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Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/[dt.sub.max]) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents ([I.sub.Na]) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of [I.sub.Na]. All of these treatments rescued [I.sub.Na] to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of [I.sub.Na]. TO assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice. sodium current; transgenic mice; ryanodine receptor

Experiments were performed to investigate the properties of adenosine-triphosphate-dependent potassium channels (ATP-dependent K+) in myocytes from diabetic rat hearts. The action potentials of diabetic ventricular myocytes were recorded and analyzed. The findings suggest a strong influence of potassium ATP channels on the action potential configuration in the diabetic heart. This implies the resistance of the diabetic heart to the effects of ischemia.

A study of the effects of size and rate on the steady state (Iss) and transient (Ito) outward potassium currents in ventricular myocytes isolated from hypo-, hyper- and euthyroid rats using the whole cell, suction electrode voltage clamp technique reveals that thyroid status influences potassium currents in rat ventricle. A lower metabolic rate accompanies the reduction in Ito size under hypothyroid conditions. Excess thyroid hormone does not exert any general, nonspecific modulation of potassium channels.

A study of the alpha-adrenergic modulation of transient outward potassium current during hyperthyroidism is presented. Previous studies have shown that alpha-adrenoceptors are abundant in cardiac muscles and thus mediate positive inotropic effects. The study protocol involved measurements of K+ currents in single cardiac myocytes isolated from normal and from hyperthyroid rabbits. The results suggest that hyperthyroidism reduces the modulation of K+ currents, possibly by reducing the number and/or the affinity of alpha-adrenergic receptors.

A sustained [K.sup.+] current ([I.sub.ss]) is attenuated in ventricular cells from streptozotocin (STZ)-induced diabetic rats. The in vitro addition of insulin to isolated cells augments [I.sub.ss] in a process that is blocked by disrupting either actin microfilaments (with cytochalasin D) or microtubules (with colchicine). When these agents are added at progressively later times, the effect of insulin becomes evident in a time-dependent manner. [I.sub.ss] is also augmented by insulin in control cells in a cytoskeleton-dependent manner. However, in contrast to diabetic cells, cytoskeleton-dependent augmentation of [I.sub.ss] by insulin occurs at a considerably faster rate in control cells. Immunofluorescent labeling shows a reduced density of [Beta]-tubulin in diabetic cells, particularly in perinuclear regions. In vitro insulin replacement or in vivo insulin injections given to STZ-treated rats enhances [Beta]-tubulin density. These results suggest an impairment of cytoskeleton function and structure under insulin-deficient conditions, which may have implications for cardiac function. cardiac potassium channels; microtubules; actin microfilaments

Shimoni, Y., D. Severson, and H. S. Ewart. Insulin resistance and the modulation of rat cardiac [K.sup.+] currents. Am J Physiol Heart Circ Physiol 279: H639-H649, 2000.-- [K.sup.+] currents were measured using a whole cell voltage-clamp method in enzymatically isolated rat ventricular myocytes obtained from two hyperinsulinemic, insulin-resistant models. Fructose-fed rats as well as genetically obese rats, both of which are resistant to the metabolic effects of insulin, were used. The normal augmentation of a calcium-independent sustained [K.sup.+] current was reduced or abolished in insulin-resistant states. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadyl sulfate (3-4 wk treatment or after 5-6 h in vitro) enhanced the sustained [K.sup.+] current. The in vitro effect of vanadyl was blocked by cycloheximide. Insulin resistance of the [K.sup.+] current was not reversed by vanadyl sulfate. The results show that insulin resistance is expressed in terms of insulin actions on ion channels, in addition to its actions on metabolism. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadate compounds, which mimic the effects of insulin on metabolism, also mimic the augmenting effects of insulin on a cardiac [K.sup.+] current in a manner suggesting synthesis of new channels. cardiac potassium channels; diabetes mellitus; vanadate; metformin

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/dtmax) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents (INa) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of INa. All of these treatments rescued INa to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of INa. To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice. [ABSTRACT FROM AUTHOR]

Presents information on a study which tested the hypothesis that cytoskeleton-dependent effects of insulin on potassium currents follow a different time course in cardiac myocytes from control and diabetic rats. Methodology; Results and discussion.

Reports that in insulin resistance there is an attenuation or abolition of the augmenting effect of insulin on the steady-state K[sup +] current I[sub ss]. Separation of currents; Fructose-fed model; Reversal of insulin resistance by insulin-sensitizing drugs; Modulation of insulin sensitivity.

Hearts from streptozotocin (STZ)-induced diabetic rats have previously been shown to have impaired intercellular electrical coupling, due to reorganization (lateralization) of connexin43 proteins. Due to the resulting reduction in conduction reserve, conduction velocity in diabetic hearts is more sensitive to conditions that reduce cellular excitability or intercellular electrical coupling. Diabetes is a known risk factor for cardiac ischemia, a condition associated with both reduced cellular excitability and reduced intercellular coupling. Activation of Ca(2+)-independent phospholipase A(2) (iPLA(2)) is known to be part of the response to acute ischemia and may contribute to the intercellular uncoupling by causing increased levels of arachidonic acid and lysophosphatidyl choline. Normally perfused diabetic hearts are known to exhibit increased iPLA(2) activity and may thus be particularly sensitive to further activation of these enzymes. In this study, we used voltage-sensitive dye mapping to assess changes in conduction velocity in response to acute global ischemia in Langendorff-perfused STZ-induced diabetic hearts. Conduction slowing in response to ischemia was significantly larger in STZ-induced diabetic hearts compared with healthy controls. Similarly, slowing of conduction velocity in response to acidosis was also more pronounced in STZ-induced diabetic hearts. Inhibition of iPLA(2) activity using bromoenol lactone (BEL; 10 μM) had no effect on the response to ischemia in healthy control hearts. However, in STZ-induced diabetic hearts, BEL significantly reduced the amount of conduction slowing observed beginning 5 min after the onset of ischemia. BEL treatment also significantly increased the time to onset of sustained arrhythmias in STZ-induced diabetic hearts but had no effect on the time to arrhythmia in healthy control hearts. Thus, our results suggest that iPLA(2) activation in response to acute ischemia in STZ-induced diabetic hearts is more pronounced than in control hearts and that this response is a significant contributor to arrhythmogenic conduction slowing.

The incidence of diabetes mellitus is increasing. Cardiac dysfunction often develops, resulting in diverse arrhythmias. These arise from ion channel remodeling or from altered speed and pattern of impulse propagation. Few studies have investigated impulse propagation in the diabetic heart. We previously showed a reduced conduction reserve in the diabetic heart, with associated changes in intercellular gap junctions. The present study investigated whether these effects are sex specific. Hearts from control and streptozotocin-diabetic male and female rats were used. Optical mapping was performed with the voltage-sensitive dye di-4-ANEPPS, using Langendorff-perfused hearts. Isolated ventricular cells and tissue sections were used for immunofluorescent labeling of the gap junction protein connexin43 (Cx43). The gap junction uncoupler heptanol (0.75 mM) or elevated K(+) (9 mM, to reduce cell excitability) produced significantly greater slowing of propagation in diabetic males than females. In ovariectomized diabetic females, 9 mM K(+) slowed conduction significantly more than in nonovariectomized females. The subcellular redistribution (lateralization) of the gap junction protein Cx43 was smaller in diabetic females. Pretreatment of diabetic males with the angiotensin-converting enzyme inhibitor quinapril reduced Cx43 lateralization and the effects of 9 mM K(+) on propagation. In conclusion, the slowing of cardiac impulse propagation in type 1 diabetes is smaller in female rats, partly due to the presence of female sex hormones. This difference is (partly) mediated by sex differences in activation of the cardiac renin-angiotensin system.

The autocrine modulation of cardiac K(+) currents was compared in ventricular and atrial cells (V and A cells, respectively) from Type 1 diabetic rats. K(+) currents were measured by using whole cell voltage clamp. ANG II was measured by ELISA and immunofluorescent labeling. Oxidative stress was assessed by immunofluorescent labeling with dihydroethidium, a measure of superoxide ions. In V cells, K(+) currents are attenuated after activation of the renin-angiotensin system (RAS) and the resulting ANG II-mediated oxidative stress. In striking contrast, these currents are not attenuated in A cells. Inhibition of the angiotensin-converting enzyme (ACE) also has no effect, in contrast to current augmentation in V cells. ANG II levels are enhanced in V, but not in A, cells. However, the high basal ANG II levels in A cells suggest that in these cells, ANG II-mediated pathways are suppressed, rather than ANG II formation. Concordantly, superoxide ion levels are lower in diabetic A than in V cells. Several findings indicate that high atrial natriuretic peptide (ANP) levels in A cells inhibit RAS activation. In male diabetic V cells, in vitro ANP (300 nM-1 muM, >5 h) decreases oxidative stress and augments K(+) currents, but not when excess ANG II is present. ANP has no effect on ventricular K(+) currents when the RAS is not activated, as in control males, in diabetic males treated with ACE inhibitor and in diabetic females. In conclusion, the modulation of K(+) currents and oxidative stress is significantly different in A and V cells in diabetic rat hearts. The evidence suggests that this is largely due to inhibition of RAS activation and/or action by ANP in A cells. These results may underlie chamber-specific arrhythmogenic mechanisms.

Gender differences were studied in ventricular myocytes from insulin-deficient (Type 1) diabetic rats. Cells were obtained by enzymatic dispersion of hearts from control male and female rats and from rats made diabetic with streptozotocin (100 mg/kg) 7-14 days before experiments. ANG II content, measured by ELISA, was augmented in diabetic males but unaltered in diabetic females. In diabetic ovariectomized females, ANG II levels were augmented as in males. ANG II affects multiple cellular pathways including activation of protein kinase C (PKC) and several tyrosine kinases as well as inhibition of protein kinase A (PKA). The involvement of these pathways in modulating outward K(+) currents was studied. Transient and sustained outward K(+) currents were measured using the whole cell voltage-clamp method. In males, these currents are attenuated under diabetic conditions but are augmented by the ANG II-converting enzyme inhibitor quinapril. Activation of PKA by 8-bromo-cAMP enhanced both K(+) currents in cells from diabetic males. The augmentation of these currents by quinapril was blocked when PKA inhibition was maintained with the Rp isomer of 3',5'-cyclic monophosphorothioate. Inhibition of tyrosine kinases by genistein also augmented K(+) currents in cells from diabetic males. Action potentials were abbreviated by 8-bromo-cAMP and genistein. However, both genistein and 8-bromo-cAMP had no effect on K(+) currents in cells from diabetic females. In cells from ovariectomized diabetic females, 8-bromo-cAMP and genistein enhanced these K(+) currents as in males. Inhibition of PKC augmented the transient and sustained K(+) currents in cells from diabetic males and females. A contribution of non-ANG II-dependent activation of PKC is suggested. These results describe some of the mechanisms that may underlie gender-specific differences in the development of cardiac disease and arrhythmias.

Hearts from insulin-resistant type 2 diabetic db/db mice exhibit features of a diabetic cardiomyopathy with altered metabolism of exogenous substrates and reduced contractile performance. Therefore, the effect of chronic oral administration of 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH), a novel ligand for peroxisome proliferator-activated receptor-gamma that produces insulin sensitization, to db/db mice (30 mg/kg for 6 wk) on cardiac function was assessed. COOH treatment reduced blood glucose from 27 mM in untreated db/db mice to a normal level of 10 mM. Insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db hearts. Working perfused hearts from COOH-treated db/db mice demonstrated metabolic changes with enhanced glucose oxidation and decreased palmitate oxidation. However, COOH treatment did not improve contractile performance assessed with ex vivo perfused hearts and in vivo by echocardiography. The reduced outward K+ currents in diabetic cardiomyocytes were still attenuated after COOH. Metabolic changes in COOH-treated db/db hearts are most likely indirect, secondary to changes in supply of exogenous substrates in vivo and insulin sensitization.

Transient and sustained K(+) currents were measured in isolated rat ventricular myocytes obtained from control, steptozotocin-induced (Type 1) diabetic, and hypothyroid rats. Both currents, attenuated by the endocrine abnormalities, were significantly augmented by in vitro incubation (>6 h) with the angiotensin-converting enzyme inhibitor quinapril or the angiotensin II (ANG II) receptor blocker saralasin. Western blots indicated a parallel increase in Kv4.2 and Kv1.2, channel proteins that underlie the transient and (part of the) sustained currents. Under diabetic and hypothyroid conditions, both currents were also augmented by an endothelin receptor blocker (PD142893) or by an endothelin-converting enzyme inhibitor. Kv4.2 density was also enhanced by PD142893. Incubation (>5 h) with the PKC inhibitor bis-indolylmaleimide augmented both currents, whereas the PKC activator dioctanoyl-rac-glycerol (DiC8) prevented the augmentation of currents by quinapril. DiC8 also prevented the augmentation of Kv4.2 density by quinapril. Specific peptides that activate PKC translocation indicated that PKC-epsilon and not PKC-delta is involved in ANG II action on these currents. In control myocytes, quinapril and PD142893 augmented the sustained late current but had no effect on peak current. It is concluded that an autocrine release of angiotensin and endothelin in diabetic and hypothyroid conditions attenuates K(+) currents by suppressing the synthesis of some K(+) channel proteins, with the effects mediated at least partially by PKC-epsilon.