Aged Rat Heart Research Articles

Age-dependent differences of ATP breakdown and ATP-catabolite release in ischemic and reperfused hearts

The hearts of young (6 months) and aged (24 months) rats, paced at a frequency of 300 bpm, were perfused by the Langendorff technique and subjected to: 20 min of equilibration perfusion, 30 min of global ischemia (95% reduction of the coronary flow) and 20 min of reperfusion. The control group was equilibrated for 20 min and then aerobically perfused for 50 min. After 20 min of stabilization, ATP and ADP levels and the adenine nucleotide pool were significantly higher in young than aged hearts (15% increase), but no modifications were found between the two age groups after 50 min of aerobic perfusion. Even the energy charge did not change under aerobic conditions. At the end of the ischemic period the levels of ATP and ADP decreased to a similar extent in young and aged hearts. After 20 min of reperfusion the myocardial level of ATP remained lower in comparison to the preischemic and control values in both age groups. At the end of the reperfusion there was a decrease in energy charge and creatine phosphate levels in the aged group in respect to the young group. The concentrations of adenosine, hypoxathine and xanthine in coronary effluents did not change during ischemia and reperfusion irrespective of the age of the animals. On the contrary, the release of uric acid during ischemia and reperfusion was greater in aged than young hearts (90% increase). Moreover, the level of inosine in perfusates during the ischemic period was significantly lower in the 24-month-old group (30% decrease). These results are in accordance with the increased purine nucleoside phosphorylase activity and the decreased hypoxanthine phosphorybosyl-transferase activity found in the myocardium of the aged vs. young rats at the end of the reperfusion period. These data indicate that in the aged rat hearts, when exposed to ischemic and reperfusion conditions, there is a modification of purine breakdown which leads to a greater production of uric acid in respect to that found in young hearts.

Incorporation of [ 14C]hypoxanthine into cardiac adenine nucleotides: effect of aging and post-ischemic reperfusion.

In order to investigate whether the ‘hypoxanthine salvage’ pathway of the cardiac muscle is modified with age, we aerobically perfused isolated hearts of 4-month- and 22-month-old male Wistar rats for 20 min with 0.18 μM [ 14C]hypoxanthine. A second group of hearts was subjected to a 30-min ischemic perfusion (95% reduction of the coronary flow), followed by 20 min of reperfusion. In this last 20 min, the perfusate contained the same concentration of [ 14C]hypoxanthine used under the aerobic condition. After 20 min of aerobic perfusion the myocardial levels of ATP were significantly lower (15%) in aged than young rat hearts, whilst no age-related differences were observed at the end of the reperfusion. In the young rats the incorporation of the isotope into ATP, ADP, and AMP was significantly higher (192%, 226%, and 300%, respectively), after 20 min of reperfusion with respect to the aerobic values. On the contrary, in the aged hearts, no significant change in the rate of [ 14C]-incorporation into ATP was observed during reperfusion, despite an increase of the [ 14C]-incorporation into ADP and AMP. Moreover, the content of each labeled adenine nucleotide was significantly higher in aged than young hearts at the end of the aerobic period, whereas the incorporation of the labeled hypoxanthine was not affected by age after 20 min of reperfusion. The release of uric acid into coronary effluents was greater (50%) in aged than young rats during the reperfusion period, but no age-dependent differences in the isotope incorporation into uric acid were observed. These data indicate that in the aged rat heart, perfused under aerobic conditions, there is an increased incorporation of hypoxanthine into ATP, although it does not further increase during postischemic reperfusion.

Analysis by two-dimensional electrophoresis of the cause of myocardial dysfunction in aging rat hearts

The severity and frequency of atherosclerosis, diabetes, and ischemic heart disease, which affect cardiac function, increase with aging. Although there are many reports about hemodynamic and histopathological studies about aging hearts, there are very few studies on changes in structural proteins in aging hearts. We investigated the contractile proteins of the left ventricles in rats aged 6, 12 and 125 weeks using two-dimensional electrophoresis. There were no difference in structural proteins in heart between 6-week and 12-week-old rats. The contents of myosin heavy chain, myosin light chain 2, actin, troponin-I in 125-week-old rats decreased compared with those of 12-week-old rats. Myosin heavy chain, which is one component of myosin, interacts with actin and changes chemical energy to mechanical energy. Therefore its decrease leads to a decline in myocardial contractility. These results seem to indicate one of the most important changes in the aging rat heart, as well as impairment in relaxation by the increase of interstitial fibrosis and decline of Ca uptake by sarcoplasmic reticulum.

Age-related decrease in omega conotoxin binding to rat cardiac synaptosomes.

A cardiac synaptosomal preparation developed by this laboratory was used to study neuronal calcium channels in aging rat heart. Ca2+ channels were quantified by measuring binding of iodinated omega conotoxin, which is reported to specifically block neuronal Ca2+ channels. We determined the binding of [125I]-omega conotoxin GVIA to a synaptosomal preparation from the hearts of 6- and 24-month-old male Fisher 344 rats. The maximum number of binding sites (Bmax +/- SD, fmol/mg protein) is lower in preparations from 24-month (2.2 +/- 0.6) than from 6-month (3.4 +/- 0.7)-old rats. This decrease in number of binding sites suggests an age-related reduction in the number of neuronal calcium channels. Since calcium is essential for exocytotic release of norepinephrine and is made available intracellularly through neuronal calcium channels, the reduction in neuronal calcium channel number may explain, in part, our previous observations of diminished release of norepinephrine in senescent hearts.

Norepinephrine content in cardiovascular tissues from the aged Fischer 344 rat.

The major aim of the present study was to examine cardiovascular tissues and to determine if norepinephrine (NE) stores declined in aged (22-30 months old) male Fischer 344 (F344) rats. NE content was measured by HPLC in the whole heart, ventricle, atria, caudal artery, renal arteries, portal vein and kidney. The release of [3H]NE from the caudal artery of adult and aged rats was assessed as a functional index of sympathetic innervation. Renal alpha 2-adrenergic binding sites were also determined using 2.0 nM [3H]rauwolscine. In general, total NE content per organ was not altered in aged rats. The exception was the ventricle of aged rats which exhibited a 25% decrease in NE content. In contrast, NE content per gram of tissue weight was significantly decreased in the whole heart, atria, ventricle, kidney and caudal artery of aged rats when compared to that of adults (6-8 months old). Neither basal nor potassium-stimulated NE release was altered in the caudal artery of aged rats. Presynaptic regulation of NE release by taurine and alpha 2-adrenoceptors was also not affected by age. Renal alpha 2-adrenergic binding sites were decreased 23% in aged rats. Tissue growth that occurs as a function of aging does not appear to receive a concomitant increase in sympathetic growth as indexed by NE content. The findings of the present study would lead to the conclusion that with the exception of the ventricle, sympathetic nerve terminals of the aged male F344 rat are relatively intact.

On the Fibrinolytic System in Aged Rats, and Its Reactivity to Endotoxin and Cytokines

Aged rats are more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation, than are young rats. To investigate whether changes in the fibrinolytic system might be involved, we investigated the fibrinolytic activity in plasma euglobulin fractions and tissues (lung and heart) of young (6-months old) and aged (24-months old) rats under baseline conditions and after challenge with endotoxin. Aged rats had lower plasma levels of tissue-type plasminogen activator (t-PA) and of urokinase-type PA (u-PA) activity. PA inhibitor (PAI) activity was higher in the plasma of aged rats, as was t-PA activity in lung and heart. Rats were treated with either a low dose (1 microgram/kg) or a high dose (10 mg/kg) of endotoxin. Both treatments induced a transient phase of increased blood fibrinolytic activity, as evidenced by higher levels of tissue-type plasminogen activator (t-PA) activity and decreased levels of PA inhibitor (PAI) activity. Over time, the fibrinolytic activity decreased, probably due to increased levels of PA inhibitor. Both the early increase in t-PA activity, and the subsequent increase in PAI activity, were more pronounced in the aged rats, as compared with the younger rats, after the high dose of endotoxin. The aged rats also responded to an injection of interleukin-1 beta or tumor necrosis factor-alpha with a larger increase of PAI activity than did the younger rats. Together the data suggest that, compared to young rats, aged rats have a decreased base-line plasma fibrinolytic activity, while their fibrinolytic system is more responsive to challenge by endotoxin and cytokines.

Alterations in β adrenergic and muscarinic receptors in aged rat heart. Effects of chronic administration of propranolol and atropine

The cardiac responses to sympathetic and vagal stimulations are attenuated with ageing. To understand these findings, the densities of β adrenergic (βR) and muscarinic (MR) receptors in the left ventricles have been quantitated in parallel in male Wistar rats (4- and 24-month-old) using [ 125I]iodocyanopindolol and [ 3H]quinuclidinyl benzilate as specific radioligands. The homologous regulation of these receptor densities was also explored after a 7-day continuous infusion of propranolol or atropine. As compared to young rats, the βR and MR densities in aged animals were decreased (from 31 ± 23 to 23 ± 2 fmol/mg protein, P < 0.05 for βR; from 104 ± 7 to 54 ± 3 fmol/mg protein, P < 0.001 for MR) but the diminution in MR was more pronounced (−48%) than that in βR (−26%), resulting in a drop in the β/MR ratio. Continuous infusion of propranolol or atropine up-regulated the βR and MR densities (respectively +50%, P < 0.01 and +33%, P < 0.05) in aged but not in young adult rats. We therefore conclude: (i) that the diminution of the cardiac response to the sympathetic and vagal stimulations during ageing may be partly explained by a decrease in the corresponding receptor density; (ii) these changes are reversible and the density of these two groups of receptors can return to adult control values by chronic administration of the appropriate antagonist.

Severe myocardial dysfunction induced by ventricular remodeling in aging rat hearts

To determine if aging engenders alterations in the functional properties of the myocardium and ventricular remodeling, the hemodynamic performance and structural characteristics of the left ventricle of male Fischer 344 rats at 4, 12, 20, and 29 mo of age were studied by quantitative physiology and morphology. In vivo assessment of cardiac pump function showed no change up to 20 mo, whereas left ventricular end-diastolic pressure was increased at 29 mo. Moreover, peak rates of pressure rise and decay, stroke volume, ejection fraction, and cardiac output were depressed at the later age interval, demonstrating the presence of ventricular failure at this time. The measurements of chamber size and wall thickness showed that ventricular end-diastolic and end-systolic volumes progressively increased with age with the greatest change occurring at 20-29 mo. Aging was also accompanied by a marked augmentation in the volume fraction of fibrotic areas in the ventricular myocardium that was due to an increase in their number and cross-sectional area with time. These architectural rearrangements, in combination with the abnormalities in ventricular function, resulted in an elevation in the volume of wall stress throughout the cardiac cycle. Wall stress increased by 64, 44, and 50% from 4 to 12, 12 to 20, and 20 to 29 mo of age. In conclusion, aging leads to a continuous rise in wall stress that is not normalized by ventricular remodeling. These two independent processes appear to be responsible for the onset of heart failure in the senescent rat.

Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart.

To determine the effects of age on the myocardium, the functional and structural characteristics of the heart were studied in rats at 4, 12, 20, and 29 months of age. Mean arterial pressure, left ventricular pressure and its first derivative (dP/dt), and heart rate were comparable in rat groups up to 20 months. During the interval from 20 to 29 months, elevated left ventricular end-diastolic pressure and decreased dP/dt indicated that a significant impairment of ventricular function occurred with senescence. In the period between 4 and 12 months, a reduction of nearly 19% in the total number of myocytes was measured in both ventricles. In the subsequent ages, similar decreases in myocyte cell number were found in the left ventricle, whereas in the right ventricle, the initial loss was fully reversed by 20 months. Moreover, from 20 to 29 months, a 59% increase in the aggregate number of myocytes occurred in the right ventricular myocardium. In the left ventricle, a 3% increment was also seen, but this small change was not statistically significant. These estimations of myocyte cellular hyperplasia, however, were complicated by the fact that cell loss continued to take place with age. The volume fraction of collagen in the tissue, in fact, progressively increased from 8% and 7% at 4 months to 16% and 22% at 29 months in the left and right ventricles, respectively. In conclusion, myocyte cellular hyperplasia tends to regenerate the ventricular mass being lost with age in the adult mammalian rat heart.

Alterations in β adrenergic and muscarinic receptors in aged rat heart. Effects of chronic administration of propranolol, xamoterol, and atropine

Alterations in β adrenergic and muscarinic receptors in aged rat heart. Effects of chronic administration of propranolol, xamoterol, and atropine

Increased myocardial adenosine production and reduction of beta-adrenergic contractile response in aged hearts.

The contractile response of the aged adult heart to beta-adrenergic stimulation is known to be reduced compared with the young adult heart. Since endogenous adenosine exerts an antiadrenergic action in the heart, this study was undertaken to determine if the basal endogenous level of myocardial adenosine increases with age and whether this increase mediates the reduced responsiveness of aged heart to beta-adrenergic stimulation. Young (3-5 months) and aged (12-22 months) Sprague-Dawley adult rat hearts of CD and SD stock were perfused at constant pressure and paced at 270 contractions/min. The two age groups had a similar level of +dP/dtmax (index of contractility) under control conditions. Adenosine release into the coronary effluent was 30 +/- 3 nmol/min/g dry wt from young and 54 +/- 9 nmol/min/g dry wt from aged hearts. Inosine release was also greater from the aged hearts. Isoproterenol (10(-8) M) stimulation increased contractile state by 113% in young hearts and only 69% in aged hearts. Isoproterenol further increased the adenosine and inosine release from both age groups. Theophylline (5 x 10(-5) M), an adenosine antagonist, prevented the difference in the contractile response to isoproterenol stimulation between the young and aged hearts. Elevation of external calcium from 2 to 4 mM increased contractility equally in both age groups without influencing adenosine release. Myocardial oxygen consumption, coronary effluent PO2, oxygen supply-demand ratio, and lactate release were similar for both age groups, indicating that under the conditions studied the elevated release of adenosine by the aged hearts was not due to hypoxia. Aged (10-14 months) adult guinea pig hearts also displayed a reduced responsiveness to the isoproterenol stimulation and released more adenosine compared with young (3-4 months) adult guinea pig hearts. These findings suggest that enhanced adenosine levels that are present in the aged myocardium are responsible, in part, for the reduced contractile responsiveness of the older adult heart to beta-adrenergic stimulation.

Mitochondrial function and superoxide generation from submitochondrial particles of aged rat hearts

A decrease in heart function with ageing might be related to an impairment of mitochondrial function, since these organelles produce the greatest fraction of ATP in the myocyte. Mitochondria extracted from Wistar rat hearts at 3, 14, 18 and 24 months of age were employed to evaluate the changes of the respiratory activity during lifetime. A slight decrease of the respiratory rate (QO 2) was observed in the 14 month group with respect to the 3 month group when succinate was used as substrate, whereas the respiratory control index (RCI) in the presence of glutamate or succinate increased in the 24 month group. The latter result may be related to a condition of moderate hypertrophy that generally occurs in the ageing heart. Submitochondrial particles (SMP) were also prepared to study the superoxide radicals (O ⨪ 2) production at the level of rotenone or antimycin-inhibited regions of the respiratory chain. A strong elevation in the O ⨪ 2 generation was observed in the antimycin-inhibited region at 14 months of age; on the contrary, the rate of O ⨪ 2 production remained unchanged in the 24 month group in comparison to the youngest group. These observations correlate well with the enhanced tissue level of oxidized glutathione that was observed at 14 and 18 months of age. The products of lipid peroxidation (TBARS) did not change in the rat heart at any of the ages measured, whereas the levels of fluorescent substances progressively increased beginning from 18 months of age, with a greater extent in the mitochondrial compartment. The present study suggests that age does not substantially affect mitochondrial respiration and energy output in the rat heart, while a greater production by cardiac mitochondria of superoxide anions in the adult rats (14 months) might accelerate the fluorescent pigment formation.

Cardiac adaptations to aortic constriction in adult and aged rats.

Myocardial function in vitro and myosin heavy chains (MHCs) were studied in control and hypertrophied hearts of adult (9-10 mo) and aged (25-28 mo) female Fischer 344 rats 7 days after aortic constriction. Aortic constriction increased left ventricular mass to 110 and 112% of the control values of 484 +/- 12 and 617 +/- 18 (SE) mg in adult and aged rats, respectively. After aortic constriction, there was a significant age-related difference in the adaptation of peak pressure development in vitro, as peak left ventricular systolic pressure increased and decreased in hearts of adult and aged rats. The maximum rate of pressure development in control hearts of aged rats was 79% of the adult value of 11,264 +/- 1,527 mmHg/s; hypertrophy did not alter values of either age group. alpha-MHC accounted for 82 +/- 1 and 48 +/- 3% of the total left ventricular MHC for the adult and aged control groups, respectively, and values were not altered by aortic constriction. With hypertrophy 7 days after aortic constriction, there is an impairment in the adaptation of left ventricular function in hearts of aged compared with adult rats. This impairment is not explained by alterations in MHC isoform.

Control of low K m cyclic adenosine 3′,5′-monophosphate-phosphodiesterase in prostate and heart of adult and aged rats

The activity of low K m cAMP-phosphodiesterase (PDE) was determined in extracts of prostate and heart of adult (10–12 months old) and aged (32–35 months old) Sprague—Dawley rats; the enzyme's response to endogenous inhibitors extracted from the two organs was analyzed by kinetic studies. Different mechanisms of inhibition, non-competitive in prostate and competitive in heart extracts, indicate organ-specificity of the inhibitor. The decreased PDE activity in organ extracts of aged rats and its continued sensitivity to the endogenous inhibitor suggest an age-associated impairment of the tissues' ability to terminate cAMP-mediated signals.

Enzyme histochemistry of monoamine oxidase in the heart of aged rats

The distribution of monoamine oxidase types A and B in the heart of young (3-month-old) and aged (26-month) Wistar rats was studied by histochemical methods. In young rats, MAO activity was higher in the left ventricle than in the right. Enzyme reactivity was present within both muscle cells and the blood vessel wall. Histochemical staining was abolished by clorgyline administration. In the heart of aged rats, we observed a very remarkable increase of clorgyline sensitive MAO activity primarily at the level of myocardial cells. The right ventricle showed the highest increase of MAO reactivity.

Effect of chronic exercise on regional substrate oxidation in aged rat hearts

Effect of chronic exercise on regional substrate oxidation in aged rat hearts

Myocyte cell loss and myocyte hypertrophy in the aging rat heart

To determine the effects of age on the myocardium, the functional and structural characteristics of the heart were studied in rats at 3, 10 to 12 and 19 to 21 months of age. Systemic arterial pressure, left ventricular pressure and its first derivative (dP/dt) and heart rate were comparable in the three animal groups. In the interval between 3 and 10 to 12 months, mean myocyte cell volume per nucleus increased 53 and 26% in the left and the right ventricle, respectively. The total number of myocyte nuclei remained constant in either ventricle. In the following period, between 10 to 12 and 19 to 21 months, a 39% further cellular hypertrophy on the left side of the heart was found in association with an 18% loss of cells in the ventricle. Cell loss was accompanied by discrete areas of interstitial and replacement fibrosis in the subendocardium. In contrast, no myocardial damage was observed in the right ventricle, and the measured 35% additional enlargement of myocytes occurred without a change in cell number. Thus, the aging left ventricle is composed of a smaller number of hypertrophied cells. Cellular hypertrophy may explain the unaltered cardiac function of the aged myocardium.

Alteration of 6-phosphofructo-1-kinase isozyme pools during heart development and aging.

The nature of 6-phosphofructo-1-kinase isozyme pools in fetal, neonatal, young adult (3 months), and aged (30 months) rat hearts was studied using chromatographic and immunological techniques. Furthermore, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. Although all three subunit types were expressed in heart throughout life, total activity and the nature of the isozyme pools varied during neonatal development and in aged heart. In fetal heart, the complex tetramers containing all three subunits appeared to be the major isozyme types. As the heart matured to the young adult stage, the M-type subunit increased over 6-fold; whereas the changes in the other two subunits were considerably less. These data indicate that during neonatal heart maturation the isozymic pools progressively exhibited increased amounts of the tetrameric forms containing two or more M-type subunits. In aged heart relative to the young adult (3 months) heart, the total activity and proportion of M-type subunit in the isozymes were decreased; and consequently, the amounts of the M-rich isozymes were decreased. The shifts in the types of isozymes during heart maturation and subsequent aging were primarily due to changes in availability of the M-type subunit to participate in random assembly of the tetrameric isozymes.

Calmodulin regulation of the cholinergic receptor in the rat heart during ontogeny and senescence

The contents of calmodulin and cholinergic muscarinic receptor binding sites in the hearts of fetal, adult and aged rats have been examined. A biphasic pattern of calmodulin development was observed. A relatively high level of calmodulin appeared on gestational days 14–15 followed by a steady but significant decrease at birth and during the first week of postnatal life. The level of calmodulin then increased significantly during the first month followed by a decrease at 6 and 26 months of age. Calmodulin contents were significantly higher in the atrium than in the ventricle in the age groups of 1–26 months. The number of [ 3H] QNB binding sites showed a steady increase during the gestational periods studied, reaching a peak at 9 days after birth and followed by a significant ( P < 0.05) decline at 6 and 26 months of age. A good correlation between the levels of [ 3H]. QNB binding and calmodulin was observed from day 9 of the postnatal period to 26 months of age. In the presence of calcium, calmodulin induced a dose-dependent receptor binding loss in the hearts of postnatal, young adult and aged rats under phosphorylating conditions. These findings support the suggestion that calmodulin may regulate cholinergic functions during ontogeny and senescence.

Functional and metabolic properties in hearts from aged spontaneously hypertensive rats.

The effect of long-term pressure overload on myocardial functional and metabolic alterations was investigated in hearts from spontaneously hypertensive rats of 16 weeks (young SHR) and 44 weeks (aged SHR) and age matched normotensive Wistar Kyoto strain rats (young WKY, aged WKY). The hearts were perfused by working heart mode and whole heart ischemia was induced by one-way valve. Following 20 min of ischemia, the hearts were reperfused for 30 min. The heart-body weight ratio in both SHR groups was significantly higher than in the respective age-matched WKY groups. Coronary flow relative to heart weight in both SHR groups was significantly lower than that of the respective age-matched WKY during both preischemic and reperfused periods. There was no significant difference in the recovery rate of cardiac output between young and aged WKY, whereas the young and aged SHR revealed significantly less recovery than their respective age-matched WKY. Tissue creatine phosphate and energy charge in both aged groups were significantly lower than in the young groups. These results indicate that long-term pressure overload increases susceptibility to ischemia and decreases the myocardial reserve presumably resulting from relative ischemia, whereas deterioration was minimal in the normotensive aged rat heart.