Mechanisms of positive inotropic action of flosequinan, hydralazine, and milrinone on mammalian myocardium.

Flosequinan is an arterial and venous dilator that also has a positive inotropic effect at relatively higher doses. The purpose of this study was to determine the mechanism of this positive inotropic effect in ferret papillary muscles loaded with the Ca2+ indicator, aequorin. Over the range of doses from 10(-6) to 10(-3) M, flosequinan produced a 61 +/- 9% increase in peak tension that was accompanied by a corresponding increase in [Ca2+]i. This positive inotropic effect was not selectively blocked by addition to the perfusate of procaine 0.6 microM, tetrodotoxin 10(-6) M or by verapamil, 5 x 10(-8) M. In contrast, the positive inotropic effect of flosequinan, but not milrinone or hydralazine, was potentiated by prior addition of ouabain 3 nM to enhance intracellular Ca2+ via reduction of the Na+/Ca2+ exchange. Moreover, antagonists of Na+/Ca2+ exchange, including cadmium 10 microM, amiloride 600 microM and choline substitution for 1/3 Na+ in the perfusate, blocked the response to flosequinan but not hydralazine or milrinone. These results indicate that flosequinan produces a positive inotropic effect by reduction of Na+/Ca2+ exchange in mammalian myocardium. Moreover, flosequinan has the potential to interact synergistically with other positive inotropic agents such as digoxin that affect Na+/Ca2+ exchange by direct or indirect actions.

Endothelin-1 does not alter Ca2+ responsiveness in saponin-skinned ferret papillary muscles.

Endothelin is a potent vasoconstrictor and a positive inotropic agent in myocardium. Endothelin has been reported to increase myocardial contractility with little or no increase in intracellular Ca2+, thus apparently enhancing myofilament responsiveness to Ca2+. We investigated the effects of endothelin on tension development and Ca2+ responsiveness in both intact and saponin-skinned ferret right ventricular papillary muscles. Isolated ferret papillary muscles were stimulated for 2 h in the presence or absence of endothelin (100 nM). The muscles were then chemically skinned with saponin and exposed to relaxing and contracting solutions containing varying amounts of Ca2+, and the developed force of contraction was measured. The [Ca2+] required for half-maximal activation (pCa50) was determined by fitting force versus Ca2+ data to the Hill equation. In isometrically contracting muscles, endothelin (100 nM) caused a mean percent increase in developed tension of 34.7% +/- 11.3% (mean +/- S.E.). In muscles that were exposed to endothelin for 2 h and then skinned, neither the pCa50 nor the maximal Ca(2+)-activated force (Fmax) were significantly different from control skinned papillary muscles. After skinning, when endothelin (100 nM) was added to the Ca2+ buffers, both pCa50 and Fmax were significantly decreased. When papillary muscles were pretreated with phorbol 12-myristate 13-acetate (PMA) and then skinned, there was a significant increase in the pCa50. These results indicate that endothelin acts directly on the myofilaments to impair force development by directly decreasing the Ca2+ responsiveness of myofilaments.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone effects on intracellular calcium and inotropic responses of rat ventricular myocardium.

To examine the mechanisms by which thyroid hormone modulates the inotropic state of rat myocardium, we studied the effects of thyroid state on isolated rat left ventricular papillary muscle function and intracellular calcium transients in the baseline state and in response to calcium and isoproterenol. Marked differences in contractile state of papillary muscles from hypothyroid and thyroid hormone-treated rats seen under baseline conditions (1.0 mM bath calcium, 30 degrees C, stimulation rate 12/min) do not appear to be due to differences in intracellular calcium concentration ([Ca2+]i) or to changes in myofilament calcium sensitivity but correlate with shifts in myosin isozyme distribution. In response to superimposed inotropic interventions (calcium, 0.625-5.0 mM, or isoproterenol, 10(-8)-10(-6) M), myocardial thyroid state modulates peak [Ca2+]i and inotropy, both of which are increased in thyroid hormone-treated relative to hypothyroid myocardium. The change in inotropy appears to be proportional to peak [Ca2+]i, whether mediated directly by calcium or as a result of beta-adrenergic stimulation. Thus, whereas baseline differences between hypothyroid and thyroid hormone-treated myocardium appear to be due to differences in myosin isozymes and presumed changes in adenosinetriphosphatase activity and cross-bridge cycling, superimposed inotropic responses appear to be mediated by changes in [Ca2+]i.

Excitation-contraction coupling in isolated myocardium from dogs with compensated left ventricular hypertrophy.

To examine the relationship between left ventricular (LV) function and intracellular calcium modulation in the presence of myocyte hypertrophy, we compared LV muscles from nine dogs with compensated LV hypertrophy (LVH) induced by chronic aortic banding with 11 controls. Mechanical properties were studied in LV muscles (control, n = 25; LVH, n = 23) stretched to the length at which maximal isometric tension developed at 30 degrees C and stimulated at 0.33 Hz; a subset of LVH muscles was loaded with the intracellular calcium indicator aequorin. In LV myocardium from dogs with compensated LVH, both the contraction duration and calcium transients were prolonged at baseline, and the response to phosphodiesterase inhibitors was impaired in keeping with findings in both human and experimental models of pressure-overload hypertrophy and failure. However, in contrast to findings in the failing myocardium, the positive inotropic response to increasing intracellular calcium was preserved in myocardium from dogs with compensated LVH.

Alterations in contractility and intracellular Ca2+ transients in isolated bundles of skeletal muscle fibers from rats with chronic heart failure.

To determine if chronic heart failure (CHF) leads to functional or structural alterations of skeletal muscle, we compared intracellular Ca2+ signaling, contractility, and the rate of fatigue development, together with electron microscopy (EM), in skeletal muscle preparations from rats with myocardial infarction-induced CHF versus sham-operated control rats. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control (n = 13) and CHF (n = 19) rats and were either loaded with aequorin or fixed for EM. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches (1.4 +/- 0.2 versus 2.8 +/- 0.7 g/mm2, P < .05) and maximal tetani (5.3 +/- 1.4 versus 10.7 +/- 2.4 g/mm2, P < .05). Depressed tension in CHF was accompanied by reduced quantitative [Ca2+]i release during twitches (0.7 +/- 0.1 versus 0.4 +/- 0.1 microM, P < .05) and during maximal tetani (1.8 +/- 0.3 versus 0.9 +/- 0.2 microM, P < .05). Skeletal muscle from CHF rats also demonstrated prolonged intracellular Ca2+ transients during twitches and tetani and accelerated fatigue development. EM revealed a lack of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling unrelated to cellular atrophy. These findings indicate that CHF is a condition accompanied by EDL skeletal muscle dysfunction.

Cellular mechanism of the positive inotropic effect of hydralazine in mammalian myocardium.

1. The purpose of this study was to elucidate the cellular mechanism of the positive inotropic effect of hydralazine, a vasodilator widely used for afterload reduction in patients with heart failure that has also been reported to have positive inotropic effects on the heart. After isolation, right ventricular papillary muscles from the ferret were maintained in bicarbonate-buffered salt solution (30 degrees C). A concentration-response relationship was obtained for hydralazine (10(-6) to 10(-3) M). In order to mimic different levels of catecholamine release found in heart failure, we utilized two methods of stimulation: (a) threshold punctate pulses and (b) suprathreshold punctate stimulation with voltage approximately 10% above threshold. 2. In a first group of muscles (n = 16), a maximally effective concentration of hydralazine (10(-3) M) increased peak isometric tension by 39 +/- 9% (P < 0.05). Doses lower than 10(-5) M had no significant effect. The bioluminescent Ca2+ indicator, aequorin, was loaded into a subset of these muscles (n = 7). A significant increase in peak light (i.e., intracellular Ca2+) developed, concurrently with an increase in peak tension (38 +/- 5% to 66 +/- 8%). This inotropic response was associated with a decrease in time to peak tension (ms), 221 +/- 7 to 186 +/- 5 (P < 0.05), and time to peak light, 65 +/- 4 to 52 +/- 2 (P < 0.05). These effects were markedly attenuated by pretreatment with autonomic blocking agents. 3. In a second group of muscles (n = 12), histamine was used to stimulate cyclic AMP production in the presence of propranolol. Hydralazine (3 x 10-4 M) led to a shift in the pD2 (i.e. the negative log of the concentration of histamine producing 50% of the maximal response) from 6.1 +/- 0.1 to 5.9 +/- 0.1(P <0.05), thus increasing the sensitivity of the muscles to histamine. Hydralazine also increased maximum tension from 160 +/- 77% to 195 +/- 57% (P <0.05) above baseline. Thus, hydralazine altered the potency and efficacy of histamine despite the presence of beta-adrenoceptor blockade.4. A third group of muscles were chemically skinned to examine the effects of hydralazine on myofilament Ca2+ responsiveness. Pretreatment of ferret papillary muscles with hydralazine (10-3 M)before skinning did not shift the force-pCa curve after skinning (n = 16). However, hydralazine added to previously skinned fibres desensitized the myofilaments, as indicated by a rightward shift of the force-pCa curve (n = 12). Maximum tension development was not changed.5. The pharmacological effects of hydralazine are characteristic of inotropic drugs that act mainly via cyclic AMP; however, the increase in peak tension demonstrated with histamine in the presence of hydralazine also suggests an effect on cyclic AMP-independent second messenger pathways. These data are consistent with reports that large doses of hydralazine may increase cellular levels of cyclic AMP, as well as other second messengers, by direct cardiac and indirect neuronal mechanisms.

Negative inotropic and relaxant effects of cocaine on myopathic human ventricular myocardium and epicardial coronary arteries in vitro.

OBJECTIVE: Cocaine produces both vascular and myocardial effects that can lead to serious cardiovascular complications in man. Tissue catecholamine stores are known to be depleted in the advanced stages of heart failure. The effects of cocaine on cardiac and coronary smooth muscle isolated from patients with end stage heart failure was tested in order to evaluate the direct actions of this drug on human tissue. METHODS: Effects of cocaine HCl were studied on cardiac ventricular trabeculae carneae and epicardial coronary artery segments obtained at heart transplantation from patients with end stage heart failure. Muscles were placed in organ baths under physiological conditions for recording isometric tension; a subset of muscles was loaded intracellularly with the bioluminescent calcium indicator, aequorin. Cardiac muscles were stimulated with threshold pulses delivered via a punctate electrode at 0.33 Hz. Coronary segments were studied under basal conditions and after contraction with 60 mM KCl. RESULTS: In both cardiac muscle and vascular smooth muscle preparations, cocaine (10(-6)-10(-3) M) produced dose related negative inotropic and relaxant effects, respectively; positive inotropic actions and vasoconstriction were not seen. In cardiac muscle, the negative inotropic actions were associated with a simultaneous decrease in peak intracellular calcium levels. In contrast, cocaine induced relaxation of potassium contracted vascular smooth muscle was not associated with a fall in peak intracellular calcium levels, a result consistent with decreased myofilament calcium responsiveness. CONCLUSIONS: These results indicate that the depressant effects of cocaine on cardiac versus vascular smooth muscle occur by different mechanisms and suggest the need for specific therapeutic approaches to managing cardiac depression versus vasodilation when these occur in cocaine intoxicated patients.

Cellular basis of negative inotropic effect of 2,3-butanedione monoxime in human myocardium.

2,3-Butanedione monoxime (BDM) exerts a marked negative inotropic effect and has been shown to have protective actions on human myocardial force production that may be of clinical use. To determine the underlying mechanisms, we studied the effects of BDM on chemically skinned and aequorin-loaded myopathic human myocardium from transplant recipients. Eighteen muscles were chemically skinned with saponin (250 micrograms/ml) and then subjected to activation-relaxation cycles, with and without 5 mM BDM. Contracture force vs. Ca2+ data were fitted to a modified Hill equation, and values for 50% maximal activation (pCa50) and maximal Ca(2+)-activated force (Fmax) were obtained. pCa50 was decreased by 0.2 pCa units, indicating myofilament Ca2+ desensitization, and Fmax was reduced by 48% in 5 mM BDM. A second group of intact muscles (n = 8) was loaded with aequorin to monitor intracellular calcium (Cai2+) transients (peak light) and twitch force in the presence of BDM (1-30 mM). Over a range of 1-20 mM, BDM depressed peak light by 3-49% while force was depressed by 10-82%. This was accompanied by an abbreviation of the duration of the twitch but not of the Cai2+ transient. At a concentration of 30 mM, BDM completely inhibited force generation, but an Cai2+ transient was still present. We conclude that in human myocardium, 5 mM BDM predominantly affects cross-bridge force production and Ca2+ sensitivity and has a less pronounced effect on Cai2+.

Differential inotropic effects of flosequinan in ventricular muscle from normal ferrets versus patients with end-stage heart failure.

1. In right ventricular papillary muscles from control ferrets, flosequinan (10(-7)-10(-4) M) produced a concentration-dependent positive inotropic effect (10(-5) M = 153 +/- 24, 10(-4) M = 198 +/- 44% increase in isometric tension; control tension = 100%; n = 11) associated with a corresponding increase in amplitude of the intracellular Ca2+ ([Ca2+]i) transient recorded with aequorin (10(-5) M = 133 +/- 11, 10(-4) M = 187 +/- 36% increase in [Ca2+]i transient; n = 11). 2. The positive inotropic effect of flosequinan in control ferret ventricular muscle was neither blocked by propranolol (6 x 10(-7) M), nor associated with the abbreviation of the [Ca2+]i transient and contraction that is typical of catecholamines. 3. Neither flosequinan (n = 12) nor BTS 53 554, its sulphone metabolite (n = 6) produced a positive inotropic effect or altered the time course of contraction in myocardium from the hearts of patients with end-stage failure. 4. In contrast to milrinone, which produces a positive inotropic effect via phosphodiesterase inhibition, the unresponsiveness of myopathic human myocardium to flosequinan was not restored after intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were increased by prior treatment with forskolin (n = 13). 5. Taken together, these data indicate that flosequinan has a direct positive inotropic effect that is Ca(2+)-dependent, but independent of changes in intracellular cyclic AMP concentrations. 6. The positive inotropic effect may be species-dependent or altered by the presence of hypertrophy and/or heart failure. However, when used therapeutically in patients with severe heart failure, our data suggest that flosequinan should not adversely affect myocardial oxygen consumption through direct or catecholamine-mediated actions on the heart.

Effects of endothelin on intracellular Ca2+ and contractility in single ventricular myocytes from the ferret and human.

We investigated the role of endothelin-1 on peak intracellular Ca2+ ([Ca2+]i) and peak shortening of ventricular myocytes (loaded with indo-1/AM) from failing human hearts. 10 nM of ET-1 significantly increased the cell peak shortening (84 +/- 29%, P less than 0.05) without significantly increasing the peak [Ca2+]i (15 +/- 7%, P greater than 0.05). Further studies on ferret cardiac myocytes indicated that in addition to producing dose-dependent (0.1-10 nM) significant increases in peak shortening (max 55 +/- 6% P less than 0.01) and non-significant increases in peak [Ca2+]i (max 35 +/- 19%, P greater than 0.05), endothelin-1 significantly shifted the peak [Ca2+]i-peak shortening curve upward. The results suggest that endothelin-1 acts directly on human and ferret cardiac myocytes to produce a positive inotropic effect that may predominantly be due to an enhanced myofilament Ca2+ responsiveness.

Abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure.

24 d of rapid ventricular pacing induced dilated cardiomyopathy with both systolic and diastolic dysfunction in conscious, chronically instrumented dogs. We studied mechanical properties and intracellular calcium (Ca2+i) transients of trabeculae carneae isolated from 15 control dogs (n = 32) and 11 dogs with pacing-induced cardiac failure (n = 26). Muscles were stretched to maximum length at 30 degrees C and stimulated at 0.33 Hz; a subset (n = 17 control, n = 17 myopathic) was loaded with the [Ca2+]i indicator aequorin. Peak tension was depressed in the myopathic muscles, even in the presence of maximally effective (i.e., 16 mM) [Ca2+] in the perfusate. However, peak [Ca2+]i was similar (0.80 +/- 0.13 vs. 0.71 +/- 0.05 microM; [Ca2+]o = 2.5 mM), suggesting that a decrease in Cai2+ availability was not responsible for the decreased contractility. The time for decline from the peak of the Cai2+ transient was prolonged in the myopathic group, which correlated with prolongation of isometric contraction and relaxation. However, similar end-diastolic [Ca2+]i was achieved in both groups (0.29 +/- 0.05 vs. 0.31 +/- 0.02 microM), indicating that Cai2+ homeostasis can be maintained in myopathic hearts. The inotropic response of the myopathic muscles to milrinone was depressed compared with the controls. However, when cAMP production was stimulated by pretreatment with forskolin, the response of the myopathic muscles to milrinone was improved. Our findings provide direct evidence that abnormal [Ca2+]i handling is an important cause of contractile dysfunction in dogs with pacing-induced heart failure and suggest that deficient production of cAMP may be an important cause of these changes in excitation-contraction coupling.

A chemical method for intracellular loading of the calcium indicator aequorin in mammalian skeletal muscle.

The bioluminescent calcium indicator aequorin was loaded into bundles of skeletal muscle fibers from the rat extensor digitorum longus by macroinjection, a technique previously applied only to cardiac muscle. After loading, the amplitude and time course of the twitch returned to control values, indicating lack of damage to the fibers. Individual light signals (i.e., calcium transients) were recorded during each twitch or tetanus without the need for signal averaging. The calcium transients obtained were qualitatively and quantitatively similar to those reported previously with microinjection of aequorin. Our data suggest that macroinjection may be the method of choice for loading aequorin into mammalian skeletal muscle.

Pathophysiology of cardiac hypertrophy and failure of human working myocardium: abnormalities in calcium handling.

Abnormal intracellular calcium ([Ca2+]i) handling appears to be a major cause of both systolic and diastolic dysfunction in animals and human beings with hypertrophy and/or heart failure. We utilized the bioluminescent calcium indicator aequorin to examine the cyclical variations in intracellular calcium levels during isometric contractions. Studies of ventricular muscle from patients with end-stage heart failure exhibited three physiologic findings not seen in preparations taken from normal hearts including: 1) abnormalities in calcium handling; 2) deficient production of cyclic AMP; and 3) a reversed force-frequency relationship. These observations have important implications with regard to the pathogenesis and therapeutics of heart failure in man.

Differential activation of myofibrils during fatigue in phasic skeletal muscle cells.

In fatigued muscles the T-system is swollen; thus the action potential may fail to travel along the T-system or the T-tubule terminal cisternae signal may fail to bring about TC Ca2+ release. This would lead to a decrease in the number of myofibrils activated and in force development, but if fatigue is the result of a generalized process, all the myofibrils would be affected equally leading to a lower activation of all of them. We have investigated this possibility in isolated twitch muscle fibres by giving them repetitive tetanic stimulations until fatigue developed. The behaviour of myofibrils was followed with cinemicrophotography. Before fatigue, no lack of shortening of myofibrils could be found. During fatigue groups of myofibrils became wavy. When exposed to caffeine, the wavy myofibrils disappeared and tension similar to the control developed. The tension-caffeine concentration relationship was shifted to the left after development of fatigue. In low Na+ solution fatigue developed faster and after reintroducing normal Ringer, tension recovered substantially. K-contractures were smaller during fatigue. These results indicate that in this type of fatigue, a step in the EC coupling chain of events is involved in its development.

Intracellular calcium transients in myocardium from spontaneously hypertensive rats during the transition to heart failure.

To investigate the mechanism of impaired myocardial function after long-term pressure overload, we studied cardiac muscle mechanical contraction and intracellular calcium transients using the bioluminescent indicator aequorin. Left ventricular papillary muscle preparations were examined from three groups of rats: 1) aging spontaneously hypertensive rats (SHR) with clinical and pathological evidence suggesting heart failure (SHR-F group), 2) age-matched SHRs with no evidence of heart failure (SHR-NF group), and 3) age-matched normotensive Wistar-Kyoto rats (WKY group). Isometric force development was depressed in both SHR groups relative to the WKY group. Resting [Ca2+]i was lower in the SHR-F group, and the time to peak [Ca2+]i was prolonged in this group. The relative increases in peak [Ca2+]i with the inotropic interventions of increased [Ca2+]o and the addition of isoproterenol were similar among groups. Although inotropy increased in all groups with increased [Ca2+]o, after isoproterenol, inotropy increased only in the WKY group. Thus, in SHR myocardium, [Ca2+]i increased after isoproterenol, but inotropy failed to increase. Myosin isozymes were shifted toward the V3 isoform in both SHR groups; the V3 isoform was virtually 100% in papillary muscles from the SHR-F group. These changes may reflect events directly contributing to the development of heart failure or represent adaptive changes to chronic pressure overload and heart failure.

The cellular basis of contraction and relaxation in cardiac and vascular smooth muscle.

Changes in cytoplasmic levels of free-ionized calcium regulate the contraction and relaxation of cardiac and vascular smooth muscle. Aside from changing the intrinsic rate of energy use within the cell, an intervention that alters the strength of contraction of cardiac or vascular smooth muscle must in general alter either the intracellular calcium level, change the calcium requirements of the contractile apparatus, or exert both effects. Intracellular calcium handling is modulated by at least three other important second messengers: cyclic adenosine monophosphate, cyclic guanosine monophosphate, and inositol 1,4,5 triphosphate. These substances modulate intracellular calcium handling at multiple steps in the excitation-contraction coupling scheme. In vascular smooth muscle, diacylglycerol appears to play an important role in the regulation of myofilament calcium requirements. Intracellular calcium homeostasis is maintained through the action of sarcolemmal mechanisms that extrude calcium into the extracellular space; these mechanisms include a sodium-calcium exchange mechanism and an energy-dependent calcium pump. Abnormalities in electrophysiologic properties, sarcoplasmic reticulum function, energy use and supply, and contractile element interaction have been identified in experimental studies of animals and patients with various cardiovascular diseases. These abnormalities may be differentially affected by therapeutic agents that act on the heart or vasculature; therefore it is important to understand the underlying cellular abnormalities and subcellular actions of inotropic, vasoconstrictor, and vasodilatory drugs to apply rational therapeutics in the clinical setting.

Effects of cocaine on intracellular calcium handling in cardiac and vascular smooth muscle.

The inotropic and lusitropic (i.e., relaxant) actions of cocaine on the heart appear to be caused primarily by changes in intracellular calcium handling. The positive inotropic and lusitropic effects of low and moderate concentrations (i.e., less than or equal to 10(-5)M) are mediated by catecholamines; the negative inotropic effects of higher concentrations appear to be due to the direct local anesthetic effects of cocaine on excitation-contraction coupling mechanisms. The relevance of these findings to humans is suggested by the fact that blood levels of cocaine in excess of 10(-5) M have been described in patients abusing this drug (Van Dyke et al. 1976; Paly et al. 1982). Blood vessels from certain vascular beds, including the epicardial coronary arteries of humans and swine, show little or no constrictor response to low concentrations of cocaine and relax at higher concentrations. In contrast to the effects on the heart, the relaxant effects of cocaine on vascular smooth muscle appear to be related to marked changes in myofilament calcium responsiveness, which may be mediated by the protein kinase-C system. These results at least indicate that the depressant effects of cocaine on cardiac vs. vascular smooth muscle occur by different mechanisms and suggest the need for specific therapeutic approaches to managing cardiac depression vs. vasodilatation when they occur in cocaine-intoxicated individuals. Moreover, these data provide evidence supporting the hypothesis that the net effects of cocaine in the intact organism are highly dependent on the underlying level of sympathetic adrenergic activity.

The effects of cocaine on intracellular Ca2+ handling and myofilament Ca2+ responsiveness of ferret ventricular myocardium.

1. When ferret right ventricular papillary muscles were stimulated with threshold punctate pulses (0.33 Hz; 30 degrees C), cocaine, 10(-5) M, increased peak tension development from 815 +/- 120 to 1125 +/- 180 mg (P less than 0.05) and increased the rate of relaxation from peak tension (time to 80% decline from peak tension decreased from 155 +/- 11 to 144 +/- 11 ms; P less than 0.05). These changes in the twitch were associated with comparable changes in the amplitude and time course of the calcium transient measured with aequorin (amplitude increased from 62 +/- 4 to 90 +/- 7% (P less than 0.05) of maximal values; time to 80% decline from peak amplitude decreased from 84 +/- 8 to 64 +/- 3 ms; P less than 0.05). These effects were markedly attenuated in the presence of the beta-adrenoceptor-blocking agent, propranolol, 6 x 10(-7) M, or by maximization of catecholamine release from the adrenergic nerve endings with field pulses of suprathreshold strength, indicating that catecholamine release from the adrenergic nerve endings is responsible for the positive inotropic and lusitropic responses to low and moderate doses of cocaine (i.e., less than or equal to 10(-5) M). 2. High doses of cocaine (i.e., greater than 10(-5) M) produced negative inotropic and lusitropic effects that were associated with a decreased amplitude and prolonged duration of the calcium transient. 3. In aequorin-loaded intact fibres, cocaine 10(-5) M did not affect the force-calcium relationship unless catecholamines were present. Cocaine, 10(-5) M, significantly shifted the force-calcium relationship of saponin-skinned muscles (pCa50 = 6.14 +/- 0.05 versus 5.92 +/- 0.07; P less than 0.05), indicating reduced responsiveness of the myofilaments to calcium. F. (maximal Ca2+-activated force) was reduced to 58% of control in the presence of 10- M cocaine, while the slope of the calcium-force curve remained unchanged. These data indicate that cocaine may also decrease myofilament calcium sensitivity and maximal calciumactivated force, via mechanisms independent of catecholamines, when cellular diffusion barriers are eliminated.

Effect of exercise conditioning on excitation-contraction coupling in aged rats.

We studied aged (24-26 mo) Fischer 344 rats after they underwent 8 wk of moderate exercise conditioning. Right ventricular papillary muscles were loaded with the calcium indicator aequorin. Electrophysiological recordings were also performed. Time to peak isometric tension in muscles from exercised aged rats (EAR) was shorter than in those from unexercised aged rats (UAR) (126 +/- 7 vs. 167 +/- 7 ms; P less than 0.01). Time to 50% relaxation from peak isometric tension was also shorter in EAR than in UAR (88 +/- 3 vs. 119 +/- 12 ms; P less than 0.05). There was a trend toward decrease in time to peak light and a significant decrease in time to 50% decline from peak light (33 +/- 4 ms in EAR vs. 59 +/- 17 ms in UAR; P = 0.001). Action potential amplitude was smaller in EAR than in UAR (67 +/- 4 vs. 82 +/- 3 mV; P = 0.003); however, action potential duration was longer (137 +/- 6 ms in EAR vs. 100 +/- 10 ms in UAR; P = 0.005). Right ventricular-to-body weight ratios revealed no evidence of hypertrophy in EAR compared with UAR. Cardiac tissue norepinephrine content was significantly greater in EAR than in UAR (1,212 +/- 25 vs. 630 +/- 105 ng/tissue; P = 0.02). In summary, exercise reversed the age-related prolongation of isometric contraction and associated intracellular calcium transient in the aged rat while it prolonged the transmembrane action potential. In addition, exercise in aged rats resulted in an increase in cardiac norepinephrine content.

Differential effects of cardiac hypertrophy and failure on right versus left ventricular calcium activation.

We studied calcium responsiveness of skinned muscle preparations from the right and left ventricles of rats with cardiac hypertrophy and cardiac hypertrophy plus failure. To test the hypothesis that differences in contractile function are due to changes in myofilament calcium responsiveness, we compared preparations from spontaneously hypertensive rats with cardiac failure, spontaneously hypertensive rats without cardiac failure, and age-matched normotensive Wistar-Kyoto control rats 18-24 months of age. Rats with failure had pleural/pericardial effusions, left atrial thrombi, and right and left ventricular hypertrophy. Muscles were skinned by saponin (250 micrograms/ml) and activated with a series of calcium buffers. Data were plotted as pCa (-log[Ca2+]) versus isometric force and then fit to a modified Hill equation. Values for 50% maximal activation (calcium sensitivity), maximal calcium-activated force, and the slope of the calcium-force relation were compared. Our data indicate that with the development of hypertrophy, calcium sensitivity of left ventricular muscles remains unaffected, but maximal calcium-activated force is increased. In contrast, maximal calcium-activated force declines toward control levels with the development of left ventricular failure, despite the continued presence of significant hypertrophy. In the normotensive rats, the left ventricle is more sensitive to calcium than the right ventricle (pCa50 = 6.0 +/- 0.05 versus 5.7 +/- 0.09; p less than 0.05); however, both the calcium sensitivity and maximal calcium-activated force of the right ventricle increase with the development of compensatory hypertrophy secondary to left ventricular failure. These changes that occur in rats with cardiac hypertrophy and failure may represent important physiological adaptive mechanisms.