Reduced conduction reserve in the diabetic rat heart: role of iPLA2 activation in the response to ischemia.

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.

Simulations of reduced conduction reserve in the diabetic rat heart: response to uncoupling and reduced excitability.

Experimental results have shown that action potential (AP) conduction in ventricular tissue from streptozotocin-diabetic (STZ) rats is compromised. This was manifest as increased sensitivity of conduction velocity (CV) to the gap junction uncoupler heptanol, as well as increased sensitivity of CV to reduced cellular excitability due to elevated extracellular K(+) concentration, in the STZ hearts. This "reduced conduction reserve" has been suggested to be due to lateralization of connexin43 (Cx43) proteins, rendering them nonfunctional, resulting in compromised intercellular electrical coupling. In this study, we have used computer simulations of one-dimensional AP conduction in a model of rat ventricular myocytes to verify this interpretation. Our results show that compromised intercellular coupling indeed reduces conduction reserve and predict a response to gap junction uncoupling with heptanol that is consistent with experiments. However, our simulations also show that compromised intercellular coupling is insufficient to explain the increased sensitivity to reduced cellular excitability. A thorough investigation of possible underlying mechanisms, suggests that subtle alterations in the voltage-dependence of steady-state gating for the Na(+) current (I (Na)), combined with compromised intercellular coupling, is a likely mechanism for these observations.

Sex-dependent impairment of cardiac action potential conduction in type 1 diabetic rats.

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.

K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association.

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292-296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

Skeletal and cardiac ryanodine receptors exhibit different responses to Ca2+ overload and luminal ca2+.

Spontaneous Ca(2+) release occurs in cardiac cells during sarcoplasmic reticulum Ca(2+) overload, a process we refer to as store-overload-induced Ca(2+) release (SOICR). Unlike cardiac cells, skeletal muscle cells exhibit little SOICR activity. The molecular basis of this difference is not well defined. In this study, we investigated the SOICR properties of HEK293 cells expressing RyR1 or RyR2. We found that HEK293 cells expressing RyR2 exhibited robust SOICR activity, whereas no SOICR activity was observed in HEK293 cells expressing RyR1. However, in the presence of low concentrations of caffeine, SOICR could be triggered in these RyR1-expressing cells. At the single-channel level, we showed that RyR2 is much more sensitive to luminal Ca(2+) than RyR1. To identify the molecular determinants responsible for these differences, we constructed two chimeras between RyR1 and RyR2, N-RyR1(1-4006)/C-RyR2(3962-4968) and N-RyR2(1-3961)/C-RyR1(4007-5037). We found that replacing the C-terminal region of RyR1 with the corresponding region of RyR2 (N-RyR1/C-RyR2) dramatically enhanced the propensity for SOICR and the response to luminal Ca(2+), whereas replacing the C-terminal region of RyR2 with the corresponding region of RyR1 (N-RyR2/C-RyR1) reduced the propensity for SOICR and the luminal Ca(2+) response. These observations indicate that the C-terminal region of RyR is a critical determinant of both SOICR and the response to luminal Ca(2+). These chimeric studies also reveal that the N-terminal region of RyR plays an important role in regulating SOICR and luminal Ca(2+) response. Taken together, our results demonstrate that RyR1 differs markedly from RyR2 with respect to their responses to Ca(2+) overload and luminal Ca(2+), and suggest that the lack of spontaneous Ca(2+) release in skeletal muscle cells is, in part, attributable to the unique intrinsic properties of RyR1.

Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon beta-adrenergic stimulation in normal and failing hearts.

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

Differential autocrine modulation of atrial and ventricular potassium currents and of oxidative stress in diabetic rats.

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.

Dexamethasone and cardiac potassium currents in the diabetic rat.

Experiments were designed to compare effects of dexamethasone on transient (Ipeak) and sustained (Isus) K+ currents in control and diabetic rat myocytes. Ventricular myocytes were isolated from control or type 1 streptozotocin (STZ)-induced diabetic male and female rats. Currents were measured using whole-cell voltage-clamp methods. Incubation of cells from control males or females with 100 nM dexamethasone (5-9 h) significantly (P<0.005) augmented Isus (by 28-31%). Ipeak was unchanged. Isus augmentation was abolished by cycloheximide or cytochalasin D, but not by inhibition of protein kinases A or C. Inhibition of tyrosine kinases by genistein (but not its inactive analog genistin) prevented the increase of Isus by dexamethasone. In marked contrast, dexamethasone had a significantly (P<0.015) smaller effect on Isus (11% increase) in cells from male STZ-diabetic rats, as compared to control cells. However, Isus augmentation in cells from female STZ-diabetic rats was normal (31% increase). In ovariectomized-diabetic rats, Isus was unchanged by dexamethasone. The reduced effect in diabetic males might be due to preactivation of tyrosine kinases linking dexamethasone to current modulation. In conclusion, type I diabetes is associated with gender-specific changes in sensitivity of K+ currents to glucocorticoids, linked to alterations in tyrosine-phosphorylated proteins.

Gender differences in ANG II levels and action on multiple K+ current modulation pathways in diabetic rats.

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.

Gender-dependent attenuation of cardiac potassium currents in type 2 diabetic db/db mice.

Single ventricular myocytes were prepared from control db/+ and insulin-resistant diabetic db/db male mice at 6 and 12 weeks of age. Peak and sustained outward potassium currents were measured using whole-cell voltage clamp methods. At 6 weeks currents were fully developed in control and diabetic mice, with no differences in the density of either current. By 12 weeks both currents were significantly attenuated in the diabetic mice, but could be augmented by in vitro incubation with the angiotensin-converting enzyme (ACE) inhibitor quinapril (1 microM, 5-9 h). In cells from female db/db mice (12 weeks of age), K(+) currents were not attenuated and no effects of quinapril were observed. To investigate whether lack of insulin action accounts for these gender differences, cells were also isolated from cardiomyocyte-specific insulin receptor knockout (CIRKO) mice. Both K(+) currents were significantly attenuated in cells from male and female CIRKO mice, and action potentials were significantly prolonged. Incubation with quinapril did not augment K(+) currents. Our results demonstrate that type 2 diabetes is associated with gender-selective attenuation of K(+) currents in cardiomyocytes, which may underlie gender differences in the development of some cardiac arrhythmias. The mechanism for attenuation of K(+) currents in cells from male mice is due, at least in part, to an autocrine effect resulting from activation of a cardiac renin-angiotensin system. Insulin is not involved in these gender differences, since the absence of insulin action in CIRKO mice diminishes K(+) currents in cells from both males and females.

Treatment of type 2 diabetic db/db mice with a novel PPARgamma agonist improves cardiac metabolism but not contractile function.

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.

Sex differences in the modulation of K+ currents in diabetic rat cardiac myocytes.

A transient (Ipeak) and a sustained (Isus) outward K+ current were measured, using whole-cell voltage-clamp methods, in isolated rat ventricular myocytes obtained by enzymatic dispersion. A comparison was made between male and female rats following induction of (insulin-deficient) diabetes with streptozotocin (STZ). In control (non-diabetic) rats, both currents were smaller in cells obtained from females, as compared to males (P<0.005). However, whereas inducing diabetes in male rats significantly attenuated both Ipeak and Isus (P<0.005), Ipeak was unchanged in female diabetic rats. Isus was significantly (P<0.005) reduced, but the extent of reduction was smaller (P<0.02) than in males. The formation of angiotensin II (ATII) or endothelin-1 (ET-1) was blocked using inhibitors of angiotensin-converting enzyme (ACE) and endothelin-converting enzyme (ECE), respectively. In cells from diabetic males both inhibitors significantly (P<0.005) enhanced K+ currents. In contrast, no effect was observed in cells from female diabetic rats. However, in ovariectomized (Ovx) diabetic females the in vitro inhibition of ATII and ET-1 formation augmented the two K+ currents, but not when oestradiol was administered in vivo prior to cell isolation. In cells from diabetic males, incubation with 100 nM 17beta-oestradiol significantly (P<0.005) enhanced both Ipeak and Isus. This effect was blocked if ATII or ET-1 was added to the medium. These results show that autocrine modulation of K+ currents by renin-angiotensin and endothelin systems is attenuated or absent in female diabetic rats. Oestradiol plays a key role in reducing this modulation. These results may underlie some of the sex differences associated with development of cardiac arrhythmias.

Role of PKC in autocrine regulation of rat ventricular K+ currents by angiotensin and endothelin.

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.