Cardiac contractility modulation with the impulse dynamics signal: studies in dogs with chronic heart failure.

The intravenous use of positive inotropic agents, such as sympathomimetics and phosphodiesterase inhibitors, in heart failure is limited by pro-arrhythmic and positive chronotropic effects. Chronic use of these agents, while eliciting an improvement in the quality of life of patients with advanced heart failure, has been abandoned because of marked increase in mortality when compared to placebo. Nevertheless, patients with advanced heart failure can benefit from long-term positive inotropic support if the therapy can be delivered 'on demand' and in a manner that is both safe and effective. In this review, we will examine the use of a novel, non-stimulatory electrical signal that can acutely modulate left ventricular (LV) contractility in dogs with chronic heart failure in such a way as to elicit a positive inotropic support. Cardiac contractility modulation (CCM) with the Impulse Dynamic(trade mark) signal was examined in dogs with chronic heart failure produced by intracoronary microembolizations. Delivery of the CCM signal from a lead placed in the great coronary vein for periods up to 10 minutes resulted in significant improvements in cardiac output, LV peak+dP/dt, LV fractional area of shortening and LV ejection fraction measured angiographically. Discontinuation of the signal resulted in a return of all functional parameters to baseline values. In cardiomyocytes isolated from dogs with chronic heart failure, application of the CCM signal resulted in improved shortening, rate of change of shortening and rate of change of relengthening suggesting that CCM application is associated with intrinsic improvement of cardiomyocyte function. The improvement in isolated cardiomyocyte function after application of the CCM signal was accompanied by an increase in the peak and integral of the Ca(2+) transient suggesting modulation of calcium cycling by CCM application. In a limited number of normal dogs, intermittent chronic delivery of the CCM signal for up to 7 days showed chronic maintenance of LV functional improvement. In conclusion, pre-clinical results to date with the Impulse Dynamics CCM signal indicate that this non-pharmacologic therapeutic modality can provide short-term positive inotropic support to the failing heart and as such, may be a useful adjunct in the treatment of advanced heart failure. Additional, long-term studies in dogs with heart failure are needed to establish the safety and efficacy of this therapeutic modality for the chronic treatment of this disease syndrome.

Short peptide-based tolerogens without self-antigenic or pathogenic activity reverse autoimmune disease.

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in brain lesions of multiple sclerosis (MS), and this peptide can trigger experimental autoimmune encephalomyelitis (EAE). We designed truncated peptides based on this pathogenic 13-mer that are not antigenic. These short peptides reduced production of IFN-gamma and TNF-alpha in vivo. Moreover, paraplegic rats given the 7-mer FKNIVTP in soluble form showed total reversal of paralysis in 24 h. Truncated peptides that are too small to stimulate antigenic responses to pathogenic regions of myelin basic protein are nevertheless effective tolerogens and are able to anergize autoreactive T cells. Short peptide-based tolerogens, devoid of immunogenic and pathogenic potential, may be attractive for therapy of autoimmune diseases.

Fas (CD95/Apo-1)-mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from perforin-deficient mice: a major role for inositol 1,4,5-trisphosphate.

Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene-knockout (P-/-) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca2+]i transients, and contractions of myocytes conjugated to P-/- PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P-/- PELs were, respectively, as follows: Vm, -73.2+/-1.5 and -53.6+/-6.4 mV (mean+/-SEM); action potential amplitude, 117.9+/-3.9 and 74.3+/-21.2 mV; and action potential duration at 80% repolarization, 17+/-6 and 42+/-13 milliseconds (all P<.05). P-/- PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca2+]i increased during the cytocidal interaction with P-/- PELs, from a fluorescence ratio of 0.82+/-0.05 (n=7) to 1.98+/-0.09 (n=13) (P<.05). All of the effects caused by P-/- PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 microg/mL), an antagonist of inositol trisphosphate (IP3)-operated sarcoplasmic reticulum Ca2+ channels, or U-73122 (2 micromol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P-/- PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP3. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.

Involvement of the IP3 cascade in the damage to guinea-pig ventricular myocytes induced by cytotoxic T lymphocytes.

We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro model for heart transplant rejection, results in electrophysiological and morphological alterations indicative of overload of the intracellular [Ca2+] ([Ca2+]i). Since these deleterious effects cannot be accounted for by increased L-type Ca2+ current (ICa,L), we hypothesize that [Ca2+]i overload due to Ca2+ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol trisphosphate (IP3) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and [Ca2+]i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting potential was reduced (compared with the nonconjugated state) from -80.9 +/- 0.7 to -59.9 +/- 2.5 mV, action potential amplitude from 139.5 +/- 1.4 to 80.6 +/- 1.7 mV and action potential duration to 50% repolarization (APD50) from 797 +/- 97 to 52 +/- 12 ms. The ratio of fluorescence at 340 and 380 nm (R340/380) increased from a control value (in nonconjugated myocytes) of 0.71 +/- 0.02 to 2.07 +/- 0.03, 30 min, after conjugate formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 micrograms/ml), an antagonist of IP3-induced Ca2+ release from SR channels, or U-73122 (2 microM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution), prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced increase in [Ca2+]i; after 60 min of PEL-myocyte interaction, R340/380 was 1.15 +/- 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage to ventricular myocytes is, at least partially, mediated by PLC activation and IP3-induced Ca2+ release from intracellular stores. Pharmacological targeting of IP3 in heart transplant rejection is thus suggested.

Experimental iron deficiency in rats: mechanical and electrophysiological alterations in the cardiac muscle.

1. Our aim was to investigate the effect of experimental iron deficiency on cardiac functional properties. We recorded ventricular isometric twitch, action potentials and the L-type Ca2+ current in isolate ventricular myocytes from iron-deficient rats and control rats. 2. Twitch tension and maximal rates of tension activation and relaxation were reduced in iron-deficient compared with control rats, whereas twitch duration was prolonged. Isoproterenol (10-(6) mol/l) augmented tension in iron-deficient rats (P < 0.05), but only moderately affected control rats. In contrast, maximal rates of tension activation and relaxation were increased equally by isoproterenol in the two groups. 3. To determine the mechanism(s) responsible for the reduced mechanical function in iron-deficient rats, action potentials and the L-type Ca2+ current (with or without isoproterenol) were recorded in both groups. 4. The L-type Ca2+ current was smaller in ventricular myocytes from control rats than in those from iron-deficient rats; at a membrane potential of 0 mV, L-type Ca2+ current amplitudes were -1.44 +/- 0.18 and -0.97 +/- 0.07 nA in myocytes from control and iron-deficient rats respectively (P < 0.05). 5. Action potential duration was markedly shortened in myocytes from iron-deficient compared with control rats; action potential duration at 50% repolarization was 12.0 +/- 1.6 and 7.2 +/- 1.4 ms in myocytes from control and iron-deficient rats respectively (P < 0.01). These iron deficiency-induced electrophysiological alterations most probably contribute to the depressed mechanical function in iron-deficient rats. 6. The L-type Ca2+ current was augmented equally by isoproterenol in the two groups, suggesting that the enhanced inotropic responsiveness in iron-deficient rats was not due to an increased response of the L-type Ca2+ current. 7. These results may have an important implication for anaemic (iron-deficient) patients; the attenuation of their cardiac mechanical performance may be compensated by an increased reactivity to beta-adrenergic stimulation.

Interaction of cytotoxic T lymphocytes and guinea pig ventricular myocytes. Pharmacological modulation by blocking K+ currents in cytotoxic T lymphocytes.

Infiltrating cytotoxic T lymphocytes (CTLs) are important immune effectors that damage the myocardium during heart transplant rejection as well as in cardiomyopathy and Chagas' heart disease. We have previously shown that in an in vitro model of murine-derived peritoneal exudate CTL (PEL)-guinea pig ventricular myocyte interaction, PEL induced in conjugated myocytes reduction of resting membrane potential and action potential (AP) amplitude, shortening of AP duration, delayed afterdepolarizations (DADs), and myocyte contracture and destruction. Since these findings indicated that cytotoxicity was largely caused by [Ca2+]i overload, in the present study we tested the hypothesis that blocking the L-type Ca2+ current (ICa,L) in the myocyte will eliminate the trigger for Ca2+ release from intracellular stores and will reduce [Ca2+]i overload and subsequent myocyte deterioration. CoCl2 (3 mmol/L) prevented PEL-induced AP changes, induction of DADs, and myocyte destruction. Since verapamil (2 mumol/L) was ineffective, indicating that the CoCl2 protection was not due to block of ICa,L, we tested whether the different action of these Ca2+ channel blockers was due to their differential effect on the PEL's K+ current (IK), previously shown to participate in lymphocyte activation and cytotoxicity. In agreement with their protective efficacy, CoCl2 but not verapamil blocked IK in PELs, suggesting that this is the mechanism for the protection provided by CoCl2. To support this notion, we tested the effect of the scorpion-derived peptide margatoxin (10 nmol/L), a specific K+ channel blocker in lymphocytes, on PEL-myocyte interaction and on PEL's IK; margatoxin prevented PEL-induced cytotoxicity and also blocked IK in PEL. Based on these findings, an alternative modality for attenuating CTL-induced lymphocytotoxicity is proposed.

Mechanisms whereby cytotoxic T lymphocytes damage guinea-pig ventricular myocytes in vitro.

We studied possible mechanisms whereby cytotoxic T lymphocytes (CTL) damage the myocardium during the immunological rejection of the transplanted heart, by investigating the in vitro interaction between CTL and cardiac myocytes. We utilized the patch-clamp technique to record membrane currents and action potentials from concanavalin A-treated guinea-pig ventricular myocytes conjugated to mouse peritoneal exudate CTL (PEL). PEL-myocyte interaction reduced action potential duration at 50% repolarization (APD50) from 731.7 +/- 57.8 to 195.3 +/- 58.0 ms, action potential amplitude from 134.9 +/- 1.9 to 104.2 +/- 6.2 mV and resting membrane potential (Vm) from -80.9 +/- 0.5 to 72.5 +/- 1.5 mV. These changes were accompanied by generation of delayed afterdepolarizations, indicative of intracellular [Ca2+] overload. The electrophysiological alterations were associated with myocyte shortening (within 28.9 +/- 2.8 min) followed by complete cell destruction (within 43.5 +/- 4.3 min). To determine whether intracellular Ca2+ stores were involved in PEL-induced myocyte damage, the protective effects of ryanodine and caffeine were investigated. While ryanodine (10 microM) delayed the electrophysiological and morphological alterations, caffeine (5 mM) provided significant protection, suggesting that Ca2+ release from intracellular stores contributes to PEL-induced damage to the myocytes. Based on our findings, we suggest that the functional derangements seen in myocyte-lymphocyte conjugates can contribute to the overall decline in cardiac function during heart transplant rejection.

Effects of purified perforin and granzyme A from cytotoxic T lymphocytes on guinea pig ventricular myocytes.

OBJECTIVE: Involvement of cytotoxic T lymphocytes (CTL) in heart transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes. METHODS: Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes. RESULTS: Resembling the effects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects. CONCLUSIONS: As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules.

Developmental changes in the myosin composition of guinea pig ventricular muscle. Relation to thyroid state and mechanical properties.

In a variety of mammalian species, thyroid hormone regulates the contractile properties of the heart as well as the expression of the alpha and beta heavy chains of myosin. We have previously shown that the plasma levels of thyroid hormone reach a peak immediately after birth in guinea pigs and decline with maturation. We therefore studied age-related changes in the expression of the myosin heavy chains in the guinea pig ventricle in relation to the ventricular mechanical properties and the levels of thyroid hormone. The composition of the myosin heavy chains was characterized by gel electrophoresis and immunoblotting. Anti-beta-chain antibody stained equally myosins from newborns (0-5 days) and adults (75-90 days), while anti-alpha-chain positively decorated only the myosins of euthyroid newborns or of hyperthyroid adults, but not myosins of embryos, hypothyroid newborns or hypothyroid adults. Myosin of euthyroid adults was faintly stained by anti-alpha-chain. The alterations in the composition of myosin corresponded with the "thyroid state" of these groups. The plasma levels of total T3 were 24.3 +/- 2.7, 9.04 +/- 1.2 and 139.0 +/- 9.3 ng/dl (mean +/- SEM) in the euthyroid, hypothyroid and hyperthyroid adults, respectively. In euthyroid and hypothyroid newborns, the plasma levels of T3 were 56.5 +/- 11.9 and 26.5 +/- 9.8 ng/dl, respectively. Within each age group the thyroid state corresponded with maximal twitch tension (Tmax), rates of development of tension and relaxation, time to peak tension and rate of activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in ventricular action potential properties in guinea-pigs are modulated by age-related changes in the thyroid state.

Previous studies have demonstrated that in different cardiac preparations action potential duration (APD) increases with age. As in various species, thyroid hormone levels increase developmentally, and since hyperthyroidism shortens APD while hypothyroidism prolongs it, we hypothesized that developmental changes in APD result from age-related variations in the thyroid state. The hypothesis was tested by analysing ventricular action potentials and total T4 (TT4) levels in guinea-pigs in the age range of 0 days to 3 months (adult), and in hyperthyroid and hypothyroid newborns (0-5 days old). We found that APD50 increased exponentially with age with a time constant of 6.7 days, from 100.6 +/- 3.4 ms in newborns (0-5 days old) to 147.4 +/- 5.2 ms in adults (P less than 0.001). TT4 decreased exponentially with age with a time constant of 4.8 days, from 3.9 +/- 0.4 micrograms/dl in newborns to less than 1.0 microgram/dl in adults. In the age range studied, APD50 and TT4 were linearly correlated: Y = -12.13X + 142, r - 0.865. In contrast to the marked changes in APD, resting potential and action potential amplitude were age-independent, and Vmax only slightly increased with age. Alterations in the thyroid state in newborns affected ventricular action potentials as predicted by the hypothesis. In euthyroid (TT4 = 3.9 +/- 0.4 micrograms/dl), hypothyroid (TT4 = 1.6 +/- 0.4 micrograms/dl) and hyperthyroid (TT4 = 39.8 +/- 10.8 micrograms/dl) newborns, APD50 was: 100.6 +/- 3.4 ms, 117.7 +/- 4.2 ms and 63.7 +/- 7.4 ms, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of thyroid state in age-dependent cardiac effects of ouabain in guinea pigs.

We tested the hypothesis that the decrease in the thyroid state, with age, contributes to the age-related increase in myocardial responsiveness to cardiac glycosides. Thyroid hormone levels (reflecting the thyroid state): total T4 (microgram/dl) and total T3 (ng/dl) in the 3 groups of guinea pigs were (mean +/- SEM): adults (3 months old): less than 1.0 and 22.6 +/- 1.1; euthyroid newborns (0-5 days old): 3.9 +/- 0.4 and 56.5 +/- 11.9; hypothyroid newborns, (0-5 days old): 1.5 +/- 0.3 and 26.5 +/- 9.8. In euthyroid newborns, T4 and T3 levels were significantly higher than in adults (p less than 0.01 for T4 and p less than 0.05 for T3) and in hypothyroid newborns (p less than 0.05). Isometric twitch was recorded from right ventricular papillary muscles by means of a force transducer. Ouabain 10(-6) M increased isometric twitch tension in adults (tension = 0.66 +/- 0.18 g/mm2) by 123.6 +/- 18.2%, in euthyroid newborns (tension = 0.19 +/- 0.04 g/mm2) by 83.6 +/- 14.5%, and in hypothyroid newborns (tension = 0.12 +/- 0.01 g/mm2) by 170.9 +/- 33.8% (p less than 0.01). Ouabain dose-response curve in the range of 10(-7) M-0.5 x 10(-5) M was significantly different (compared by two-way ANOVA) between euthyroid newborns and hypothyroid newborns (p less than 0.01), and between euthyroid newborns and adults (p less than 0.01). Toxic effects of ouabain reflected by the generation of aftercontractions were also age related and were augmented by hypothyroidism in newborns.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological effects of thyroid hormones in guinea-pig ventricular muscle: time course and relationships to blood levels.

Chronic alterations in the thyroid state have been shown to induce marked changes in action potential characteristics, the most pronounced being shortening of action potential duration (APD) by hyperthyroidism and an increase in duration by hypothyroidism. In the present study our major objectives were to investigate the time course of the effect of thyroid hormone on action potential characteristics, to examine the relationships between thyroid hormone levels and these changes, and to test whether the electrophysiological alterations are induced both by thyroxine (T4) and triiodothyronine (T3). The major findings were that a single dose of either hormone (100 micrograms/kg) caused a marked shortening of APD, while resting potential, action potential amplitude and Vmax were unchanged. APD shortening was observed promptly after ip T4 or T3 administration, with maximal effect occurring within 2 to 3 hrs. Having determined thyroid hormone levels in the guinea-pigs used for the electrophysiological experiments, we found a close temporal association and inverse linear relationships (r = -0.82) between total T4 levels and APD. To determine whether APD shortening is induced due to a direct effect of thyroid hormone on the myocardium, euthyroid papillary muscles were superfused with 10(-6) M T3; within 3 hrs of superfusion with T3, APD was shortened from 148.8 +/- 4.7 ms to 117.7 +/- 6.4 ms (P less than 0.01), an effect blocked by the protein synthesis inhibitor cycloheximide. Our results demonstrate that thyroid hormones affect the duration of the ventricular action potential in a concentration dependent fashion. Furthermore, the study suggests that thyroid hormones affect the myocardium directly via mechanism(s) that are probably associated with thyroid hormone-related protein synthesis.

Inter-species variations in myocardial responsiveness to cardiac glycosides: possible relations to the thyroid status.

Mammalian species differ in their myocardial responsiveness to cardiac glycosides; whereas glycosides induce a marked positive inotropic effect in species such as dog, rabbit and guinea-pig, the rat myocardium is virtually insensitive. We investigated the physiological basis for this phenomenon by testing the hypothesis that the inter-species variations in the response of the myocardium to cardiac glycosides results, at least in part, from species-related differences in the "thyroid status". In the present study we focused on the toxic effects of the glycosides, and studied ouabain-induced delayed afterdepolarizations (DAD): (1) in guinea-pigs, rats and mice, which encompass a wide range of thyroid statuses, as indicated by their O2 consumption and thyroid hormone levels; (2) in guinea-pigs and rats in which the thyroid status was decreased by propylthiouracil treatment or increased by thyroxine administration (in the former species only). DAD were readily induced in guinea-pigs after 40 to 60 min superfusion with 10(-6) M ouabain and 5.4 mM Ca2+. In rats, DAD were induced only when the Ca2+ concentration was raised to 8.1 mM, but were absent in mice even after 2 h of superfusion with ouabain and 8.1 mM Ca2+. In guinea-pigs and rats (at cycle length = 500 ms), DAD amplitude was (means +/- S.E.): 2.8 +/- 0.7 mV and 1.1 +/- 0.4 mV, respectively. The slope of the DAD ascending limb (dV/dt) in guinea-pigs was 47.6 +/- 8.6 mV/s and in rats was 8.1 +/- 3.4 mV/s. In both species DAD characteristics were altered by the thyroid status. In eu-, hyper- and hypothyroid guinea-pigs, DAD amplitude and dV/dt (cycle length = 500 ms) were as follows: 2.8 +/- 0.7 mV and 47.6 +/- 8.6 mV/s; 1.2 +/- 0.4* mV and 12.6 +/- 3.9* mV/s; 7.5 +/- 0.6* mV and 204.0 +/- 18.4* mV/s, respectively (*, P less than 0.005, compared to euthyroid guinea-pigs). The occurrence of triggered beats was also dependent on the thyroid status. They occur more frequently in hypothyroidism and less frequently in hyperthyroidism. Hypothyroidism in rats augmented ouabain toxicity as reflected by an increase in DAD amplitude and dV/dt by 109% and 105%, respectively (P less than 0.05, as compared to euthyroid rats). In conclusion, we suggest that species-related differences in the thyroid status may contribute to the inter-species (as well as for the intra-species) variations in the myocardial responsiveness to cardiac glycosides.