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Aging alters the force-frequency relationship and toxicity of oxidative stress in rabbit heart
Life Sciences, Vol. 48, pp. 1769-1777 Printed in the U.S.A
Pergamon Press
AGING ALTERS THE FORCE-FREQUENCYRELATIONSHIP AND TOXICITY OF OXIDATIVE STRESS IN RABBIT HEART
Barry J. Cusacki'z'4, Philljp S. Mushlin3, Tomasz AndrejukI'2, Louis D. Voulelis ~, and Richard D. Olson1'z. The Clinical Pharm@cology and Gerontology Unit, V.A. Medical Center, Boise, ID 83702". Department of Medicine, School of Medicine, University of Washington, Seattle, WA 98]95 . Department of Anesthesia~ Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115°. (Received in final form February 27, 1991)
Summary Adult (6 months) and senescent (> 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of I/sec, cardiac relaxation (go% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P < 0.05). Additional experiments indicated that HzOz (500 /I/4),. a powerful oxidant, increased resting force and decreaseo developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 8] ± 6% and senescent = 42 ± 27% of pre-H~O~ values; P < 0.05). Age-related differences in effects 6f-HzO2 did not result simply from a decreased a b i l i t y of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H202mediated decreases in GSH were similar in adult ~6d senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway. Effects of aging on cardiac function have not been fully elucidated. Senescence appears to impair cardiac relaxation without appreciably altering contractility or resting force (a measure of muscle stiffness or compliance) (5,10). Such changes in cardiac mechanics may relate to dysfunction of sarcoplasmic reticulum (SR), with perturbations of calcium sequestration or release (4,9,13). Regulation of calcium handling by SR depends, in part, on the oxidative state of c r i t i c a l SH groups (3,18), which may be modified by aging (6). In rodents, aging appears to decrease the concentration of reduced glutathione and the activities of glutathione peroxidase and reductase (7,8,17) in several tissues. 4To whom correspondence should be addressed: VA Medical Center, 500 West Fort St., Boise, ID 83702 0024-3205/91 $3.00 + .00
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On this basis, the senescent myocardium may have a decreased capacity to buffer oxidative stress. Alternatively, senescence, by mechanisms unrelated to c e l l u l a r redox state, may decrease capacity to remove calcium from the cytoplasm, thereby predisposing to myocardial dysfunction whenever cytoplasmic calcium fluxes increase or oxidative injury occurs. We therefore evaluated effects of aging on I) the force-frequency relationship (cytoplasmic calcium increases as contraction frequency increases), and 2) contractile and biochemical responses (glutathione pathway) to an oxidative stress (H202). Methods Animals Adult New Zealand White rabbits of either sex, approximately 6 months of age and weighing 2.5-3.5 kg, were obtained from Dorothy's Rabbit Co., New Plymouth, ID. Each animal was housed in a stainless steel cage and received food (Purina Rabbit Chow) and water ad libitum. Senescent New Zealand White rabbits of either sex (7 females, 2 males) were greater than 5 years of age and weighed approximately 3.2-5.2 kg. These rabbits were retired breeders purchased from several rabbitries in the northwestern United States; they were transferred to Dorothy's Rabbit Co., New Plymouth, ID at least I year prior to this study. We observed rabbits for one week or longer in our f a c i l i t y prior to study to rule out overt i l l n e s s . During this time, senescent animals were housed as described for adult rabbits above. Post mortem examinations failed to reveal gross pathological lesions, except for uterine fibroids in a few senescent females. No attempts were made to characterize cardiovascular status of rabbits prior to euthanization. Protocol Adult and senescent New Zealand White rabbits were euthanized by captive bolt discharge to the cranium and hearts were removed and placed in buffer. Left a t r i a l tissue was rapidly excised for studies of cardiac function, and the right ventricular free wall was excised for glutathione studies. Atrial strips from adult and senescent rabbits were similar in size and weighed approximately 150 mg each. Each s t r i p was suspended in a Krebs-bicarbonate buffer (pH 7.4, 30°C) that was bubbled with a 95% 02, 5% CO2mixture. The buffer contained 127 mM NaCl, 2.5 mM CaCl2, 2.3 B~MKCl, 25 mMNaHCO3, 1.3 mM KH2P04, 0.6 mM MgSO4, and 5.6 mM glucose. Baseline values of cardiac function were recorded after preparations had stabilized. In some studies, contractile function was monitored following addition of 500 ~ H202to the buffer. Soluble sulfhydryl content, glutathione peroxidase a c t i v i t y aria glutathione reductase a c t i v i t y were measured in thin right ventricular strips (100 - 300 mg, t y p i c a l l y less than I mmin thickness) that had been suspended in the buffer (as above). These strips were removed at 19, 35, and 75 minutes following addition of 500 p~MH202. Cardiac Function Studies Left a t r i a l strips were attached to an isometric force transducer (Kulite BG25) and e l e c t r i c a l l y stimulated (Grass S88) to contract isometrically using a square wave pulse (3 msec) at 10% above threshold voltage. Data were recorded on a Gould 2400S oscillographic recorder. Each s t r i p was stretched to a resting force of 0.5 g and allowed to stabilize for at least 30 minutes before determining baseline cardiac variables. Variables measured included resting force (RF), developed force (DF), maximum rate of force development ( i . e . , dF/dt, referred to as c o n t r a c t i l i t y ) , time interval from onset of contraction to peak force (TTPF), and time interval from peak force until DF had decreased
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by90%of i t s peak value (90% relaxation time; 90% RT). Measurementswere made at stimulation rates of I, 2, and 3 beats/second. Glutathione Studies Tissue glutathione from right ventricular free wall strips was assayed as soluble sulfhydryl (-SH) concentrations, using the method of Sedlack and Lindsay (16). This method is based on the reduction of Ellman's reagent ( d i t h i o - b i s nitrobenzoic acid) by sulfhydryl groups in the supernatant fraction to form a mercaptobenzoic acid with an intense yellow color (measured at 412 nm). Data were expressed as ~g soluble sulfhydryl content/g wet weight of tissue. Glutathione peroxidase a c t i v i t y was assessed in right ventricular free wall strips using the technique of Paglia and Valentine (14) with more recent modifications (8,15). Tissue samples were added to 0.25 M sucrose (4°C) to obtain concentrations of 10%, 5% and 2.5%w/v. Homogenateswere centrifuged at 9,000 x g for 20 minutes; supernatant was removed and recentrifuged at 9,000 x g for 20 minutes. Supernatant (0.05 ml) was vortexed with 0.95 ml of buffer (pH 7.5) containing final concentrations of 24 mMHEPES, 1.15 mMEDTA, 0.46 mMNaN3 and excess GSH (1.0 mM), NADPH (0.2 mM) and glutathione reductase (1.0 units). The mixture was allowed to stand at room temperature for 10 minutes. The reaction was started by addition of 50 #l of 5 mM H20z solution. Oxidation of NADPH was assessed every 15 seconds for 1.5 minutes at 340 nm to obtain glutathione peroxidase a c t i v i t y expressed as nmol NADPH oxidized/min per mg protein. Glutathione peroxidase a c t i v i t i e s for 10%, 5% and 2.5% tissue-sucrose concentrations were combined and averaged. A control assay was obtained using buffer without homogenate to correct for non-enzymatic oxidation of NADPH. Glutathione reductase a c t i v i t y was assessed in right ventricular free wall strips using the technique described by Massey and Williams (11). Tissue was homogenized and the supernatant obtained as described for the glutathione peroxidase assay. Supernatant (50 #l) was added to 0.95 ml buffer (pH 7.5; final concentrations of 40 mM K~HPO 4, 2.0 mM EDTA, 4 mM oxidized glutathione [GSSG] and 1.0 mM FAD+), vortexe~ and allowed to stand at room temperature for 10 minutes. The assay for glutathione reductase a c t i v i t y was i n i t i a t e d by addition of 50 #l of 2.0 mMNADPH, and oxidation of NADPHwas assessed every 15 seconds for 1.5 minutes at 340 nm; glutathione reductase a c t i v i t y was expressed as nmol NADPHoxidized/min per mg protein. Glutathione reductase a c t i v i t i e s for tissue/sucrose concentrations of 10%, 5%, and 2.5% w/v were combined and averaged as described in the glutathione peroxidase assay. A control assay was obtained using buffer without homogenate to correct for non-enzymatic oxidation of NADPH. Protein assay was performed by the Bradford technique (2). Statistical Methods Cardiac function data w i t h i n groups were analyzed using randomized block analysis of variance with Duncan's New M u l t i p l e Range t e s t . Between group comparisons employed one way analysis of variance with Duncan's New M u l t i p l e Range t e s t . Data obtained from studies assessing soluble s u l f h y d r y l concentrations and a c t i v i t i e s of GSH peroxidase and GSH reductase were analyzed by one way analysis of variance and Student-Neuman-Keuls t e s t . Effects of H2O2 on c o n t r a c t i l e function in young and senescent rabbits (Table I) were compared using Students unpaired t - t e s t with correction for unequal sample sizes and unequal variances. The n u l l hypothesis was rejected i f P < 0.05.
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Results Effects of frequency of contraction on changes in resting force (RF; changes in muscle stiffness), developed force (DF; strength of contraction) and dF/dt ( c o n t r a c t i l i t y ) of atria from adult and senescent rabbits are shown in figure I. There were no age-related differences in RF or dF/dt at any contraction frequency. Developed force was similar in the two groups at I contraction/sec (adult was 0.46 ± 0.06 g; senescent was 0.72 ± 0.17 g) but was s i g n i f i c a n t l y greater in senescent than young adult atria at higher frequencies (1.76 ± 0.28 g vs. 0.95 ± 0.10 g at 2 contractions/sec and 1.93 ± 0.29 g vs. 1.01 ± 0.11 g at 3 contractions per second). Cardiac relaxation time (90% RT) was unaffected by senescence at a contraction frequency of I/sec (figure 2). However, as contraction frequency increased, 90% RT was s i g n i f i c a n t l y shortened (P < 0.01) in adult (106 ± 3 msec at I beat/sec vs. 96 ¢ 3 and 93 ± 3 msec at 2 and 3 beat/sec, respectively) but not senescent atria (113 ± 8, 120 ± 8 and 111 ± 6 msec at I, 2 and 3 contraction/sec respectively). Direct comparisons between adult and senescent atria revealed s t a t i s t i c a l l y significant differences in 90% RT at frequencies of 2 beats/sec (96 ± 3 msec vs 120 ± 8 msec; p < 0.01) and 3 beats/sec (93 ¢ 3 msec vs. 111 ± 6 msec; p < 0.05) (figure 2). Time to peak force (TTPF) was shorter in adult than senescent atria (P < 0.01) at all contraction frequencies (adult = 78 ± 3, 74 ± 3 and 71 ± 3 msec; senescent = 92 ± 5, 89 ± 3 and 85 ¢ 3 msec at I, 2 and 3 beats/sec, respectively; figure 2). Treatment with H)O2 caused striking alterations in resting force and developed force (dF/dt, TTPFand 90% RT were not measured in these experiments, owing to exclusive use of slow oscillographic tracings). The time interval between addition of H202 and the f i r s t detectable change in either RF or DF was defined as "onset latency" ( i n i t i a l changes in RF and DF occurred simultaneously in all preparations). In several preparations, increases in resting force and decreases in developed force culminated in contracture (high resting force without force development). The time interval from addition of H202 to the maximal (peak) H20~effect was termed "peak latency." Onset latency was 6 times greater in adult than senescent atria (19.0 ± 1.5 min vs. 3.2 ± 0.9 min; P < 0.001) (table I ) , despite similar peak latencies in both groups (33.6 ± 5.8min in adult atria, 29.0 ± 1.0 min in senescent a t r i a ) . Maximal increases of RF were comparable in the two groups (adult = 75 ± 11% and senescent = 89 ± 6% of pre H~O2 values), but maximal depressions of D~ were approximately 2 times greater-in adult than in senescent atria (adult 81 ± 6% and senescent = 42 ± 27% of pre HzOz values; P < 0.05). Concentration of soluble sulfhydryl groups and a c t i v i t i e s of glutathione peroxidase and reductase were similar in senescent and adult rabbit heart (figure 3). Following addition of 500 /d~ H202, soluble sulfhydryl concentrations decreased comparably in both age groups during the 75 minute study period. In adult heart, soluble SH levels had decreased from the baseline by 22 ± 3%, 34 ± 4% and 38 ± 8% at 19 min, 35 min and 75 min after H202, and by 32 ± 4%, 34 ± 15% and 33 ± 9% in senescent heart at the above times. Basal glutathione peroxidase a c t i v i t i e s were comparable in adult and senescent heart (47 ± 3 vs. 50 ± 4 nmol NADPH/min per mg protein, respectively). H202did not s i g n i f i c a n t l y alter glutathione peroxidase a c t i v i t i e s in either age group (figure 3). Similarly, basal glutathione reductase a c t i v i t i e s did not d i f f e r between adult and senescent preparations (27 ± 2 vs. 25 ± 3 nmole NADPH/min per mg protein) andeH20~thdid not alter glutathione reductase a c t i v i t y of either age group (with exception of a decrease in a c t i v i t y at 75 minutes in the senescent animals).
Vol.
48,
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18,
1991
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FIG. 1. Effects of age on resting force (Panel A), developed force (Panel B), and dF/dt (Panel C) in l e f t atrial strips from young adult (open circles, n = 10) and senescent (closed circles, n = g) r~bbits. S t u d i e s w e r e performed at ], 2, and 3 contractions/second. Circles and bars show mean ± SE. **P < 0.001 for young vs. old preparations. Discussion The present study provides the f i r s t evidence that senescence alters the function of the rabbit heart. Age-related alterations of basal myocardial function (figures I and 2) in rabbits were similar to those previously reported in the rat (4, 10). For example, aging did not alter myocardial contractility (dF/dt; figure 1) but did impair cardiac relaxation (TTPF, 90% RT, figure 2). Relaxation was enhanced as frequency of contraction increased in adult but not senescent atria (figure 2). Senescent atria developed more force than adult atria, primarily reflecting prolongation of contraction rather than an alteration of the inotropic state (DF = dF/dt x TTPF) Thus, impairment of relaxation was the most prominent age-related alteration of myocardial function. Effects of aging on cardiac function may relate to alterations of calcium metabolism. Aging does not appear to affect the rate of calciumdeliveryto the myofibrillar apparatus (12), which may account for an absence of age-related effects on contractility (dF/dt; figure I; Ig). However, aqueorin studies indicate that aging prolongs the time to attainment of peak cytoplasmic calcium level and slows the decline from the peak level (5). This may be explained by a prolongation of the transmembrane action potential or a decrease in velocity of calcium uptake and Ca-Mg ATPase a c t i v i t y of cardiac SR. Whatever the mechanism, a prolonged availability of calcium for contraction (i.e. increased TTPF) allowed more time for senescent hearts to develop force (increased DF,
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figure 1). The age-related impairment of cardiac relaxation was most evident at higher contraction frequencies (figure 2), suggesting that aging decreases the reserve of mechanisms that clear the increased cytoplasmic calcium resulting from increased heart rate. Altered calcium handling with aging may relate to an increased exposure to oxidative stresses that cause dysfunction of SR ( I ) . A g i n g may impair mechanisms that scavenge free radicals, predisposing to free radical injury. The glutathione pathway plays an important protective role against oxidative stress by detoxifying oxidants via a reaction that converts GSH to GSSG. Age-related declines in glutathione peroxidase a c t i v i t y , glutathione reductase a c t i v i t y and GSH have been reported (7,8,17). A deficiency of GSH could predispose to an increased proportion of enzymes with oxidized sulfhydryl groups, such as those on calcium release channels of SR. As a result, a residual leakage of calcium from terminal cisternae may occur (18). Alternatively, aging may increase calcium leakage into the cytoplasm via mechanisms independent of oxidative stresses or by mechanisms involving oxidative injuries that f a i l to perturb the glutathione system (e.g., l i p i d peroxidation of membranes). The mechanical consequencesof enhancedcytoplasmic calcium influxes would be magnified by a coexisting impairment of calcium sequestration.
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FIG. 2. Effects of age on 90% relaxation time (go% RT) and time to peak force (TTPF) in l e f t atrial strips from young adult (open circles; n = 10) and senescent (closed circles; n = g) rabbits. Studies were performed at I, 2, and 3 contractions/second. Circles and bars show mean ± SE. *P < 0.05 and **P < 0.01 for young vs. old preparations.
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TABLE I Effects of H202 (500 #m) on Function of Left Atrial Strips from Young Adult and Senescent (Old) Rabbits. GROUPS
Baseline8
Latencyb
Peak Effect
RF
DF
Onset
Peak
RF
(g)
(g)
(min)
(min)
(g)
Young Mean 0.53 (n,14) ± SE 0.02
0.41 0.05
19.0 1.5
33.6 5.8
0.91 0.06
74.9 1).3
0.08 0.03
81.1 5.8
Old (n,3)
0.53 0.17
29.0 1.0
0.87 0.04
89.0 5.5
0.32" 0.18
42.3" 27.0
Mean 0 . 4 6 ± SE 0 . 0 3
3.2"* 0.9
Increasec DF in RF (%) (g)
Decrease c in DF (%)
aContraction frequency of I/sec. bTime intervals between addition of HzO2 and i n i t i a l changes (onset latency) or maximal changes (peak latency) in function. CPercentage change from baseline. *P < 0.05, and **P < 0.001 for differences between age groups. This study determined effects of aging on the glutathione pathway in cardiac tissue subjected to an oxidant stress. The glutathione pathway was evaluated in right ventricular free wall preparations prior to and after exposure to HzO2. (Thin right ventricular free wall strips rather than atrial preparations were used to obtain sufficient quantities of tissue for assays of glutathione pathways). Basal GSH concentrations and glutathione peroxidase and reductase activities did not d i f f e r between the two groups, suggesting, that in our rabbit model, the capacity to metabolize peroxides by this pathway was unaffected by age. When the glutathione pathway was challenged by treatment with H20z, GSH concentrations decreased comparably in both age groups (figure 3). In contrast, the effects of HzOz on myocardial function were highly age-dependent (tablel). Treatment with HzO2 increased resting force and decreased developed force in both senescent and-adult atria, but these changes were accelerated in senescent atria (3 ± I vs. 19 ± 2 min respectively; P < 0.001). This suggests that aging increases susceptibility to oxidative injury. Developed force, however, was decreased more in adult than senescent atria (81 ± 6% vs. 42 ± 27% decrease from pre-HzO z value), despite similar maximum increases of RF {75 ± II vs. 89 ± 6% increase from pre-HzO ~ values) in the two groups. Thus, advanced age may be associated with adaptlve mechanisms that better preserve contractile function in the presence of an oxidative stress. The subcellular mechanism of the rapid onset of H202 toxicity in senescent atria may involve the release of large quantities of calcium from terminal cisternae, coupled with a severely limited capacity to sequester calcium into longitudinal SR. In summary, the primary effect of aging on myocardial function was an impairment of r e l a x a t i o n r a t h e r than c o n t r a c t i l i t y . Senescent a t r i a did not r e l a x as r a p i d l y as adult a t r i a when frequency of c o n t r a c t i o n was increased. An o x i d a t i v e stress (H2Oz) caused a more rapid onset of t o x i c i t y in senescent than in adult a t r i a . However, senescent a t r i a were b e t t e r able than adult a t r i a to maintain c o n t r a c t i l e f u n c t i o n in the presence of an o x i d a t i v e stress.
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Age-related differences in t o x i c i t y of HzOz were probably unrelated to detoxification capacity of the glutathione pathway because H20z comparably decreased soluble sulfhydryl concentrations in both age groups. Further studies w i l l be required to elucidate mechanisms by which aging alters effects of an oxidative stress and modifies the force-frequency relationship.
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FIG. 3 Effects of 500 #M HzOz on soluble sulfhydryl concentrations (panel A), glutathione peroxidase a c t i v i t y (panel B) and glutathione reductase a c t i v i t y (panel C) of right ventricular strips from young adult (open bars) and senescent (closed bars) rabbits. Bars represent mean ± SE. The number of preparations per group is shown inside bars. *P < 0.05, and**P < 0.01 for baseline (time = O) vs. post-treatment values within age groups. Acknowledqements The authors would l i k e to thank Gloria Kent and Sally Sellers for preparation of the manuscript; Steve Young, Joni Strander and Matthew Parks for expert technical assistance. This work was supported by grants from the Department of Veteran's Affairs and the American Federation for Aging Research, Inc. References 1. 2. 3.
J. ABRAHAHSONand G. SALAMA, Mol. C e l l . Biochem. 8 2 : 8 1 - 8 4 (1988). M.M. BRADFORD, Anal. Biochem. 7 2 : 2 4 8 - 2 5 4 (1976). D.G. BRUNDER, C. DETTBARN and P. PALADE, J. B i o l . Chem. 263: 18785-18792 (1988).
4.
J . M . CAPASSO, A. MALHOTRA, R.M. REMILY, J. SCHEUER and E.H. SONNENBLICK, Am. J. Physiol. 245:H72 - H79 (1983). J.P. FROELICH, E.G. LAKATTA, E. BEARD, H.A. SPURGEON,M.L. WEISFELDT and G. GERSTENBLITH, J. Mol. Cell. Cardiol. ]0:427-438 (1978). S. GOLDSTEIN, N. Engl. J. Med. 285:1120-1121 (1971).
5. 6.
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7. 8. 9. 10. 11. ]2. 13. 14. 15. ]6. 17. ]8. 19. 20.
Cardiac Function and Aging
G.A. HAZELTON and C.A. LANG, Biochem. J. ]88:25-30 (1980). G.A. HAZELTON and C.A. LANG, Mech. Age Dev. 29:71-81 (]985). E.G. LAIC~TTAand F.C.P. YIN, Am. J. Physiol. 242:H927-H941 (1982). E.G. LAKATTA, G. GERSTENBLITH, C.S. AGNELL, N.W. SHOCK, and M.L. WEISFELDT, J. Clin. Invest. 5_55:61-68 (1975). V. MASSEYand C.H. WILLIAMS, J. Biol. Chem. 240:]58-]69 (1965). D.W. MAUGHAN, E.S. LOW and N.R. ALPERT. J. Gen. Physiol. 71:431-451 (1978). N. NARAYANA, Biochem. Biophys. Acta 678:442-459 (1981). D.E. PAGLIA and W.N. VALENTINE, J. Lab. Clin. Med. 70:158-169 (1967). J.R. PROHASKA, S-H OH, W.G. HOKSTRA and H.E. GANTHER, Biochem. 8iophys. Res. Comun. 74: 64-7] (1977). J. SEDLAK and R.H. LINDSAY, Anal. Biochem. 25:192-205 (]968). J, STOHS, J.N. HASSING, W.A. AL-TURK and A.M. HASOUD, Age 3 : 1 1 - 1 4 (1980). J.L. TRINH, G. SALAMAand J.J. ABRAMSON, J. Biol. Chem. 261: ]609216098 (]986). G.D. WALFORD, H.A. SPURGEONand E.G. LAKATTA, Circ. Res. 63:502-511 (1988). M.L. WEISFELDT, E.G. LAKATTA and G. GERSTENBLITH, in Heart Disease, Ed. E. Braunwald, p. 1650-1662, Saunders, Philadelphia (1988).
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Pergamon Press
AGING ALTERS THE FORCE-FREQUENCYRELATIONSHIP AND TOXICITY OF OXIDATIVE STRESS IN RABBIT HEART
Barry J. Cusacki'z'4, Philljp S. Mushlin3, Tomasz AndrejukI'2, Louis D. Voulelis ~, and Richard D. Olson1'z. The Clinical Pharm@cology and Gerontology Unit, V.A. Medical Center, Boise, ID 83702". Department of Medicine, School of Medicine, University of Washington, Seattle, WA 98]95 . Department of Anesthesia~ Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115°. (Received in final form February 27, 1991)
Summary Adult (6 months) and senescent (> 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of I/sec, cardiac relaxation (go% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P < 0.05). Additional experiments indicated that HzOz (500 /I/4),. a powerful oxidant, increased resting force and decreaseo developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 8] ± 6% and senescent = 42 ± 27% of pre-H~O~ values; P < 0.05). Age-related differences in effects 6f-HzO2 did not result simply from a decreased a b i l i t y of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H202mediated decreases in GSH were similar in adult ~6d senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway. Effects of aging on cardiac function have not been fully elucidated. Senescence appears to impair cardiac relaxation without appreciably altering contractility or resting force (a measure of muscle stiffness or compliance) (5,10). Such changes in cardiac mechanics may relate to dysfunction of sarcoplasmic reticulum (SR), with perturbations of calcium sequestration or release (4,9,13). Regulation of calcium handling by SR depends, in part, on the oxidative state of c r i t i c a l SH groups (3,18), which may be modified by aging (6). In rodents, aging appears to decrease the concentration of reduced glutathione and the activities of glutathione peroxidase and reductase (7,8,17) in several tissues. 4To whom correspondence should be addressed: VA Medical Center, 500 West Fort St., Boise, ID 83702 0024-3205/91 $3.00 + .00
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On this basis, the senescent myocardium may have a decreased capacity to buffer oxidative stress. Alternatively, senescence, by mechanisms unrelated to c e l l u l a r redox state, may decrease capacity to remove calcium from the cytoplasm, thereby predisposing to myocardial dysfunction whenever cytoplasmic calcium fluxes increase or oxidative injury occurs. We therefore evaluated effects of aging on I) the force-frequency relationship (cytoplasmic calcium increases as contraction frequency increases), and 2) contractile and biochemical responses (glutathione pathway) to an oxidative stress (H202). Methods Animals Adult New Zealand White rabbits of either sex, approximately 6 months of age and weighing 2.5-3.5 kg, were obtained from Dorothy's Rabbit Co., New Plymouth, ID. Each animal was housed in a stainless steel cage and received food (Purina Rabbit Chow) and water ad libitum. Senescent New Zealand White rabbits of either sex (7 females, 2 males) were greater than 5 years of age and weighed approximately 3.2-5.2 kg. These rabbits were retired breeders purchased from several rabbitries in the northwestern United States; they were transferred to Dorothy's Rabbit Co., New Plymouth, ID at least I year prior to this study. We observed rabbits for one week or longer in our f a c i l i t y prior to study to rule out overt i l l n e s s . During this time, senescent animals were housed as described for adult rabbits above. Post mortem examinations failed to reveal gross pathological lesions, except for uterine fibroids in a few senescent females. No attempts were made to characterize cardiovascular status of rabbits prior to euthanization. Protocol Adult and senescent New Zealand White rabbits were euthanized by captive bolt discharge to the cranium and hearts were removed and placed in buffer. Left a t r i a l tissue was rapidly excised for studies of cardiac function, and the right ventricular free wall was excised for glutathione studies. Atrial strips from adult and senescent rabbits were similar in size and weighed approximately 150 mg each. Each s t r i p was suspended in a Krebs-bicarbonate buffer (pH 7.4, 30°C) that was bubbled with a 95% 02, 5% CO2mixture. The buffer contained 127 mM NaCl, 2.5 mM CaCl2, 2.3 B~MKCl, 25 mMNaHCO3, 1.3 mM KH2P04, 0.6 mM MgSO4, and 5.6 mM glucose. Baseline values of cardiac function were recorded after preparations had stabilized. In some studies, contractile function was monitored following addition of 500 ~ H202to the buffer. Soluble sulfhydryl content, glutathione peroxidase a c t i v i t y aria glutathione reductase a c t i v i t y were measured in thin right ventricular strips (100 - 300 mg, t y p i c a l l y less than I mmin thickness) that had been suspended in the buffer (as above). These strips were removed at 19, 35, and 75 minutes following addition of 500 p~MH202. Cardiac Function Studies Left a t r i a l strips were attached to an isometric force transducer (Kulite BG25) and e l e c t r i c a l l y stimulated (Grass S88) to contract isometrically using a square wave pulse (3 msec) at 10% above threshold voltage. Data were recorded on a Gould 2400S oscillographic recorder. Each s t r i p was stretched to a resting force of 0.5 g and allowed to stabilize for at least 30 minutes before determining baseline cardiac variables. Variables measured included resting force (RF), developed force (DF), maximum rate of force development ( i . e . , dF/dt, referred to as c o n t r a c t i l i t y ) , time interval from onset of contraction to peak force (TTPF), and time interval from peak force until DF had decreased
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by90%of i t s peak value (90% relaxation time; 90% RT). Measurementswere made at stimulation rates of I, 2, and 3 beats/second. Glutathione Studies Tissue glutathione from right ventricular free wall strips was assayed as soluble sulfhydryl (-SH) concentrations, using the method of Sedlack and Lindsay (16). This method is based on the reduction of Ellman's reagent ( d i t h i o - b i s nitrobenzoic acid) by sulfhydryl groups in the supernatant fraction to form a mercaptobenzoic acid with an intense yellow color (measured at 412 nm). Data were expressed as ~g soluble sulfhydryl content/g wet weight of tissue. Glutathione peroxidase a c t i v i t y was assessed in right ventricular free wall strips using the technique of Paglia and Valentine (14) with more recent modifications (8,15). Tissue samples were added to 0.25 M sucrose (4°C) to obtain concentrations of 10%, 5% and 2.5%w/v. Homogenateswere centrifuged at 9,000 x g for 20 minutes; supernatant was removed and recentrifuged at 9,000 x g for 20 minutes. Supernatant (0.05 ml) was vortexed with 0.95 ml of buffer (pH 7.5) containing final concentrations of 24 mMHEPES, 1.15 mMEDTA, 0.46 mMNaN3 and excess GSH (1.0 mM), NADPH (0.2 mM) and glutathione reductase (1.0 units). The mixture was allowed to stand at room temperature for 10 minutes. The reaction was started by addition of 50 #l of 5 mM H20z solution. Oxidation of NADPH was assessed every 15 seconds for 1.5 minutes at 340 nm to obtain glutathione peroxidase a c t i v i t y expressed as nmol NADPH oxidized/min per mg protein. Glutathione peroxidase a c t i v i t i e s for 10%, 5% and 2.5% tissue-sucrose concentrations were combined and averaged. A control assay was obtained using buffer without homogenate to correct for non-enzymatic oxidation of NADPH. Glutathione reductase a c t i v i t y was assessed in right ventricular free wall strips using the technique described by Massey and Williams (11). Tissue was homogenized and the supernatant obtained as described for the glutathione peroxidase assay. Supernatant (50 #l) was added to 0.95 ml buffer (pH 7.5; final concentrations of 40 mM K~HPO 4, 2.0 mM EDTA, 4 mM oxidized glutathione [GSSG] and 1.0 mM FAD+), vortexe~ and allowed to stand at room temperature for 10 minutes. The assay for glutathione reductase a c t i v i t y was i n i t i a t e d by addition of 50 #l of 2.0 mMNADPH, and oxidation of NADPHwas assessed every 15 seconds for 1.5 minutes at 340 nm; glutathione reductase a c t i v i t y was expressed as nmol NADPHoxidized/min per mg protein. Glutathione reductase a c t i v i t i e s for tissue/sucrose concentrations of 10%, 5%, and 2.5% w/v were combined and averaged as described in the glutathione peroxidase assay. A control assay was obtained using buffer without homogenate to correct for non-enzymatic oxidation of NADPH. Protein assay was performed by the Bradford technique (2). Statistical Methods Cardiac function data w i t h i n groups were analyzed using randomized block analysis of variance with Duncan's New M u l t i p l e Range t e s t . Between group comparisons employed one way analysis of variance with Duncan's New M u l t i p l e Range t e s t . Data obtained from studies assessing soluble s u l f h y d r y l concentrations and a c t i v i t i e s of GSH peroxidase and GSH reductase were analyzed by one way analysis of variance and Student-Neuman-Keuls t e s t . Effects of H2O2 on c o n t r a c t i l e function in young and senescent rabbits (Table I) were compared using Students unpaired t - t e s t with correction for unequal sample sizes and unequal variances. The n u l l hypothesis was rejected i f P < 0.05.
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Vol. 48, No. 18, 1991
Results Effects of frequency of contraction on changes in resting force (RF; changes in muscle stiffness), developed force (DF; strength of contraction) and dF/dt ( c o n t r a c t i l i t y ) of atria from adult and senescent rabbits are shown in figure I. There were no age-related differences in RF or dF/dt at any contraction frequency. Developed force was similar in the two groups at I contraction/sec (adult was 0.46 ± 0.06 g; senescent was 0.72 ± 0.17 g) but was s i g n i f i c a n t l y greater in senescent than young adult atria at higher frequencies (1.76 ± 0.28 g vs. 0.95 ± 0.10 g at 2 contractions/sec and 1.93 ± 0.29 g vs. 1.01 ± 0.11 g at 3 contractions per second). Cardiac relaxation time (90% RT) was unaffected by senescence at a contraction frequency of I/sec (figure 2). However, as contraction frequency increased, 90% RT was s i g n i f i c a n t l y shortened (P < 0.01) in adult (106 ± 3 msec at I beat/sec vs. 96 ¢ 3 and 93 ± 3 msec at 2 and 3 beat/sec, respectively) but not senescent atria (113 ± 8, 120 ± 8 and 111 ± 6 msec at I, 2 and 3 contraction/sec respectively). Direct comparisons between adult and senescent atria revealed s t a t i s t i c a l l y significant differences in 90% RT at frequencies of 2 beats/sec (96 ± 3 msec vs 120 ± 8 msec; p < 0.01) and 3 beats/sec (93 ¢ 3 msec vs. 111 ± 6 msec; p < 0.05) (figure 2). Time to peak force (TTPF) was shorter in adult than senescent atria (P < 0.01) at all contraction frequencies (adult = 78 ± 3, 74 ± 3 and 71 ± 3 msec; senescent = 92 ± 5, 89 ± 3 and 85 ¢ 3 msec at I, 2 and 3 beats/sec, respectively; figure 2). Treatment with H)O2 caused striking alterations in resting force and developed force (dF/dt, TTPFand 90% RT were not measured in these experiments, owing to exclusive use of slow oscillographic tracings). The time interval between addition of H202 and the f i r s t detectable change in either RF or DF was defined as "onset latency" ( i n i t i a l changes in RF and DF occurred simultaneously in all preparations). In several preparations, increases in resting force and decreases in developed force culminated in contracture (high resting force without force development). The time interval from addition of H202 to the maximal (peak) H20~effect was termed "peak latency." Onset latency was 6 times greater in adult than senescent atria (19.0 ± 1.5 min vs. 3.2 ± 0.9 min; P < 0.001) (table I ) , despite similar peak latencies in both groups (33.6 ± 5.8min in adult atria, 29.0 ± 1.0 min in senescent a t r i a ) . Maximal increases of RF were comparable in the two groups (adult = 75 ± 11% and senescent = 89 ± 6% of pre H~O2 values), but maximal depressions of D~ were approximately 2 times greater-in adult than in senescent atria (adult 81 ± 6% and senescent = 42 ± 27% of pre HzOz values; P < 0.05). Concentration of soluble sulfhydryl groups and a c t i v i t i e s of glutathione peroxidase and reductase were similar in senescent and adult rabbit heart (figure 3). Following addition of 500 /d~ H202, soluble sulfhydryl concentrations decreased comparably in both age groups during the 75 minute study period. In adult heart, soluble SH levels had decreased from the baseline by 22 ± 3%, 34 ± 4% and 38 ± 8% at 19 min, 35 min and 75 min after H202, and by 32 ± 4%, 34 ± 15% and 33 ± 9% in senescent heart at the above times. Basal glutathione peroxidase a c t i v i t i e s were comparable in adult and senescent heart (47 ± 3 vs. 50 ± 4 nmol NADPH/min per mg protein, respectively). H202did not s i g n i f i c a n t l y alter glutathione peroxidase a c t i v i t i e s in either age group (figure 3). Similarly, basal glutathione reductase a c t i v i t i e s did not d i f f e r between adult and senescent preparations (27 ± 2 vs. 25 ± 3 nmole NADPH/min per mg protein) andeH20~thdid not alter glutathione reductase a c t i v i t y of either age group (with exception of a decrease in a c t i v i t y at 75 minutes in the senescent animals).
Vol.
48,
No.
18,
1991
Cardiac
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FIG. 1. Effects of age on resting force (Panel A), developed force (Panel B), and dF/dt (Panel C) in l e f t atrial strips from young adult (open circles, n = 10) and senescent (closed circles, n = g) r~bbits. S t u d i e s w e r e performed at ], 2, and 3 contractions/second. Circles and bars show mean ± SE. **P < 0.001 for young vs. old preparations. Discussion The present study provides the f i r s t evidence that senescence alters the function of the rabbit heart. Age-related alterations of basal myocardial function (figures I and 2) in rabbits were similar to those previously reported in the rat (4, 10). For example, aging did not alter myocardial contractility (dF/dt; figure 1) but did impair cardiac relaxation (TTPF, 90% RT, figure 2). Relaxation was enhanced as frequency of contraction increased in adult but not senescent atria (figure 2). Senescent atria developed more force than adult atria, primarily reflecting prolongation of contraction rather than an alteration of the inotropic state (DF = dF/dt x TTPF) Thus, impairment of relaxation was the most prominent age-related alteration of myocardial function. Effects of aging on cardiac function may relate to alterations of calcium metabolism. Aging does not appear to affect the rate of calciumdeliveryto the myofibrillar apparatus (12), which may account for an absence of age-related effects on contractility (dF/dt; figure I; Ig). However, aqueorin studies indicate that aging prolongs the time to attainment of peak cytoplasmic calcium level and slows the decline from the peak level (5). This may be explained by a prolongation of the transmembrane action potential or a decrease in velocity of calcium uptake and Ca-Mg ATPase a c t i v i t y of cardiac SR. Whatever the mechanism, a prolonged availability of calcium for contraction (i.e. increased TTPF) allowed more time for senescent hearts to develop force (increased DF,
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Cardiac Function and Aging
Vol. 48, No. 18, 1991
figure 1). The age-related impairment of cardiac relaxation was most evident at higher contraction frequencies (figure 2), suggesting that aging decreases the reserve of mechanisms that clear the increased cytoplasmic calcium resulting from increased heart rate. Altered calcium handling with aging may relate to an increased exposure to oxidative stresses that cause dysfunction of SR ( I ) . A g i n g may impair mechanisms that scavenge free radicals, predisposing to free radical injury. The glutathione pathway plays an important protective role against oxidative stress by detoxifying oxidants via a reaction that converts GSH to GSSG. Age-related declines in glutathione peroxidase a c t i v i t y , glutathione reductase a c t i v i t y and GSH have been reported (7,8,17). A deficiency of GSH could predispose to an increased proportion of enzymes with oxidized sulfhydryl groups, such as those on calcium release channels of SR. As a result, a residual leakage of calcium from terminal cisternae may occur (18). Alternatively, aging may increase calcium leakage into the cytoplasm via mechanisms independent of oxidative stresses or by mechanisms involving oxidative injuries that f a i l to perturb the glutathione system (e.g., l i p i d peroxidation of membranes). The mechanical consequencesof enhancedcytoplasmic calcium influxes would be magnified by a coexisting impairment of calcium sequestration.
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FIG. 2. Effects of age on 90% relaxation time (go% RT) and time to peak force (TTPF) in l e f t atrial strips from young adult (open circles; n = 10) and senescent (closed circles; n = g) rabbits. Studies were performed at I, 2, and 3 contractions/second. Circles and bars show mean ± SE. *P < 0.05 and **P < 0.01 for young vs. old preparations.
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TABLE I Effects of H202 (500 #m) on Function of Left Atrial Strips from Young Adult and Senescent (Old) Rabbits. GROUPS
Baseline8
Latencyb
Peak Effect
RF
DF
Onset
Peak
RF
(g)
(g)
(min)
(min)
(g)
Young Mean 0.53 (n,14) ± SE 0.02
0.41 0.05
19.0 1.5
33.6 5.8
0.91 0.06
74.9 1).3
0.08 0.03
81.1 5.8
Old (n,3)
0.53 0.17
29.0 1.0
0.87 0.04
89.0 5.5
0.32" 0.18
42.3" 27.0
Mean 0 . 4 6 ± SE 0 . 0 3
3.2"* 0.9
Increasec DF in RF (%) (g)
Decrease c in DF (%)
aContraction frequency of I/sec. bTime intervals between addition of HzO2 and i n i t i a l changes (onset latency) or maximal changes (peak latency) in function. CPercentage change from baseline. *P < 0.05, and **P < 0.001 for differences between age groups. This study determined effects of aging on the glutathione pathway in cardiac tissue subjected to an oxidant stress. The glutathione pathway was evaluated in right ventricular free wall preparations prior to and after exposure to HzO2. (Thin right ventricular free wall strips rather than atrial preparations were used to obtain sufficient quantities of tissue for assays of glutathione pathways). Basal GSH concentrations and glutathione peroxidase and reductase activities did not d i f f e r between the two groups, suggesting, that in our rabbit model, the capacity to metabolize peroxides by this pathway was unaffected by age. When the glutathione pathway was challenged by treatment with H20z, GSH concentrations decreased comparably in both age groups (figure 3). In contrast, the effects of HzOz on myocardial function were highly age-dependent (tablel). Treatment with HzO2 increased resting force and decreased developed force in both senescent and-adult atria, but these changes were accelerated in senescent atria (3 ± I vs. 19 ± 2 min respectively; P < 0.001). This suggests that aging increases susceptibility to oxidative injury. Developed force, however, was decreased more in adult than senescent atria (81 ± 6% vs. 42 ± 27% decrease from pre-HzO z value), despite similar maximum increases of RF {75 ± II vs. 89 ± 6% increase from pre-HzO ~ values) in the two groups. Thus, advanced age may be associated with adaptlve mechanisms that better preserve contractile function in the presence of an oxidative stress. The subcellular mechanism of the rapid onset of H202 toxicity in senescent atria may involve the release of large quantities of calcium from terminal cisternae, coupled with a severely limited capacity to sequester calcium into longitudinal SR. In summary, the primary effect of aging on myocardial function was an impairment of r e l a x a t i o n r a t h e r than c o n t r a c t i l i t y . Senescent a t r i a did not r e l a x as r a p i d l y as adult a t r i a when frequency of c o n t r a c t i o n was increased. An o x i d a t i v e stress (H2Oz) caused a more rapid onset of t o x i c i t y in senescent than in adult a t r i a . However, senescent a t r i a were b e t t e r able than adult a t r i a to maintain c o n t r a c t i l e f u n c t i o n in the presence of an o x i d a t i v e stress.
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Age-related differences in t o x i c i t y of HzOz were probably unrelated to detoxification capacity of the glutathione pathway because H20z comparably decreased soluble sulfhydryl concentrations in both age groups. Further studies w i l l be required to elucidate mechanisms by which aging alters effects of an oxidative stress and modifies the force-frequency relationship.
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FIG. 3 Effects of 500 #M HzOz on soluble sulfhydryl concentrations (panel A), glutathione peroxidase a c t i v i t y (panel B) and glutathione reductase a c t i v i t y (panel C) of right ventricular strips from young adult (open bars) and senescent (closed bars) rabbits. Bars represent mean ± SE. The number of preparations per group is shown inside bars. *P < 0.05, and**P < 0.01 for baseline (time = O) vs. post-treatment values within age groups. Acknowledqements The authors would l i k e to thank Gloria Kent and Sally Sellers for preparation of the manuscript; Steve Young, Joni Strander and Matthew Parks for expert technical assistance. This work was supported by grants from the Department of Veteran's Affairs and the American Federation for Aging Research, Inc. References 1. 2. 3.
J. ABRAHAHSONand G. SALAMA, Mol. C e l l . Biochem. 8 2 : 8 1 - 8 4 (1988). M.M. BRADFORD, Anal. Biochem. 7 2 : 2 4 8 - 2 5 4 (1976). D.G. BRUNDER, C. DETTBARN and P. PALADE, J. B i o l . Chem. 263: 18785-18792 (1988).
4.
J . M . CAPASSO, A. MALHOTRA, R.M. REMILY, J. SCHEUER and E.H. SONNENBLICK, Am. J. Physiol. 245:H72 - H79 (1983). J.P. FROELICH, E.G. LAKATTA, E. BEARD, H.A. SPURGEON,M.L. WEISFELDT and G. GERSTENBLITH, J. Mol. Cell. Cardiol. ]0:427-438 (1978). S. GOLDSTEIN, N. Engl. J. Med. 285:1120-1121 (1971).
5. 6.
Vol. 48, No. 18, 1991
7. 8. 9. 10. 11. ]2. 13. 14. 15. ]6. 17. ]8. 19. 20.
Cardiac Function and Aging
G.A. HAZELTON and C.A. LANG, Biochem. J. ]88:25-30 (1980). G.A. HAZELTON and C.A. LANG, Mech. Age Dev. 29:71-81 (]985). E.G. LAIC~TTAand F.C.P. YIN, Am. J. Physiol. 242:H927-H941 (1982). E.G. LAKATTA, G. GERSTENBLITH, C.S. AGNELL, N.W. SHOCK, and M.L. WEISFELDT, J. Clin. Invest. 5_55:61-68 (1975). V. MASSEYand C.H. WILLIAMS, J. Biol. Chem. 240:]58-]69 (1965). D.W. MAUGHAN, E.S. LOW and N.R. ALPERT. J. Gen. Physiol. 71:431-451 (1978). N. NARAYANA, Biochem. Biophys. Acta 678:442-459 (1981). D.E. PAGLIA and W.N. VALENTINE, J. Lab. Clin. Med. 70:158-169 (1967). J.R. PROHASKA, S-H OH, W.G. HOKSTRA and H.E. GANTHER, Biochem. 8iophys. Res. Comun. 74: 64-7] (1977). J. SEDLAK and R.H. LINDSAY, Anal. Biochem. 25:192-205 (]968). J, STOHS, J.N. HASSING, W.A. AL-TURK and A.M. HASOUD, Age 3 : 1 1 - 1 4 (1980). J.L. TRINH, G. SALAMAand J.J. ABRAMSON, J. Biol. Chem. 261: ]609216098 (]986). G.D. WALFORD, H.A. SPURGEONand E.G. LAKATTA, Circ. Res. 63:502-511 (1988). M.L. WEISFELDT, E.G. LAKATTA and G. GERSTENBLITH, in Heart Disease, Ed. E. Braunwald, p. 1650-1662, Saunders, Philadelphia (1988).
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