- to 30-mg sample of frozen tissue eRVP group. in 0.42 M perchloric acid at -2°C. This preparation was homogenized, neutralized with KOH, and centrifuged at DISCUSSION 6,500 g (19, 27). Adenine nucleotides were measured in the extract either by high-pressure liquid chromatograChronic RVP. In this study we have observed signifiphy or by fluorometry. High-pressure liquid chromatogcant alterations in myocardial energy status as a result of raphy separation was done isocratically in a mobile phase RVP. Previous studies indicate that chronic tachycardia consisting of NH,H2P0,, tetrabutylammonium hydroresults in changes in myocardial metabolism (9, 13, 23). gen sulfate, and acetonitrile at pH 6.5 on a Waters Cl8 These studies suggested that the dog myocardium is unRadial-Pak column, as described previously (27). Fluoroable to completely satisfy the increased energy demand metric detection of ATP was done by coupling the reducof chronic pacing and that the resulting imbalance betion of NADP with the glucose-6-phosphate dehydrogetween high-energy phosphate production and utilization nase reaction at pH 8.1 (19). Lactate in the extract was may be due to a redistribution of blood flow between the measured fluorometrically by coupling the reduction of different layers of the myocardium. It is also possible NAD with the lactate dehydrogenase reaction at pH 10 in that the metabolic capacity of the myocardium, which is the presence of hydrazine (19). Glycogen was measured correlated with resting heart rate (6), is encroached on by digesting the tissue sample in 30% KOH and deterby RVP. mining glucose concentration spectrophotometrically at The perfused heart has been shown to function well at 620 nm with the anthrone reagent (25, 26). reduced absolute levels of ATP (11). Therefore the absoExperimental groups were compared using a two-way lute tissue concentrations of the different adenine nuanalysis of variance with Scheffb’s post hoc test at the cleotides provide limited insight into the metabolic sta0.05 confidence level. Data are presented as means t SE. tus of the myocardium when examined independently. For this reason, the EC was calculated for each myocardial preparation. EC is a traditional measure of the enerRESULTS getic status of the cell and is simply a measure of the Table 1 summarizes the metabolite concentrations for molar ratio of the TAN (2). EC has been related to metafull transection samples of sham and RVP myocardia. bolic regulation, function, and viability of cells (2). EC eRVP animals did not differ from pRVP animals in any was normal in sham myocardia (0.82) compared with the of these indexes. Therefore these two groups were comsignificantly lower value in RVP animals (0.62). EC of bined (cRVP) and compared with the sham group (Fig. 0.62 is approaching the value at which the viability of the Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 14, 2019.
ENERGY
STATUS
AT
CONGESTIVE
1. Metabolic parameters from transverse sections of the
TABLE Group
n
WW
WPI
Sham pRVP eRVP
4 4 6
4.951029 2.43&0.18* 2.74&0.12*
1.50~0.04 1.83k0.12 2.00~0.10
Values are means adenine nucleotides;
[TANI
0.4640.13 l-32&0.24* 1.24-t-0.04*
6,90+0.32 5.58kO.46 5.97-to.21
-t SE in pmol/g; n, no. of dogs. pRVP, placebo EC, energy charge; La, lactate; Gly, glycogen.
rapidly paced * Significantly
6 5 4 3 2 1 0I
a z 3 E '
s-[ATP]
c-[ATP]
s-[ADP]
2365
FAILURE
left ventricular
[AMP1
tissue may be threatened (2). Hence, it appears that, at peak heart failure, RVP results in severe metabolic perturbations in the dog myocardium. Although the ATPproducing pathways must be working at high levels, the adenine nucleotide concentrations are not maintained in normal proportions. ATP declines while both ADP and AMP rise at the RVP failure state. The 47% decrease in ATP seen here is considerably greater than the 16% drop reported by Coleman et al. (9). This difference appears to be related to differences in experimental protocol. In the study by Coleman et al., dogs were paced at 280 beats/ min for an average of only 19 days before tissue analysis. Furthermore those animals showed increased cardiac outputs, whereas the dogs in our study showed a significant decrease in cardiac index (1). Also in the study of Coleman et al., samples were taken while the heart rate was 150 beats/min. In this study, sampling was accomplished while pacing was maintained at 250 beats/min. Despite the significant changes that occur in the concentrations of the three adenine nucleotides in the RVP group, TAN is not different between the sham and paced groups. This is in contrast to studies of acute ischemia that show that TAN decreases as the ischemic period increases (10,12). In heart muscle, the major pathway of A
HEART
free wall EC
La1
0.82kO.02 0.6OkO,O2* 0.63+0.01*
group; eRVP, enalapril-treated different from sham, P
