George J. Rozanski

Up-regulation of K+ channels in diabetic rat ventricular myocytes by insulin and glutathione

OBJECTIVE The cardiac pathogenesis of diabetes mellitus involves oxidative stress that elicits profound changes in myocardial glutathione, an endogenous regulator of cell function. This study examined the role of glutathione in regulating K(+) channel activity in isolated ventricular myocytes from diabetic rats and its relationship to insulin signaling. METHODS AND RESULTS Colorimetric analysis of extracts of ventricular tissue from Sprague-Dawley rats showed that the basal level of reduced glutathione (GSH) was significantly less in rats with experimental diabetes compared with sham controls, consistent with oxidative stress conditions. This change in GSH status paralleled a significant decrease in the activity of gamma-glutamylcysteine synthetase, a major pathway involved in GSH homeostasis. Voltage-clamp studies confirmed that, compared with control myocytes, K(+) channels carrying the transient outward current (I(to)) are down-regulated in the diabetic state and that this electrophysiological change is reversed by in vitro treatment with insulin for 2-3 h. Incubation of diabetic rat myocytes with GSH also normalized I(to) density compared with untreated myocytes, but with a longer time course than insulin. To determine if up-regulation of I(to) by insulin was mediated by alterations in myocyte GSH, insulin-responsiveness of diabetic rat myocytes was tested in the presence of 1,3-bis-chloroethyl-nitrosourea, an inhibitor of glutathione reductase, or buthionine sulfoximine, a blocker of gamma-glutamylcysteine synthetase. Neither blocker alone altered I(to) density in diabetic rat myocytes when compared with untreated cells, but each blocked the effect of insulin to up-regulate I(to). CONCLUSIONS These data suggest that oxidative stress-induced alteration in GSH redox state plays an important role in regulating I(to) channel function and that GSH homeostasis in ventricular myocytes is functionally coupled to insulin signaling.

Exercise training during diabetes attenuates cardiac ryanodine receptor dysregulation

The present study was undertaken to assess the effects of exercise training (ExT) initiated after the onset of diabetes on cardiac ryanodine receptor expression and function. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin (STZ). Three weeks after STZ injection, diabetic rats were divided into two groups. One group underwent ExT for 4 wk while the other group remained sedentary. After 7 wk of sedentary diabetes, cardiac fractional shortening, rate of rise of left ventricular pressure, and myocyte contractile velocity were reduced by 14, 36, 44%, respectively. Spontaneous Ca(2+) spark frequency increased threefold, and evoked Ca(2+) release was dyssynchronous with diastolic Ca(2+) releases. Steady-state type 2 ryanodine receptor (RyR2) protein did not change, but its response to Ca(2+) was altered. RyR2 also exhibited 1.8- and 1.5-fold increases in phosphorylation at Ser(2808) and Ser(2814). PKA activity was reduced by 75%, but CaMKII activity was increased by 50%. Four weeks of ExT initiated 3 wk after the onset of diabetes blunted decreases in cardiac fractional shortening and rate of left ventricular pressure development, increased the responsiveness of the myocardium to isoproterenol stimulation, attenuated the increase in Ca(2+) spark frequency, and minimized dyssynchronous and diastolic Ca(2+) releases. ExT also normalized the responsiveness of RyR2 to Ca(2+) activation, attenuated increases in RyR2 phosphorylation at Ser(2808) and Ser(2814), and normalized CaMKII and PKA activities. These data are the first to show that ExT during diabetes normalizes RyR2 function and Ca(2+) release from the sarcoplasmic reticulum, providing insights into mechanisms by which ExT during diabetes improves cardiac function.

Cardiac Sympathetic Afferent Denervation Attenuates Cardiac Remodeling and Improves Cardiovascular Dysfunction in Rats with Heart Failure

The enhanced cardiac sympathetic afferent reflex (CSAR) contributes to the exaggerated sympathoexcitation in chronic heart failure (CHF). Increased sympathoexcitation is positively related to mortality in patients with CHF. However, the potential beneficial effects of chronic CSAR deletion on cardiac and autonomic function in CHF have not been previously explored. Here, we determined the effects of chronic CSAR deletion on cardiac remodeling and autonomic dysfunction in CHF. To delete the transient receptor potential vanilloid 1 receptor–expressing CSAR afferents selectively, epicardial application of resiniferatoxin (50 &mgr;g/mL), an ultrapotent analog of capsaicin, was performed during myocardium infarction surgery in rats. This procedure largely abolished the enhanced CSAR, prevented the exaggerated renal and cardiac sympathetic nerve activity and improved baroreflex sensitivity in CHF rats. Most importantly, we found that epicardial application of resiniferatoxin largely prevented the elevated left ventricle end-diastolic pressure, lung edema, and cardiac hypertrophy, partially reduced left ventricular dimensions in the failing heart, and increased cardiac contractile reserve in response to &bgr;-adrenergic receptor stimulation with isoproterenol in CHF rats. Molecular evidence showed that resiniferatoxin attenuated cardiac fibrosis and apoptosis and reduced expression of fibrotic markers and transforming growth factor-&bgr; receptor I in CHF rats. Pressure–volume loop analysis showed that resiniferatoxin reduced the end-diastolic pressure volume relationships in CHF rats, indicating improved cardiac compliance. In summary, cardiac sympathetic afferent deletion exhibits protective effects against deleterious cardiac remodeling and autonomic dysfunction in CHF. These data suggest a potential new paradigm and therapeutic potential in the management of CHF.

Regulation of glutathione in cardiac myocytes

Reduced glutathione (GSH) is an essential, multifunctional tripepetide that controls redox-sensitive cellular processes, but its regulation in the heart is poorly understood. The present study used a pharmocological model of GSH depletion to examine cellular mechanisms controlling cardiac GSH. Inhibition of GSH metabolism was elicited in normal rats by daily injections of buthionine sulfoximine (BSO), a blocker of gamma-glutamylcysteine synthetase, plus 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. After 3 d of BSO/BCNU treatment, intracellular [GSH] was measured in isolated-ventricular myocytes by fluorescence microscopy using the probe monochlorobimane. Basal [GSH] in left-ventricular myocytes from BSO/BCNU-treated rats (2.0 +/- 0.05 amol/microm(3), n = 146) was 50% less than control (4.0 +/- 0.13 amol/microm(3), n = 116; P < 0.05). Incubation of myocytes from BSO/BCNU rats with 0.1 microM insulin normalized [GSH] after a delay of 3-4 h (3.6 +/- 0.29 amol/microm(3), n = 66). This effect of insulin was blocked by pre-treating myocytes with cycloheximide. A protein tyrosine phosphatase inhibitor, bis-peroxovanadium-1,10-phenanthroline (bpV(phen), 1 microM), elicited a similar effect as insulin, while neither agent altered [GSH] in myocytes from control rats. Moreover, the effect of insulin and bpV(phen) to up-regulate GSH was blocked by inhibitors of PI 3-kinase (wortmannin, LY294002), MEK (PD98059) and p38 MAP kinases (SB203580). These data suggest that the insulin-signaling cascade regulates [GSH] in ventricular myocytes by a coordinated activation of PI 3-kinase and MAP kinase pathways. These signaling mechanisms may play essential roles in controlling intracellular redox state and normal function of cardiac myocytes.

Developmental changes in AT1 and AT2 receptor-protein expression in rats

It has long been known that angiotensin type-1 receptors (AT1R) play a critical role in sympathetic regulation, cardiovascular activity, and hormone secretion under physiological and pathological states. On the other hand, the functional significance of angiotensin type-2 receptors (AT2R) is poorly understood. In a recent study we demonstrated that, in rats with chronic heart failure, AT1R protein expression was increased but AT2R expression was decreased in the rostral ventrolateral medulla (RVLM). This imbalance of angiotensin receptors contributed to sympatho-excitation in the heart failure state. In the current experiment, we measured AT1R and AT2R protein expressions in the brainstem, kidney and liver from male foetuses (3 days before birth), male neonates (3 days after birth), male and female adults (8 weeks) and male aged (28 months) rats by Western blot analysis. In the brainstem, we found that the foetuses and neonates exhibited a significantly lower AT2R protein expression compared with adult rats (foetus 0.08 ± 0.01, neonate 0.12 ± 0.01, male adult 0.25 ± 0.01, female adult 0.22 ± 0.02; n = 4 per group, p < 0.001 foetus and neonate compared with male or female adults). In contrast, the foetuses and neonates expressed significantly higher AT1R protein than that of the adults (foetus 0.64 ± 0.09, neonate 0.56 ± 0.01, male adult 0.13 ± 0.02, female adult 0.08 ± 0.02; n = 4 each group, p < 0.001 foetus and neonate compared with male and female adults). In the liver, the AT2R protein was also higher in foetus and neonate, than in adult rats. Interestingly, the foetal liver expressed higher AT1R protein compared with that of the neonate. In the kidney, AT2R expression was significantly increased with age (foetus 0.08 ± 0.01, neonate 0.19 ± 0.02, male adult 0.49 ± 0.04, female adult 0.90 ± 0.10; n = 4 per group, p < 0.01—0.001). AT1R expression, on the other hand, was higher in the foetuses than that in both neonate and male adults. This study provides data contrary to existing dogma that AT2R expression is higher in foetal life and low in adults, suggesting an involvement of a potentially important functional role for AT2R in adult animals and AT1R in foetal development and/or physiology.

A Metabolic Mechanism For Cardiac K+ Channel Remodelling

1. Electrical remodelling of the ventricle is a common pathogenic feature of cardiovascular disease states that lead to heart failure. Experimental data suggest this change in electrophysiological phenotype is largely due to downregulation of K+ channels involved in repolarization of the action potential.

Attenuated outward potassium currents in carotid body glomus cells of heart failure rabbit: involvement of nitric oxide

It has been shown that peripheral chemoreceptor sensitivity is enhanced in both clinical and experimental heart failure (HF) and that impairment of nitric oxide (NO) production contributes to this enhancement. In order to understand the cellular mechanisms associated with the alterations of chemoreceptor function and the actions of NO in the carotid body (CB), we compared the outward K+ currents (IK) of glomus cells in sham rabbits with that in HF rabbits and monitored the effects of NO on these currents. Ik was measured in glomus cells using conventional and perforated whole‐cell configurations. IK was attenuated in glomus cells of HF rabbits, and their resting membrane potentials (−34.7 ± 1.0 mV) were depolarized as compared with those in sham rabbits (−47.2 ± 1.9 mV). The selective Ca2+‐dependent K+ channel (KCa) blocker iberiotoxin (IbTx, 100 nm) reduced IK in glomus cells from sham rabbits, but had no effect on IK from HF rabbits. In perforated whole‐cell mode, the NO donor SNAP (100 μm) increased IK in glomus cells from HF rabbits to a greater extent than that in sham rabbits (P < 0.01), and IbTx inhibited the effects of SNAP. However, in conventional whole‐cell mode, SNAP had no effect. Nω‐nitro‐L‐arginine (L‐NNA, NO synthase inhibitor) decreased Ik in sham rabbits but not in HF rabbits. The guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ) inhibited the effect of SNAP on Ik. These results demonstrate that IK is reduced in CB glomus cells from HF rabbits. This effect is due mainly to the suppression of KCa channel activity caused by decreased availability of NO. In addition, intracellular cGMP is necessary for the KCa channel modulation by NO.

Exercise training initiated after the onset of diabetes preserves myocardial function: effects on expression of β-adrenoceptors

The present study was undertaken to assess cardiac function and characterize beta-adrenoceptor subtypes in hearts of diabetic rats that underwent exercise training (ExT) after the onset of diabetes. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. Four weeks after induction, rats were randomly divided into two groups. One group was exercised trained for 3 wk while the other group remained sedentary. At the end of the protocol, cardiac parameters were assessed using M-mode echocardiography. A Millar catheter was also used to assess left ventricular hemodynamics with and without isoproterenol stimulation. beta-Adrenoceptors were assessed using Western blots and [(3)H]dihydroalprenolol binding. After 7 wk of diabetes, heart rate decreased by 21%, fractional shortening by 20%, ejection fraction by 9%, and basal and isoproterenol-induced dP/dt by 35%. beta(1)- and beta(2)-adrenoceptor proteins were reduced by 60% and 40%, respectively, while beta(3)-adrenoceptor protein increased by 125%. Ventricular homogenates from diabetic rats bound 52% less [(3)H]dihydroalprenolol, consistent with reductions in beta(1)- and beta(2)-adrenoceptors. Three weeks of ExT initiated 4 wk after the onset of diabetes minimized cardiac function loss. ExT also blunted loss of beta(1)-adrenoceptor expression. Interestingly, ExT did not prevent diabetes-induced reduction in beta(2)-adrenoceptor or the increase of beta(3)-adrenoceptor expression. ExT also increased [(3)H]dihydroalprenolol binding, consistent with increased beta(1)-adrenoceptor expression. These findings demonstrate for the first time that ExT initiated after the onset of diabetes blunts primarily beta(1)-adrenoceptor expression loss, providing mechanistic insights for exercise-induced improvements in cardiac function.

Carbonylation of myosin heavy chains in rat heart during diabetes.

Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague-Dawley rats were injected with streptozotocin (STZ). Fourteen days later the animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for 6 weeks, while the other group received no treatment. After 8 weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+dP/dt) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca(2+)- and Mg(2+)-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-alpha to MHC-beta ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-alpha and MHC-beta. Aminoguanidine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca(2+)-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +dP/dt induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes.

Early afterdepolarizations and triggered activity in rabbit cardiac Purkinje fibers recovering from ischemic-like conditions. Role of acidosis.

BackgroundThe mechanisms underlying repetitive activity during reperfusion of ischemic myocardium are thought to include triggered responses elicited at short pacing cycle lengths. The potential to generate repetitive responses at longer pacing cycle lengths under similar conditions, however, has not been explored. Thus, the present study examined the role of cycle length on the cellular electrical changes produced during recovery from ischemic-like conditions and identified the major component precipitating nondriven, repetitive activity. Methods and ResultsTransmembrane potentials were recorded in vitro from isolated rabbit Purkinje fibers exposed to hypoxia (defined as Po2<30 mm Hg, high [K+]0, and zero glucose) plus lactic acidosis (pH 6.7) for 45 minutes and during recovery in normal Tyrodes solution (pH 7.4). Compared with control, action potential duration (90% repolarization) during recovery increased transiently by 40.9±11.8 and 241.0±51.1 msec at respective basic cycle lengths of 1,000 and 3,000 msec (both p < 0.005). In 81% of preparations, action potential prolongation was accompanied by early afterdepolarizations and triggered activity generated from low (positive to -40 mV) or high (negative to -40 mV) membrane potentials. In 62% of experiments, brief periods of abnormal automaticity also occurred. Triggered responses were 1) unaffected by 1 μM ryanodine, 2) abolished by pacing at short basic cycle lengths or by exposing tissues to 2.5 μg/ml lidocaine, and 3) more easily induced at long basic cycle lengths or by superfusing 2.5 μg/ml quinidine. When tissues were conditioned with hypoxia alone (pH 7.4), action potential prolongation on recovery was comparatively small, and nondriven responses did not develop. Conversely, addition of 10-20 μM amiloride to the hypoxic, acidic test solution augmented recovery-induced action potential prolongation. ConclusionsWe conclude that acidosis, as a component of ischemia, plus slow pacing frequencies may mediate the genesis of early afterdepolarizations and triggered activity in Purkinje fibers on recovery, long after extracellular pH has been restored to normal. These data may have clinical relevance to the mechanisms of reperfusion arrhythmias in the intact human heart.