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Regional myocardial function assessment is essential for diagnosis and evaluation of heart disease. The purpose of this study was to enhance the spatial resolution of a speckle tracking echocardiography approach and enable layer-specific analysis of the myocardium. Following validation with software-implemented and mechanical phantoms versus imposed values, short-axis cines were obtained from 50 rats. The cines were post-processed by a speckle tracking commercial program, and the myocardial velocities were processed by a three-dimensional wavelet de-noising program, instead of the built-in smoothing process of the commercial program. Software-implemented phantom measurements yielded rotation errors of 7.5%, 2.9%, and 3.4%, for inner, middle, and outer layers, respectively. Analysis of a shrinking/expanding mechanical phantom yielded strain errors of 3%, 5%, and 7% for the three layers. Bland-Altman analysis showed agreement between the commercial and enhanced programs. Thus, layer-specific analysis is feasible while using echocardiography even on small animals such as rats.

Among the key effects of fluid shear stress on vascular endothelial cells is modulation of gene expression. Promoter sequences termed shear stress response elements (SSREs) mediate the responsiveness of endothelial genes to shear stress. While previous studies showed that shear stress responsiveness is mediated by a single SSRE, these endogenous promoters often encode for multiple SSREs. Moreover, hybrid promoters encoding a single SSRE rarely respond to shear stress at the same magnitude as the endogenous promoter. Thus, to better understand the interplay between the various SSREs, and between SSREs and endothelial-specific sequences (ESS), we generated a series of constructs regulated by SSREs cassettes alone, or in combination with ESS, and tested their response to shear stress and endothelial specific expression. Among these constructs, the most responsive promoter (NR1/2) encoded a combination of two GAGACC/SSREs, the Sp1/Egr1 sequence, as well as a TPA response element (TRE). This construct was four- to five-fold more responsive to shear stress than a promoter encoding a single SSRE. The expression of constructs containing other SSRE combinations was unaffected or suppressed by shear stress. Addition of ESS derived from the Tie2 promoter, either 5' or 3' to NR1/2 resulted in shear stress transcriptional suppression, yet retained endothelial specific expression. Thus, the combination and localization order of the various SSREs in a single promoter is crucial in determining the pattern and degree of shear stress responsiveness. These shear stress responsive cassettes may prove beneficial in our attempt to time the expression of an endothelial transgene in the vasculature.

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.

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to determine whether their actions can be related to the overall immunological rejection of the transplanted heart. Granule-induced myocyte morphological changes and final destruction were preceded by shortening of action potential duration (APD) and reductions of the resting potential and the action potential amplitude. APD shortening was probably caused by a granule-induced increase in outward current (most likely non-specific). Ryanodine, which blocks Ca2+ release from the sarcoplasmic reticulum, did not interfere with the morphological and electrophysiological effects of lytic granules. Fura-2 imaging indicated that [Ca2+]i initially increased about 2-fold from 90.0 +/- 11.5 nM, while cell length decreased less than 5% from a mean value of 99.0 +/- 9.0 microns. A further increase in [Ca2+]i (greater than 10 fold) was associated with progressive contracture and destruction, suggesting that the structural damage inflicted by lytic granules is caused by [Ca2+]i overload. The results indicate that the cytocidal action of CTL-derived lytic granules may be involved in immunologically induced damage, even to the extent of rejection of the transplanted heart.

Thyroid hormones have been previously shown to alter cardiac electrophysiological and mechanical properties in humans and in experimental animals. To investigate electrophysiological mechanisms responsible for some of these alterations, we recorded action potentials and membrane currents from isolated ventricular myocytes obtained from euthyroid, hypothyroid and hyperthyroid guinea-pigs. Hyperthyroidism was induced by injecting 150 micrograms/kg triiodothyronine for 8-11 days, and hypothyroidism was induced by propylthiouracil treatment for 35-45 days. We found that the slow inward current, was increased by hyperthyroidism and decreased by hypothyroidism: in euthyroid, hyperthyroid and hypothyroid myocytes peak slow inward current was (mean +/- SEM): -1.08 +/- 0.06 nA, -1.83 +/- 0.18a nA and -0.64 +/- 0.07a nA, respectively (a, p less than 0.005). In addition, the membrane potential at which peak slow inward current occurred was modified by the thyroid state and in euthyroid, hyperthyroid and hypothyroid myocytes it was (mean +/- SEM): 4.8 +/- 0.7 mV, -1.8 +/- 1.6a mV and 11.0 +/- 1.4a mV, respectively. The outward rectifying current, was also affected by the thyroid state, and in euthyroid, hyperthyroid and hypothyroid myocytes, the amplitude at VM = +60 mV was (mean +/- SEM): 0.51 +/- 0.09 nA, 1.15 +/- 0.08a nA and 0.49 +/- 0.05 nA, respectively. a, p less than 0.001 compared to euthyroid myocytes. Intraperitoneal administration of a single dose of triiodothyronine to guinea-pigs, 2 h prior to the electrophysiological experiment, increased the slow inward current amplitude, as was seen with chronic hyperthyroidism, but had no significant effect on the outward current and on the action potential.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effects of thyroid hormone on the action potential and membrane currents recorded from enzymatically dissociated guinea pig ventricular myocytes, by means of the whole-cell recording technique. Hypothyroidism was associated with an increase in action potential duration, whereas hyperthyroidism was associated with a decrease in duration. These effects are similar to those reported in multicellular preparations. Hypo- and hyperthyroidism were also associated with a decrease and an increase, respectively, in the slope of the action potential plateau. Resting potential and action potential amplitude were unaffected by the alterations in the thyroid state. The voltage-clamp experiments revealed that as compared with euthyroid myocytes, the peak calcium current was bigger in hyperthyroid myocytes and smaller in hypothyroid myocytes. The potassium outward current (at a membrane potential = 50 mV) was of similar amplitude in hypo- and euthyroid myocytes and bigger in hyperthyroid myocytes. Our major conclusion is that thyroid hormones regulate the amplitude of the calcium current and that this effect may be responsible in part for the modulation of myocardial contractility by thyroid hormones.

The effects of sodium salts of various bile acids on the contractile force and the electrophysiological properties of rat ventricular muscle were studied in vitro. Primary, conjugated, and secondary bile acids were studied in a concentration range of 10(-9)-10(-6) mol/l, which corresponds to concentrations found in the plasma of patients with cholestatic jaundice. In general, the bile acid induced a negative inotropic effect which was manifested as a reduction in active tension, maximum rate of tension activation, and maximum rate of tension relaxation. Twitch duration and time to peak tension were unaffected by the bile acids. The negative inotropism was associated with a reduction in ventricular action potential duration. Resting potential, action potential amplitude, and maximum upstroke velocity of phase 0 depolarization were unaffected. Voltage clamp experiments in rat ventricular myocytes demonstrated that sodium taurocholate decreased the slow inward current and slightly increased the outward potassium current. Hence, these effects on the membrane currents are probably responsible for the negative inotropic effect.