Differential effect of DPI 201-106 on the sensitivity of the myofilaments to Ca2+ in intact and skinned trabeculae from control and myopathic human hearts.

The effects of DPI, a new inotropic agent, were compared in trabeculae carneae from control and myopathic human hearts loaded with aequorin, a bioluminescent calcium indicator that emits light when it combines with calcium, and in saponin-skinned trabeculae carneae from the same hearts. The force-pCa curves in saponin-skinned fibers and the peak force-peak Ca2+ curves in aequorin-loaded preparations were not significantly different between the control and myopathic tissues. The force-pCa curve in the skinned and aequorin-loaded preparations from the same control hearts displayed no significant shifts with the addition of DPI. In contrast, a leftward shift was present in the force-calcium relationship in the presence of DPI in aequorin-loaded and skinned muscles from myopathic hearts, indicating an increase in the sensitivity of the myofilaments to calcium. These differences in the modulation of calcium activation between myopathic and control tissues indicate that pharmacological agents may produce differential effects in normal and diseased hearts.

Role of intracellular sodium in the regulation of intracellular calcium and contractility. Effects of DPI 201-106 on excitation-contraction coupling in human ventricular myocardium.

Experiments were performed to investigate the mechanism of action of DPI 201-106 on human heart muscle. In both control and myopathic muscles, DPI produced concentration-dependent increases in action potential duration, resting muscle tension, peak isometric tension, and duration of isometric tension. These changes were associated with increases in resting intracellular calcium and peak calcium transients as measured by aequorin. At higher concentrations of DPI, a second delayed Ca2+ transient (L') appeared. L' was inhibited by tetrodotoxin and ryanodine, suggesting that DPI acts at both the sarcolemma and the sarcoplasmic reticulum. DPI toxicity was manifested by after-glimmers and after-contractions reflecting a Ca2+-overload state: DPI effects were mimicked by veratridine, a Na+ channel agonist, and reversed by tetrodotoxin, yohimbine, and cadmium, Na+ channel antagonists. These results suggest that DPI acts primarily as a Na+ channel agonist. DPI may produce an increase in intracellular Ca2+ by increasing intracellular Na+ and altering Na+-Ca2+ exchange across the sarcolemma. DPI may also increase intracellular Ca2+ by directly altering sarcoplasmic reticulum Ca2+ handling.

Role of intracellular calcium handling in force-interval relationships of human ventricular myocardium.

Experiments were performed in human working myocardium to investigate the relationship of intracellular calcium handling and availability to alterations in the strength of contraction produced by changes in stimulation rate and pattern. Both control and myopathic muscles exhibited potentiation of peak isometric force during the postextrasystolic contraction which was associated with an increase in the peak intracellular calcium transient. Frequency-related force potentiation was attenuated in myopathic muscles compared to controls. This occurred despite an increase in resting intracellular calcium and in the peak amplitude of the calcium transient as detected with aequorin. Therefore, abnormalities in contractile function of myopathic muscles during frequency-related force potentiation are not due to decreased availability of intracellular calcium, but more likely reflect differences in myofibrillar calcium responsiveness. Sarcolemmal calcium influx may also contribute to frequency-related changes in contractile force in myopathic muscles as suggested by a decrease in action potential duration with increasing stimulation frequency which is associated with fluctuations in peak calcium transient amplitude.

Diastolic dysfunction in hypertrophic cardiomyopathy. Effect on active force generation during systole.

We tested the hypothesis that intracellular Ca++ [( Ca++]i) overload underlies the diastolic dysfunction of patients with hypertrophic cardiomyopathy. Myocardial tissue was obtained at the time of surgery or transplantation from patients with hypertrophic cardiomyopathy and was compared with control myocardium obtained from patients without heart disease. The isometric contractions and electrophysiologic properties of all myocardial specimens were recorded by standard techniques and [Ca++]i was measured with the bioluminescent calcium indicator aequorin. In contrast to the controls, action potentials, Ca++ transients, and isometric contraction and relaxation were markedly prolonged in the hypertrophic myocardium, and the Ca++ transients consisted of two distinct components. At 38 degrees C and 1 Hz pacing frequency, a state of relative Ca++ overload appeared develop, which produced a rise in end-diastolic [Ca++]i, incomplete relaxation, and fusion of twitches with a resultant decrease in active tension development. We also found that drugs with increase [Ca++]i, such as digitalis, exacerbated these abnormalities, whereas drugs that lower [Ca++]i, such as verapamil, or agents that increase cyclic AMP, such as forskolin, prevented them. These results may explain why patients with hypertrophic cardiomyopathy tolerate tachycardia poorly, and may have important implications with regard to the pharmacologic treatment of patients with hypertrophic cardiomyopathy.

Passive stiffness changes caused by upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts.

In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (approximately 3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from approximately 30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by approximately 10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.

Myofilament calcium regulation in human myocardium.

BACKGROUND: We investigated whether decreased myofilament calcium contractile activation may, in part, contribute to heart failure. METHODS AND RESULTS: Calcium concentration required for 50% activation and Hill coefficient for fibers from nonfailing and failing human hearts at pH 7.1 were not different. Maximum calcium-activated force (F(max)) was also not different. However, at pH 6.8 and 6.9, differences were seen in myofilament calcium activation between nonfailing and failing hearts. At lower pH, failing myocardium was shifted left on the calcium axis compared with nonfailing myocardium, which suggested an increase in myofilament calcium responsiveness. Increased inorganic phosphate concentration decreased maximal force development by 56% in nonfailing and 36% in failing myocardium and shifted the calcium-force relationship by 2.01+/-0.22 versus 0.86+/-0.13 micromol/L, respectively (P<0.05). Addition of cAMP resulted in a 0. 56 micromol/L shift toward higher intracellular calcium concentrations in nonfailing myocardium and a 1.04 micromol/L shift in failing myocardium. Protein kinase A in the presence of cAMP resulted in a further rightward shift in nonfailing human myocardium but did not further shift the calcium-force relationship in fibers from failing hearts. cGMP also resulted in a greater decrease in myofilament calcium sensitivity in fibers from failing hearts. CONCLUSIONS: We propose that changes at the level of the thin myofilaments result in differential responses to changes in the intracellular milieu in nonfailing versus failing myocardium.

Restoration of diastolic function in senescent rat hearts through adenoviral gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase.

BACKGROUND: Senescent hearts are characterized by diastolic dysfunction and a decrease in sarcoplasmic reticulum (SR) Ca(2+)-ATPase protein (SERCA2a). METHODS AND RESULTS: To test the hypothesis that an increase in SERCA2a could improve cardiac function in senescent rats (age 26 months), we used a catheter-based technique of adenoviral gene transfer to achieve global myocardial transduction of SERCA2a in vivo. Adult rat hearts aged 6 months and senescent rat hearts infected with an adenovirus containing the reporter gene beta-galactosidase were used as controls. Two days after infection, parameters of systolic and diastolic function were measured in open-chest rats. Cardiac SERCA2a protein and ATPase activity were significantly decreased in senescent hearts compared with adult rats (Delta -30+/-4% and -49+/-5%) and were restored to adult levels after infection with Ad.SERCA2a. At baseline, left ventricular systolic pressure and +dP/dt were unaltered in senescent hearts; however, diastolic parameters were adversely affected with an increase in the left ventricular time constant of isovolumic relaxation and diastolic pressure (Delta +29+/-9% and +38+/-12%) and a decrease in -dP/dt (Delta -26+/-11%). Overexpression of SERCA2a did not significantly affect left ventricular systolic pressure but did increase +dP/dt (Delta +28+/-10%) in the senescent heart. Overexpression of SERCA2a restored the left ventricular time constant of isovolumic relaxation and -dP/dt to adult levels. Infection of senescent hearts with Ad.SERCA2a markedly improved rate-dependent contractility and diastolic function in senescent hearts. CONCLUSIONS: These results support the hypothesis that decreased Ca(2+)-ATPase activity contributes to the functional abnormalities observed in senescent hearts and demonstrates that Ca(2+) cycling proteins can be targeted in the senescent heart to improve cardiac function.

Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase in a rat model of heart failure.

BACKGROUND: In heart failure, sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling and contractile dysfunction. We have previously shown that increasing SERCA2a expression by gene transfer improves ventricular function in a rat model of heart failure created by ascending aortic constriction. METHODS AND RESULTS: In this study, we tested the effects of gene transfer of SERCA2a on survival, left ventricular (LV) volumes, and metabolism. By 26 to 27 weeks after aortic banding, all animals developed heart failure (as documented by >25% decrease in fractional shortening) and were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a) or control virus (Ad.betagal-GFP) by use of a catheter-based technique. Sham-operated rats, uninfected or infected with either Ad.betagal-GFP or Ad.SERCA2a, served as controls. Four weeks after gene transfer, survival in rats with heart failure treated with Ad.betagal-GFP was 9%, compared with 63% in rats receiving Ad.SERCA2a. LV volumes were significantly increased in heart failure (0.64+/-0.05 versus 0.35+/-0.03 mL, P<0.02). Overexpression of SERCA2a normalized LV volumes (0.46+/-0.07 mL) in the failing hearts. (31)P NMR analysis showed a reduced ratio of phosphocreatine to ATP content in failing+Ad.betagal-GFP compared with sham+Ad.betagal-GFP (0.82+/-0.13 versus 1.38+/-0.14, P<0.01). Overexpression of SERCA2a in failing hearts improved the phosphocreatine/ATP ratio (1.23+/-0.28). CONCLUSIONS: In this study, we show that unlike inotropic agents that improve contractile function at the expense of increased mortality and worsening metabolism, gene transfer of SERCA2a improves survival and the energy potential in failing hearts.

Transplant coronary artery disease: a novel model independent of cellular alloimmune response.

BACKGROUND: Allograft coronary atherosclerosis (TxCAD) is the leading cause of death after the first year after transplantation. TxCAD is believed to be a form of chronic rejection of the cardiac allografts. This study was undertaken to determine whether TxCAD could develop in the absence of a cellular alloimmune response. METHODS AND RESULTS: Inbred lean Zucker rats (>26 generations) served as donors and recipients of the cardiac grafts. Donor hearts were explanted at 60 or 90 days. Explanted hearts were processed for coronary artery histological analysis. Cytokine expression was determined by reverse transcription-polymerase chain reaction, and the presence of T cells within the explanted hearts was evaluated by immunohistochemistry. Forty-six transplantations were made, and TxCAD developed in all but one of the transplanted hearts. Overall, one third of the vessels examined were affected by TxCAD, and in roughly half of these vessels, the disease was severe. Native hearts were free of atherosclerosis. Interleukin-2 was absent from the transplanted hearts, and T cells were present in minimal amounts (<1 per low-power field). CONCLUSIONS: TxCAD developed in the absence of a cellular alloimmune response in these genetically similar donors and recipients. The observed TxCAD was significant and comparable to what is found in rat allografting models.

An experimental model of acute and subacute viral myocarditis in the pig.

Twenty-six young pigs were infected with encephalomyocarditis virus, observed clinically, studied at intervals by noninvasive and invasive methods to assess cardiac function and eventually examined pathologically. All infected animals appeared ill, usually manifesting diminished appetite, lethargy and fever. Spontaneous mortality occurred either 1 to 4 or 20 to 21 days after infection. Electrocardiographic abnormalities, seen in the majority of animals, comprised ST-T wave changes, conduction disturbances or ventricular ectopic rhythm. The majority of animals manifested echocardiographic evidence of left ventricular dilation and decreased systolic function, which improved with time in some animals. Hemodynamic studies revealed elevation of biventricular filling pressures in 3 of 10 animals; as a group, infected animals manifested significantly elevated right ventricular filling pressures. In selected animals, the feasibility of gallium scans as well as left ventriculography and coronary angiography was demonstrated. At autopsy, heart weight/body weight ratio was significantly elevated in infected animals. The heart of all but two animals showed active myocarditis associated with fibrosis and focal calcification in the later stages. In general, the cardiovascular manifestations were parallel with those seen in acute and subacute myocarditis in humans. It is concluded that encephalomyocarditis infection in the pig is a large animal model of viral myocarditis suitable for assessing alterations in the structure and function of the cardiovascular system and the effects of interventions.

Comparison of twitch force and calcium handling in papillary muscles from right ventricular pressure overload hypertrophy in weanling and juvenile ferrets.

OBJECTIVE: The aim was to compare differences in peak twitch force occurring despite similar degrees of right ventricular hypertrophy in ferrets with pulmonary artery banding at either weanling or juvenile age. METHODS: After inducing pressure overload hypertrophy by banding the pulmonary artery of weanling and juvenile age ferrets, mechanical function (that is, isometric twitch force and passive stiffness), intracellular [Ca2+] using the calcium indicator aequorin, markers of myocardial energy supply, and quantified connective tissue content were studied. RESULTS: It was previously found that there was a reduced peak isometric twitch force despite normal [Ca2+]i in juvenile banded ferrets age 10-12 weeks with right ventricular pressure overload hypertrophy (POHj). In the present study we report findings in banded weanling ferrets (POHw) age 7 weeks. POHw animals showed a similar degree of hypertrophy to that found in the POHj. However, there was a greater peak twitch force in hypertrophied muscles at higher [Ca2+]o. There was no difference in peak [Ca2+]i: -3.1(SEM 0.1) v -3.1(0.3) (log fractional luminescence) at 16 mM [Ca2+]o for control and POHw, respectively. Connective tissue content for control animals was 10(1)% versus 10(2)% in POHw. Despite a lack of quantitative change in connective tissue content or resting [Ca2+]i in POHw, passive stiffness in papillary muscles was increased. Retrospective analysis of tissue from POHj revealed a connective tissue content of 24(6.8)% (P << 0.001). Thus the decreased peak twitch force reported in POHj might in part be due to an increase in fibrous connective tissue. In this study, lactate dehydrogenase was significantly higher (38%) in POHw animals. In contradistinction, total creatine kinase activity and total creatine content were significantly less (22%) in hearts from POHj animals, indicating differences in myocyte remodelling despite similar degrees and durations of hypertrophy. CONCLUSIONS: Comparison of POHw and POHj showed that, when there is restructuring of the extracellular space in terms of increased fibrosis, there is also molecular remodelling in the myocyte, as demonstrated by a decrease in the creatine kinase system.

Myocardial Ca(2+)- and ATP-cycling imbalances in end-stage dilated and ischemic cardiomyopathies.

OBJECTIVES: We have previously demonstrated deficiencies in myocardial cycling of Ca2+, and ATP turnover, in animals with heart failure (HF). The objective of this study was to determine the relevance of these changes to human HF. METHODS: We used the Ca2+ dye, indo-1, and the Ca(2+)-channel modulator ryanodine to examine Ca(2+)-cycling in homogenates containing 2.5% myocardium from 12 patients undergoing cardiac transplantations because of ischemic or idiopathic dilated cardiomyopathies (ISCM, DCM), and compared them to homogenates from 11 organ donors who died from noncardiac causes. Key enzymes of ATP production and utilization were also assayed. RESULTS: In HF due to either ISCM or DCM, compared to nonfailing myocardium, rate constants (x 10(-3) s-1) for sarcoplasmic reticulum Ca(2+)-pumping (41.6 +/- 16.0 versus 15.1 +/- 5.9) and Ca(2+)-channel (25.1 +/- 8.3 versus 6.2 +/- 4.1) activities were decreased by 64 and 75%, respectively. These changes in rate constants were associated with a three-fold increase in ionized Ca2+ concentration. Compared to nonfailing myocardium, activities (IU/g) of ATP turnover were also decreased in ISCM and DCM HF by 39%, 30%, and 34%, respectively, for ATP production capacity of creatine kinase (1830 +/- 130 versus 1110 +/- 411) and oxidative phosphorylation (20.0 +/- 3.3 and 14.1 +/- 4.8), and for ATP utilization (28.2 +/- 18.7 versus 18.7 +/- 4.0). Myoglobin, a key component of oxidative phosphorylation, was approximately 50% lower with HF (1.72 +/- 0.30 versus 0.97 +/- 0.20 mg/g). CONCLUSIONS: As in animal models, cycling of Ca2+ and ATP turnover were markedly impaired in human heart failure. There were no consistent biochemical differences attributable to difference in etiology, excepting that myoglobin deficiency was 33% greater in ISCM than DCM. We conclude that ATP and Ca2+ cycling are significantly impaired in human HF due to DCM and ISCM.

Relationship of abnormal intracellular calcium mobilisation to myocyte hypertrophy in human ventricular myocardium.

OBJECTIVE: Calcium transients in muscles from patients with end stage heart failure consist of two components (L1 and L2) at physiological extracellular calcium concentrations ([Ca2+]o); the second component (L2) can appear in normal human myocardium at high [Ca2+]o. In muscles from end stage heart failure patients L2 is associated with significant hypertrophy. To expand these observations a group of muscles from control patients with mild hypertrophy but without overt heart disease was studied (n = 8), in which a second calcium transient component was present during high [Ca2+]o. METHODS: Using the ratio of the two components of the calcium transient (L2/L1) seen in trabeculae from heart failure patients as a marker of intracellular calcium mobilisation, the hypothesis was tested that the extent of abnormality in transsarcolemmal calcium flux, and/or sarcoplasmic reticular calcium release and reuptake, correlates with the degree of hypertrophy present. RESULTS: In contrast to non-hypertrophied myocardium, hypertrophied myocardium from patients without heart failure often showed an increase in the L2/L1 ratio at higher [Ca2+]o. Hypertrophied myocardium from patients with failure showed a progressive increase in the L2/L1 ratio, reflecting further impairment of calcium mobilisation. There was a positive correlation between the degree of hypertrophy and calcium mobilisation alterations that was enhanced by raised [Ca2+]o. Altered [Ca2+]i mobilisation may develop early in the course of hypertrophy, before the onset of clinical signs of cardiac dysfunction.

Pathophysiological and biochemical characterisation of an avian model of dilated cardiomyopathy: comparison to findings in human dilated cardiomyopathy.

OBJECTIVE: With the recent availability of human myocardium, many animal models have been shown to be unsuitable as models of human heart failure. The aim of this study was to describe the pathophysiological changes in a model of dilated cardiomyopathy in turkey poults and to compare them to results obtained from failing human hearts. METHODS: After receiving furazolidone for 2-3 weeks, animals developed cardiomyopathy (Fz-DCM) and were studied at the whole heart and isolated muscle level. Myofibrillar ATPase activity and noradrenaline turnover were determined in tissue homogenates in failing and non-failing control hearts. RESULTS: Fz-DCM animals had greater heart weights, heart weight/body weight ratios, and end diastolic volumes. Fractional shortening of the left ventricle and systolic blood pressures were reduced (p < 0.01) in myopathic animals. Isolated perfused hearts had lower peak developed pressures (p < 0.01). Isolated muscle preparations showed no significant differences in peak twitch forces between control and Fz-DCM muscles at a 1 Hz stimulation rate. The relationship between force and frequency of stimulation was positive in control muscles up to 1.7 Hz, whereas in Fz-DCM muscles the relationship was sharply negative above 1 Hz. Time to 80% relaxation was markedly slower in the Fz-DCM muscles. Although [Ca2+]o responsiveness was similar for Fz-DCM and normal animals, responsiveness to isoprenaline was significantly reduced in Fz-DCM hearts. Cardiomyopathic animals displayed diminished noradrenaline content in the left ventricle. Fractional noradrenaline turnover was higher (p < 0.05) in the cardiomyopathic birds. In skinned fibre preparations from control and Fz-DCM hearts calcium activations were similar. Maximum myofibrillar ATPase activities were, however, significantly lower in myopathic animals and myofibrillar protein content was reduced by 25%. CONCLUSIONS: In this model of dilated cardiomyopathy: (1) relaxation is markedly prolonged; (2) the response to beta adrenergic stimulation is diminished; (3) Mg-ATPase activities and myofibrillar protein content are reduced; and (4) sympathetic activity in the heart is markedly increased with depletion of noradrenaline stores. Furthermore, a reduction in tissue noradrenaline content per se is a misleading index of the dynamic state of cardiac noradrenaline stores. With its similarities to human cardiomyopathy, this model promises to provide new insights into the pathophysiology and progression of dilated cardiomyopathy.

Effect of a high omega-3 fatty acid diet on cardiac contractile performance in Oncorhynchus mykiss.

OBJECTIVE: Omega-3 fatty acids have been implicated in the amelioration of cardiovascular disease in humans. Since these fatty acids are found in salmonid fish and are known to be essential for all salmonids, this study was undertaken to determine the effect of a high dietary intake of omega-3 fatty acids on the function of trout myocardium. METHODS: Rainbow trout (Oncorhynchus mykiss) from a single stock population were divided into two groups and fed either a diet high in omega-3 fatty acids (i.e. 4.0%) or low in omega-3 fatty acids (i.e. 2.1%) for 3 months. Heart function was studied at the whole heart and isolated muscle level. RESULTS: In whole heart preparations, peak developed pressures in freely ejecting hearts from salmonids fed the high omega-3 fatty acid diet were significantly greater than the hearts from salmonids fed the low omega-3 fatty acid diet (21 +/- 1.5 vs. 11.5 +/- 0.9 mmHg respectively, P < 0.05). These data correlated with results from isolated muscle preparations of myocardium from fish fed high and low omega-3 fatty acid diets (4.12 +/- 0.32 vs. 3.08 +/- 0.28 mN/mm2 respectively, P < 0.05). The calcium uptake rate of heart homogenates from fish fed the high omega-3 diet was slower and sarcoplasmic reticulum Ca2+ ATPase activity was lower. The myofilament force-calcium relationship in myocardium from trout fed the low omega-3 diet was shifted leftward on the calcium axis to lower intracellular calcium concentrations (delta 0.4 pCa units) compared to mammalian myocardium. This resulted in greater activation at lower intracellular calcium concentrations. However, trouts fed diets high in omega-3 fatty acids had [Ca2+] required for half maximal activation more similar to what has been reported for mammalian myocardium (delta 0.1 pCa unit). Furthermore, the myofilaments of trout hearts appear to show less cooperativity (Hill coefficient approximately 1) than has been found in mammalian myocardium (Hill coefficient > or = 2). CONCLUSIONS: Our experimental results demonstrate for the first time that dietary omega-3 fatty acid content affects myocardial force of contraction by affecting calcium metabolism and myofilament calcium-activation.

Intracellular calcium related to force development in twitch contraction of mammalian myocardium.

To characterize the relationship between force production and Ca2+ occupancy of troponin C, investigators have related peak intracellular Ca2+, measured with a variety of Ca2(+)-indicators, and peak force during twitches. Inherent in the force-[Ca2+] relationship is the responsiveness of the myofilaments to Ca2+ which can be altered by different pharmacological manipulations. In this study we compared the force-[Ca2+] relationship obtained in aequorin-injected papillary muscles and saponin skinned trabeculae from control, right ventricular pressure-overload hypertrophy (POH), and hyperthyroid ferret hearts. In POH, the twitch and [Ca2+]i transient were prolonged as compared to control. Force-[Ca2+] relationships from skinned fiber preparations were superimposable between control and POH. The peak force-peak [Ca2+]i relationship in intact muscles from POH was shifted to the left as compared to control. In hyperthyroid hearts, the twitch and [Ca2+]i were abbreviated. Force-[Ca2+]i relationships from skinned fiber preparations were superimposable between control and thyrotoxic hearts. The peak force-peak [Ca2+]i relationship in intact muscles from hyperthyroid hearts was shifted to the right as compared to control. Our findings indicate that time course changes in the calcium transient artifacturally shift the peak force-peak calcium relationship in a predictable manner. Therefore, this relationship can not be used to address changes at the level of the myofilaments as previously suggested.

Protection of turkeys against furazolidone-induced cardiomyopathy.

When fed furazolidone, 700 ppm, with their mash, most turkey poults develop dilated cardiomyopathy characterized by gross left ventricular dilatation with thinning of both the left ventricular free wall and ventricular septum. Birds fed propranolol, but not digoxin, did not develop this cardiomyopathy. It is not known what pharmacologic property of propranolol conferred protection or if mammals would receive similar protection.

The effects of milrinone and piroximone on intracellular calcium handling in working myocardium from the ferret.

The effects of milrinone and piroximone were compared to those of isoprenaline, dibutyryl adenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP), forskolin, isobutylmethylxanthine, increased extracellular calcium [( Ca2+]o) and caffeine in ferret right ventricular papillary muscles that were loaded intracellularly with aequorin, a bioluminescent calcium indicator that emits light when it combines with calcium. The positive inotropic action of each drug, except caffeine, was associated with an increase in the peak amplitude of the aequorin light signal (i.e. intracellular Ca2+ transient) reflecting an increased amount of calcium available for excitation-contraction coupling; the positive inotropic effect of caffeine appears to occur by other mechanisms. The time courses of the aequorin light signal and corresponding tension response were shortened by isoprenaline, forskolin, isobutylmethylxanthine, dibutyryl cyclic AMP, milrinone and piroximone; unchanged by increased [Ca2+]o and prolonged by caffeine, suggesting that the rates of Ca2+ release and uptake by the sarcoplasmic reticulum were respectively increased, unchanged or decreased by these groups of drugs. Relative to changes in [Ca2+]o, the ratio of the peak of the aequorin light signal to the peak of the tension response was increased by isoprenaline, milrinone and piroximone, and decreased by caffeine, indicating that the Ca2+-sensitivity of the myofilaments was respectively decreased, and increased by these drugs. The effects of milrinone and piroximone on the amplitude and time course of the aequorin light signal, as they relate to changes in uptake and release of calcium from the sarcoplasmic reticulum and to changes in the sensitivity of the myofilaments to Ca2+, are consistent with the findings that positive inotropic doses of these agents act by increasing intracellular concentrations of cyclic AMP. Higher doses of milrinone and piroximone produced negative inotropic effects that were characterized by diminution of developed tension but no change or an increase in the amplitude of the aequorin light signal, suggesting a decrease in the sensitivity of the contractile elements to Ca2+. Toxic doses of milrinone, piroximone and isoprenaline were associated with development of a Ca2+-overload state characterized by the presence of after-glimmers, after-contractions and dysrhythmias, and by decreased amplitude of both the aequorin light signal and tension response. The negative inotropic and toxic effects of milrinone and piroximone can be explained only in part by increased intracellular concentrations of cyclic AMP; we suggest that these drugs may have other cardiac actions.

Mechanisms of positive inotropic effects and delayed relaxation produced by DPI 201-106 in mammalian working myocardium: effects on intracellular calcium handling.

1. We used the bioluminescent protein aequorin, which emits light when it combines with Ca2+, to test the hypothesis that the inotropic and lusitropic actions of DPI 201-106 are due to changes in intracellular Ca2+ handling in papillary muscles from ferrets and guinea-pigs. 2. DPI 201-106 increased peak isometric tension (T) in a dose-dependent manner, with an 83% increase in T as the concentration of DPI 201-106 was increased to 1 x 10(-5) M; however, peak [Ca2+]i did not increase significantly until the concentration of DPI 201-106 reached 3 x 10(-6) M, suggesting a sensitization of the contractile apparatus to Ca2+. 3. Tetrodotoxin (1 x 10(-6) M), which did not reduce the tension response significantly before DPI 201-106, decreased both [Ca2+]i and T in the presence of 1 x 10(-5) M DPI 201-106, suggesting involvement of a sodium channel activation mechanism; however, tetrodotoxin did not completely reverse the calcium sensitization. 4. The shift of the [Ca2+]i versus T relationship was not observed in the presence of another sodium channel agonist, veratridine (3 x 10(-7)-1 x 10(-6) M). 5. In the guinea-pig, DPI 201-106 markedly prolonged relaxation of tension (increase of 60% in the time from peak to 50% tension regression), which was accompanied by the appearance of a second component in the aequorin light signal; effects on relaxation were less prominent in the ferret. 6. Tension prolongation and the second component of the [Ca2+]i transient in the guinea-pig were exacerbated by increased [Ca2+]o and decreased by tetrodotoxin. Ryanodine (3 x 10(-7) M) markedly diminished the calcium transient in controls and the initial component of the calcium transient in the presence of DPI 201-106, but had only a modest effect on the second component. 7. We conclude that although sodium agonism plays a role, sensitization of the contractile apparatus to Ca2+ is an important mechanism in the positive inotropic action of DPI 201-106. 8. The negative lusitropic action of DPI 201-106 varies between ferret and guinea-pig, possibly reflecting differences between these two species in subcellular Ca2+ handling.

Clinical correlates of the myocardial force-frequency relationship in patients with end-stage heart failure.

BACKGROUND: This study tested the hypothesis that in patients with suspected heart failure, peak oxygen consumption was the best predictor of heart muscle failure. Failing human myocardium is characterized by an abnormal force-frequency relationship, which has been previously shown to be altered in parallel with the severity of heart failure. METHODS: We examined whether seven different functional parameters of isolated electrically driven ventricular trabeculae carneae obtained from 34 explanted hearts of patients undergoing heart transplantation for end-stage heart failure correlated with any of 47 separate pretransplantation clinical parameters. The functional muscle parameters were active force at 0.33 Hz, time to 80% relaxation (RT 80%) of twitch force at 0.33 Hz, optimal frequency (OF), active force at 1.0 Hz (AF1), diastolic force at 1.0 Hz (DF1), active force at 2.0 Hz (AF2), and diastolic force at 2.0 Hz (DF2). RESULTS: Before transplantation the mean left ventricular ejection fraction was 21% +/- 10%, and all patients were in New York Heart Association class III or IV. Mean peak whole body VO2 was 10.9 +/- 3.3 ml/min/kg and percent body mass/age/sex-adjusted maximum VO2 oxygen consumption was 34.7% +/- 10.4%. Univariate analysis of VO2 yielded the following significant correlations: active force at 33 Hz, RT80%, OF, AF1, DF1, AF2, DF2; whereas univariate analysis of percent body mass/age/sex-adjusted VO2 yielded the following significant correlations: RT80%, OF, DF1, AF2, DF2. Multivariate analysis showed that OF and DF1 were independent predictors of peak VO2. CONCLUSION: In this study we show that peak oxygen uptake measured during cardiopulmonary exercise testing obtained before transplantation is correlated with the force-frequency behavior of isolated muscles at the time of transplantation. Peak VO2 seems to be a strong indicator of the severity of cardiac contractile dysfunction in patients with heart failure.