Irit Rubinstein

Effects of thyroid hormone on the action potential and membrane currents of guinea pig ventricular myocytes

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.

Management of Shock and Acute Renal Failure in Casualties Suffering from the Crush Syndrome

Widespread muscle crush injury is often associated with profound hemodynamic shock and myoglobinuric acute renal failure (ARF). The main reason for the shock is rapid uptake by the injured muscles of a substantial portion of extracellular fluid. The shock is aggravated by NO-dependent vasodilation in the injured muscles and by hyperkalemia and hypocalcemia, which suppress the entire cardiovascular tree. Treatment consists of early massive volume replacement and forced alkaline solute (mannitol) diuresis. With this regimen it is possible to increase survival of life and limbs, and prevent myoglobinuric ARF. Our preliminary experience suggests that i.v. hypertonic mannitol is protective also to the injured muscle and can be used as a noninvasive adjunct in the management of compartment syndrome in man. Moreover, by preserving muscular integrity, mannitol can conceivable reduce leakage of the nephrotoxic myoglobin and urate and thus further defend kidney function.

Effects of bile acids on ventricular muscle contraction and electrophysiological properties: studies in rat papillary muscle and isolated ventricular myocytes

SummaryThe 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 plasm 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.

Hyperbaric oxygen treatment improves GFR in rats with ischaemia/reperfusion renal injury: a possible role for the antioxidant/oxidant balance in the ischaemic kidney

Background. Ischaemic kidney injury continues to play a dominant role in the pathogenesis of acute renal failure (ARF) in many surgical and medical settings. A major event in the induction of renal injury is related to the generation of oxygen-free radicals. Hyperbaric oxygen therapy (HBO) is indicated for treatment of many ischaemic events but not for ARF. Therefore, the present study examined the effects of HBO on kidney function and renal haemodynamics in rats with ischaemic ARF. Methods. Renal ischaemia was induced by unilateral renal artery clamping (45 min) in rats. Within 24 h following ischaemia, rats were treated twice with HBO of 100% O2 at 2.5 absolute atmospheres for 90 min each (+HBO). Untreated rats (−HBO) served as a control. Forty-eight hours later, GFR, RBF and endothelial-dependent vasorelaxation were measured. In addition, the immunoreactive staining of 4-hydroxy-2-noneal (4-HNE), a major product of endogenous lipid peroxidation, and superoxide dismutase (SOD) were assessed. Results. In the −HBO group, GFR was reduced by 94% compared with the untouched normal kidney (ischaemic: 0.06 ± 0.03 ml/min, normal: 1.02 ± 0.13 ml). In contrast, in the +HBO group, GFR of the ischaemic kidney (0.36 ± 0.07 ml/min) was reduced only by 68% compared with the contralateral normal kidney (1.12 ± 0.12 ml/min). In line with these findings, HBO improved the vasodilatory response to ACh as expressed in enhancement of both total and regional renal blood flow. In addition, HBO reduced the formation of 4-HNE by 33% and 76% and increased SOD by 30% and 70% in the cortex and outer stripe region of the medulla of the ischaemic kidney, respectively. Conclusion. HBO attenuates the decline in GFR following renal ischaemia, and improves endothelial-dependent vasorelaxation, suggesting that treatment with HBO may be beneficial in the setting of ischaemic ARF.

Thyroid hormone modulates membrane currents in guinea-pig ventricular myocytes

SummaryThyroid 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 μg/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 < 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 atVM = + 60 mV was (mean ± SEM): 0.51 ± 0.09 nA, 1.15 ± 0.08a nA and 0.49 ± 0.05 nA,respectively. a,p < 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. Finally, action potential and membrane currents were not altered by superfusing euthyroid myocytes with 10−6 mol/l triiodothyronine for 30 min, or by introducing 10−7 mol/l triiodothyronine into the cell through the recording pipette.From these findings we conclude that cardiac mechanical performance may be modulated by thyroid hormones through their effect on the slow inward current, while shortening of action potential duration is brought about, at least in part, by increasing the amplitude of the outward rectifying current.

Effects of Novel Vasopressin Receptor Antagonists on Renal Function and Cardiac Hypertrophy in Rats with Experimental Congestive Heart Failure

Arginine vasopressin (AVP) plays an important role in renal hemodynamic alterations, water retention, and cardiac remodeling in congestive heart failure (CHF). The present study evaluated the acute and chronic effects of vasopressin V1a receptor subtype (V1a) and vasopressin V2 receptor subtype (V2) antagonists on renal function and cardiac hypertrophy in rats with CHF. The effects of acute administration of SR 49059 [(2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide)] (0.1 mg/kg) and SR 121463B (1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclohexane]indol-2-one, fumarate; equatorial isomer) (0.3 mg/kg), V1a and V2 antagonists, respectively, on renal function, and of chronic treatment (3.0 mg/kg/day for 7 or 28 days, via osmotic minipumps or p.o.), on water excretion and cardiac hypertrophy were studied in rats with aortocaval fistula and control rats. CHF induction increased plasma AVP (12.8 ± 2.5 versus 32.2 ± 8.3 pg/ml, p < 0.05). Intravenous bolus injection of SR 121463B to controls produced dramatic diuretic response (from 5.5 ± 0.8 to 86.3 ± 21.9 μl/min; p < 0.01). In contrast, administration of SR 49059 did not affect urine flow. Likewise, administration of SR 121463B, but not SR 49059, to rats with CHF significantly increased urinary flow rate from 20.8 ± 6.4 to 91.6 ± 26.5 μl/min (p < 0.01). The diuretic effects of SR 121463B were associated with a significant decline in urinary osmolality and insignificant change of Na+ excretion. In line with its acute effects, chronic administration of SR 121463B to CHF rats increased daily urinary volume 2 to 5-fold throughout the treatment period. Both SR 121463B and SR 49059 significantly reduced heart weight in CHF rats when administered for 4 weeks, but not 1 week. These results suggest that V2 and V1a antagonists improve water balance and cardiac hypertrophy in CHF and might be beneficial for the treatment of water retention and cardiac remodeling in CHF.

Vascular endothelial growth factor (VEGF) fails to improve blood flow and to promote collateralization in a diabetic mouse ischemic hindlimb model

BackgroundAngiogenic therapy with vascular endothelial growth factor (VEGF) has been proposed as a treatment paradigm for patients suffering from an insufficiency of collateral vessels. Diabetes is associated with increase in the production of VEGF and therefore additional VEGF may not be beneficial. Accordingly, we sought to determine the efficacy of VEGF therapy to augment collateral formation and tissue perfusion in a diabetic mouse ischemic hindlimb model.MethodsDiabetic and non-diabetic mice were studied in parallel for the efficacy of VEGF administration. Diabetes was induced with streptozotocin. Hindlimb ischemia was produced by severing the left iliac artery. An outlet tube from an osmotic infusion pump with placebo/ 500 micrograms of plasmid-DNA encoding VEGF was fenestrated and tunneled into the left quadriceps muscle.ResultsVEGF induced more rapid and complete restoration of blood flow in normal mice. However, in the setting of diabetes there was no difference between VEGF Vs. placebo in the rate or adequacy of flow restoration. There was a significant increase in smooth muscle actin and Factor-VIII antigen densities in diabetic animals and in animals which received VEGF.ConclusionsAngiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.

Immunological rejection of heart transplant : how lytic granules from cytotoxic T lymphocytes damage guinea pig ventricular myocytes

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to dertermine 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 sacroplasmic 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 μm. A further increase in [Ca2+]i (>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.

Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF

The optimal vector, regulatory sequences, and method of delivery of angiogenic gene therapy are of considerable interest. The Spalax ehrenbergi superspecies live in subterranean burrows at low oxygen tensions and its tissues are highly vascularized. We tested whether continuous perimuscular administration of Spalax vascular endothelial growth factor (VEGF) DNA could increase tissue perfusion in a murine hindlimb ischemia model. Placebo or VEGF ± internal ribosome entry site (IRES) was continuously administrated perimuscularly in the ischemic zone by using an infusion pump. None of the mice in the VEGF-treated group (>50 μg) developed visible necrosis vs. 33% of the placebo group. Microscopic necrosis was observed only in the placebo group. Spalax VEGF muscular infiltration resulted in a faster and more complete restoration of blood flow. The restoration of blood flow by VEGF was dose-dependent and more robust and rapid when using the VEGF–IRES elements. The flow restoration using continuous perimuscular infiltration was faster than single i.m. injections. Vessel density was higher in the VEGF and VEGF–IRES (−) groups compared with the placebo. Continuous perimuscular administration of angiogenic gene therapy offers a new approach to restore blood flow to an ischemic limb. Incorporation of an IRES element may assist in the expression of transgenes delivered to ischemic tissues. Further studies are needed to determine whether VEGF from the subterranean mole rat Spalax VEGF is superior to VEGF from other species. If so, 40 million years of Spalax evolution underground, including adaptive hypoxia tolerance, may prove important to human angiogenic gene therapy.

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)