Sympatho-excitatory response to pulmonary chemosensitive spinal afferent activation in anesthetized, vagotomized rats.

The sensory innervation of the lung is well known to be innervated by nerve fibers of both vagal and sympathetic origin. Although the vagal afferent innervation of the lung has been well characterized, less is known about physiological effects mediated by spinal sympathetic afferent fibers. We hypothesized that activation of sympathetic spinal afferent nerve fibers of the lung would result in an excitatory pressor reflex, similar to that previously characterized in the heart. In this study, we evaluated changes in renal sympathetic nerve activity (RSNA) and hemodynamics in response to activation of TRPV1-sensitive pulmonary spinal sensory fibers by agonist application to the visceral pleura of the lung and by administration into the primary bronchus in anesthetized, bilaterally vagotomized, adult Sprague-Dawley rats. Application of bradykinin (BK) to the visceral pleura of the lung produced an increase in mean arterial pressure (MAP), heart rate (HR), and RSNA. This response was significantly greater when BK was applied to the ventral surface of the left lung compared to the dorsal surface. Conversely, topical application of capsaicin (Cap) onto the visceral pleura of the lung, produced a biphasic reflex change in MAP, coupled with increases in HR and RSNA which was very similar to the hemodynamic response to epicardial application of Cap. This reflex was also evoked in animals with intact pulmonary vagal innervation and when BK was applied to the distal airways of the lung via the left primary bronchus. In order to further confirm the origin of this reflex, epidural application of a selective afferent neurotoxin (resiniferatoxin, RTX) was used to chronically ablate thoracic TRPV1-expressing afferent soma at the level of T1-T4 dorsal root ganglia pleura. This treatment abolished all sympatho-excitatory responses to both cardiac and pulmonary application of BK and Cap in vagotomized rats 9-10 weeks post-RTX. These data suggest the presence of an excitatory pulmonary chemosensitive sympathetic afferent reflex. This finding may have important clinical implications in pulmonary conditions inducing sensory nerve activation such as pulmonary inflammation and inhalation of chemical stimuli.

Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states.

KEY POINTS: Cardiac sympathetic afferents are considered to be essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischaemia. However, a potential contribution of the CSAR control of cardiac dysfunction in both normal and chronic heart failure (CHF) states remains unknown. We found that activation of the CSAR evokes little increase in cardiac contractility with an exaggerated peripheral vasoconstriction in the CHF state. CSAR inhibition by epicardial lidocaine decreased cardiac contractility to a greater extent in CHF rats than sham rats. Furthermore, we also found that epicardial lidocaine paradoxically decreased left ventricular end-diastolic pressure (LVEDP) and left ventricular end-diastolic volume (preload) in CHF rats, which was not observed in sham rats. Chronic ablation of the CSAR by epicardial application of the afferent neurotoxin, RTX, selectively lowered diastolic blood pressure CHF rats. The observation suggests that CSAR has a differential effect on cardiac function in normal and CHF states. CSAR activation in normal state causes significant increase in cardiac contractility and cardiac output. ABSTRACT: The enhanced 'cardiac sympathetic afferent reflex' (CSAR) critically contributes to the exaggerated global sympathetic tone in chronic heart failure (CHF). However, a potential contribution of the cardio-cardiac reflex control of cardiac function in both normal and CHF states remains unknown. In this study, we evaluated the effects of direct activation or inhibition of the CSAR on cardiac function by pressure-volume (P-V) loop analysis in ∼12-week sham-operated and myocardial infarcted (MI) rats. In sham rats, acute CSAR activation by epicardial application of bradykinin (BK) increased heart rate (HR), left ventricular systolic pressure (LVSP), the maximum first derivative of left ventricular pressure (dp/dtmax ), and the slope of the end-systolic P-V relationship (ESPVR), suggesting that acute CSAR activation in the normal state enhances myocardial contractility. CSAR activation also decreased left ventricular (LV) systolic and diastolic volumes with little effect on LV end-diastolic pressure (LVEDP) or the end-diastolic P-V relationship (EDPVR) in sham rats. Compared to sham, CHF rats exhibit a reduced increase in the slope of the ESPVR and dp/dtmax in response to BK, indicating a poor contractile response to CSAR activation. Interestingly, BK application in CHF rats increased cardiac systolic and diastolic volumes and further increased the elevated LVEDP, neither of which was seen in sham rats. Following CSAR inhibition by epicardial lidocaine, blood pressure, HR, LVSP, dp/dt, LVEDP and ESPVR decreased in CHF rats whereas lidocaine had little effect in sham rats, indicating that the CSAR is tonically active in CHF and contributes to cardiac dysfunction. Furthermore, we found that epicardial lidocaine paradoxically decreased LV end-diastolic volume (preload) in CHF rats, which was not observed in sham rats. The decreased preload by lidocaine in CHF rats may be due to a reduction in peripheral vascular resistance since epicardial lidocaine significantly lowered peripheral (renal) sympathetic nerve activity in CHF rats but not in sham rats. Furthermore, chronic ablation of CSAR by epicardial application of a selective afferent neurotoxin, resiniferatoxin, selectively lowered diastolic blood pressure both at daytime and night-time with less effect on systolic blood pressure in CHF rats. Our data suggest that there is an imbalance between cardiac and peripheral responses to CSAR in CHF animals compared to sham-operated controls.

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 µg/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 ß-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-ß 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.

Spinal cord GABA receptors modulate the exercise pressor reflex in decerebrate rats.

Neurotransmitters and neuromodulators released by contraction-activated skeletal muscle afferents into the dorsal horn of the spinal cord initiate the central component of the exercise pressor reflex (EPR). Whether γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter within the mammalian central nervous system, is involved in the modulation of the EPR at the level of dorsal horn remains to be determined. We performed local microinjection of either the GABA(A) antagonist bicuculline or the GABA(B) antagonist CGP 52432 into the ipisilateral L4/L5 dorsal horns to investigate the effect of GABA receptor blockade on the pressor response to either static contraction induced by stimulation of the peripheral end of L4/L5 ventral roots, passive stretch, or hindlimb arterial injection of capsaicin (0.1 µg/0.2 ml) in decerebrate rats. Microinjection of either bicuculline (1 mM, 100 nl) or CGP 52432 (10 mM, 100 nl) into the L4/5 dorsal horns significantly increased the pressor and cardioaccelerator responses to all stimuli. Microinjection of either bicuculline or CGP 52432 into the L5 dorsal horn significantly increased the pressor and cardioaccelerator responses to direct microinjection of l-glutatmate (10 mM, 100 nl) into this spinal segment. The disinhibitory effect of both GABA receptor antagonists on the EPR was abolished by microinjection of the broad-spectrum glutamate receptor antagonist kynurenate (10 mM/100 nl). These data suggest that 1) GABA exerts a tonic inhibition of the EPR at the level of dorsal horn; and 2) that an interaction between glutamatergic and GABAergic inputs exist at the level of dorsal horn, contributing to spinal control of the EPR.

Pyruvate restores ß-adrenergic sensitivity of L-type Ca(2+) channels in failing rat heart: role of protein phosphatase.

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to ß-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted ß-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

Carbonylation induces heterogeneity in cardiac ryanodine receptor function in diabetes mellitus.

Heart failure and arrhythmias occur at 3 to 5 times higher rates among individuals with diabetes mellitus, compared with age-matched, healthy individuals. Studies attribute these defects in part to alterations in the function of cardiac type 2 ryanodine receptors (RyR2s), the principal Ca(2+)-release channels on the internal sarcoplasmic reticulum (SR). To date, mechanisms underlying RyR2 dysregulation in diabetes remain poorly defined. A rat model of type 1 diabetes, in combination with echocardiography, in vivo and ex vivo hemodynamic studies, confocal microscopy, Western blotting, mass spectrometry, site-directed mutagenesis, and [(3)H]ryanodine binding, lipid bilayer, and transfection assays, was used to determine whether post-translational modification by reactive carbonyl species (RCS) represented a contributing cause. After 8 weeks of diabetes, spontaneous Ca(2+) release in ventricular myocytes increased ~5-fold. Evoked Ca(2+) release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca(2+)-dependent ligand [(3)H]ryanodine was significantly reduced. Western blotting and mass spectrometry revealed RCS adducts on select basic residues. Mutation of residues to delineate the physiochemical impact of carbonylation yielded channels with enhanced or reduced cytoplasmic Ca(2+) responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca(2+) release and induced Ca(2+) waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca(2+) release from the SR, reduced carbonylation of RyR2s, and increased binding of [(3)H]ryanodine to RyR2s. From these data, we conclude that post-translational modification by RCS contributes to the heterogeneity in RyR2 activity that is seen in experimental diabetes.

Role of apoptosis signal-regulating kinase-1-c-Jun NH2-terminal kinase-p38 signaling in voltage-gated K+ channel remodeling of the failing heart: regulation by thioredoxin.

c-Jun NH(2)-terminal kinase (JNK) and p38 kinase are key regulators of cardiac hypertrophy and apoptosis during pathological stress, but their role in regulating ion channels in the diseased heart is unclear. Thus, we compared the kinase profile and electrophysiological phenotype of the rat ventricle 6-8 weeks after myocardial infarction (MI). Molecular analyses showed that JNK and p38 activities were markedly increased in post-MI hearts, while parallel voltage-clamp studies in ventricular myocytes revealed a characteristic downregulation of transient outward K(+) current (I(to)) density. When post-MI myocytes were treated with JNK or p38 inhibitors, I(to) density increased to control levels. Upregulation of I(to) was also elicited by insulin-like growth factor-1, which decreased JNK/p38 activity in post-MI hearts, and these changes were blocked by the thioredoxin (Trx) reductase inhibitor auranofin. Consistent with activation of JNK-p38 signaling, binding of apoptosis signal-regulating kinase-1 with Trx1 was also markedly decreased post-MI, and was reversed by insulin-like growth factor-1 in an auranofin-sensitive manner. We conclude that expression of ventricular K(+) channels is redox regulated and that chronic impairment of the Trx system in the post-MI heart contributes to I(to) remodeling through sustained activation of apoptosis signal-regulating kinase-1-JNK-p38 signaling. The cardiac Trx system may thus be a novel therapeutic target to reverse or prevent ventricular arrhythmias in the failing heart.

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.

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.

Anion-sensitive regions of L-type CaV1.2 calcium channels expressed in HEK293 cells.

L-type calcium currents (I(Ca)) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I(Ca) and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca(2+) channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from approximately 75%-80% to approximately 50% by omitting beta subunits but unaffected by omitting alpha(2)delta subunits. Similarly, gluconate inhibition was reduced to approximately 50% by deleting an alpha1 subunit N-terminal region of 15 residues critical for beta subunit interactions regulating open probability. Omitting beta subunits with this mutant alpha1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different beta subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from approximately 75%-80% to approximately 50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to approximately 60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to approximately 25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving beta subunit interactions with the N terminus and a short C terminal region.

Role of gamma-glutamyl transpeptidase in redox regulation of K+ channel remodeling in postmyocardial infarction rat hearts.

gamma-Glutamyl transpeptidase (gamma-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH (GSH(o)). The objective of this study was to identify the role of gamma-GT in reversing pathogenic K(+) channel remodeling in the diseased heart. Chronic ventricular dysfunction was induced in rats by myocardial infarction (MI), and studies were done after 6-8 wk. Biochemical assays of tissue extracts from post-MI hearts revealed significant increases in gamma-GT activity in left ventricle (47%) and septum (28%) compared with sham hearts, which paralleled increases in protein abundance and mRNA. Voltage-clamp studies of isolated left ventricular myocytes from post-MI hearts showed that downregulation of transient outward K(+) current (I(to)) was reversed after 4-5 h by 10 mmol/l GSH(o) or N-acetylcysteine (NAC(o)), and that the effect of GSH(o) but not NAC(o) was blocked by the gamma-GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine did not prevent upregulation of I(to) by GSH(o), suggesting that intracellular synthesis of GSH was not directly involved. However, pretreatment of post-MI myocytes with an SOD mimetic [manganese (III) tetrapyridylporphyrin] and catalase completely blocked recovery of I(to) by GSH(o). Confocal microscopy using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate confirmed that GSH(o) increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, GSH(o)-mediated upregulation of I(to) was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, and AG1024) and thioredoxin reductase (auranofin and 13-cis-retinoic acid). These data suggest that GSH(o) elicits gamma-GT- and ROS-dependent transactivation of tyrosine kinase signaling that upregulates K(+) channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by gamma-GT catalysis of GSH(o) may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart.

Glutathione homeostasis in ventricular myocytes from rat hearts with chronic myocardial infarction.

The ubiquitous tripeptide glutathione (GSH) is an essential factor in many biological processes, thus its depletion has a major impact on cell function and survival. In this study, we examined regulation of GSH in cardiomyocytes under chronic oxidative stress elicited by myocardial infarction (MI). Cardiac dysfunction was induced in rats by coronary artery ligation, and experiments were conducted in myocytes isolated from non-infarcted left ventricle and septum after 6-8 weeks. Fluorescence microscopy studies using the probe monochlorobimane showed that [GSH] in myocytes from post-MI hearts was 42% less than in sham control hearts (P < 0.05). However, depleted GSH levels were normalized after 5-6 h by an insulin mimetic (bis-peroxovanadium-1,10-phenanthroline, bpV(phen); 10 micromol l(-1)) or by exogenous pyruvate (5 mmol l(-1)). The increase in [GSH] by bpV(phen) was partly inhibited by buthionine sulphoximine (BSO; 50 micromol l(-1)), a blocker of GSH synthesis, and by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU; 100 micromol l(-1)), an inhibitor of glutathione disulphide reductase. By comparison, the effect of pyruvate was not altered by BSO but was completely blocked by BCNU. Studies using inhibitors of signalling cascades indicated that upregulation of [GSH] by bpV(phen) in myocytes from post-MI hearts was mediated by mitogen activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and p38 mitogen-activated protein kinase but not by phosphatidylinositol 3-kinase. The effect of pyruvate was not altered by any kinase inhibitor tested. In cells loaded with the probe TEMPO-9-AC to monitor superoxide anion, baseline fluorescence was 2.3-fold greater in post-MI myocytes than in sham control myocytes (P < 0.05) and was markedly decreased by diphenyleneiodonium (30 micromol l(-1)), an inhibitor of NADPH oxidase, exogenous GSH (10 mmol l(-1)) or bpV(phen). In parallel studies, [GSH] in post-MI myocytes was also normalized by diphenyleneiodonium or exogenous GSH. These data show that GSH is differentially regulated by receptor tyrosine kinase-dependent and -independent agonists that maintain functional GSH levels necessary to neutralize excess generation of reactive oxygen species in the failing heart.

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.

Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system.

Redox imbalance elicited by oxidative stress contributes to pathogenic remodeling of ion channels that underlies arrhythmogenesis and contractile dysfunction in the failing heart. This study examined whether the expression of K(+) channels in the remodeled ventricle is controlled by the thioredoxin system, a principal oxidoreductase network regulating redox-sensitive proteins. Ventricular dysfunction was induced in rats by coronary artery ligation, and experiments were conducted 6-8 wk postinfarction. Biochemical assays of tissue extracts from infarcted hearts showed that thioredoxin reductase activity was decreased by 32% from sham-operated controls (P < 0.05), whereas thioredoxin activity was 51% higher postinfarction (P < 0.05). These differences in activities paralleled changes in protein abundance as determined by Western blot analysis. However, whereas real-time PCR showed thioredoxin reductase mRNA levels to be significantly decreased postinfarction, thioredoxin mRNA was not altered. In voltage-clamp studies of myocytes from infarcted hearts, the characteristic downregulation of transient-outward K(+) current density was reversed by exogenous pyruvate (5 mmol/l), and this effect was blocked by the specific inhibitors of the thioredoxin system: auranofin or 13-cis-retinoic acid. Real-time PCR and Western blot analyses of myocyte suspensions from infarcted hearts showed that pyruvate increased mRNA and protein abundance of Kv4.2 and Kv4.3 channel alpha-subunits as well as the accessory protein KChIP2 when compared with time-matched, untreated cells (P < 0.05). The pyruvate-induced increase in Kv4.x expression was blocked by auranofin, but the upregulation of KChIP2 expression was not affected. These data suggest that the expression of Kv4.x channels is redox-regulated by the thioredoxin system, which may be a novel therapeutic target to reverse or limit electrical remodeling of the failing heart.

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

Oxidoreductase regulation of Kv currents in rat ventricle.

Oxidative stress contributes to the arrhythmogenic substrate created by myocardial ischemia-reperfusion partly through a shift in cell redox state, a key modulator of protein function. The activity of many oxidation-sensitive proteins is controlled by oxidoreductase systems that regulate the redox state of cysteine thiol groups, but the impact of these systems on ion channel function is not well defined. Thus, we examined the roles of the thioredoxin and glutaredoxin systems in controlling K(+) channels in the ventricle. An oxidative shift in redox state was elicited in isolated rat ventricular myocytes by brief exposure to diamide, a thiol-specific, membrane-permeable oxidant. Voltage-clamp studies showed that diamide decreased peak outward K(+) current (I(peak)) evoked by depolarizing test pulses by 41% (+60 mV; p<0.05) while steady-state outward current (I(ss)) measured at the end of the test pulse was decreased by 45% (p<0.05). These electrophysiological effects were not prevented by protein kinase C blockers, but the tyrosine kinase inhibitors genistein or lavendustin A blocked the suppression of both K(+) currents by diamide. Moreover, inhibition of I(peak) and I(ss) by diamide was reversed by dichloroacetate and an insulin-mimetic. The effect of dichloroacetate to normalize I(peak) after diamide was blocked by the thioredoxin system inhibitors auranofin or 13-cis-retinoic acid, but I(ss) was not affected by either compound. A pan-specific inhibitor of glutaredoxin and thioredoxin systems, 1,3-bis-(2-chloroethyl)-1-nitrosourea, also blocked the dichloroacetate effect on I(peak) but only partially inhibited the recovery of I(ss). These data suggest that acute regulation of cardiac K(+) channels by oxidoreductase systems is mediated by redox-sensitive tyrosine kinase/phosphatase pathways. The pathways controlling I(peak) channels are targets of the thioredoxin system whereas those regulating I(ss) channels are likely controlled by the glutaredoxin system. Thus, cardiac oxidoreductase systems may be important regulators of ion channels affected by pathogenic oxidative stress.

Pro-oxidant effect of transforming growth factor- beta1 mediates contractile dysfunction in rat ventricular myocytes.

AIMS: Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that contributes to pathogenic cardiac remodelling via mechanisms that involve oxidative stress. However, the direct impact of TGF-beta1 on contractile function of ventricular myocytes is incompletely understood. METHODS AND RESULTS: Reactive oxygen species (ROS) production and intracellular glutathione (GSH) were measured by fluorescence microscopy in isolated rat ventricular myocytes pretreated with TGF-beta1 (0.1-10 ng/mL). In separate studies, video edge detection measurements were made to evaluate myocyte contractile function, and confocal microscopy was used to monitor evoked Ca2+ transients. TGF-beta1 (1 ng/mL) for 3-4 h significantly increased ROS production by 90% (P < 0.05) and decreased GSH by 34% (P < 0.05) compared with control. These changes paralleled a significant decrease in the rate of myocyte shortening and relaxation by 33% and 43%, respectively (0.5 Hz; P < 0.05), whereas fractional shortening was not altered. Ca2+ transients in TGF-beta1-treated myocytes were characterized by a delayed peak and slowing in the rate of decay but no change in peak Ca2+ amplitude. Increased ROS production and GSH depletion by TGF-beta1 were prevented by an NAD(P)H oxidase inhibitor or a free radical scavenger, both of which significantly mitigated TGF-beta1-induced myocyte contractile dysfunction. Moreover, pretreating myocytes with exogenous GSH or the GSH precursor N-acetylcysteine also prevented myocyte contractile impairment and abnormal Ca2+ transients elicited by TGF-beta1. CONCLUSION: Our data suggest that TGF-beta1-induced cardiomyocyte contractile dysfunction is associated with enhanced ROS production and oxidative alterations in Ca2+ handling proteins regulated by endogenous GSH.

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats.

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.

Insulin regulation of glutathione and contractile phenotype in diabetic rat ventricular myocytes.

Cardiovascular complications of diabetes mellitus involve oxidative stress and profound changes in reduced glutathione (GSH), an essential tripeptide that controls many redox-sensitive cell functions. This study examined regulation of GSH by insulin to identify mechanisms controlling cardiac redox state and to define the functional impact of GSH depletion. GSH was measured by fluorescence microscopy in ventricular myocytes isolated from Sprague-Dawley rats made diabetic by streptozotocin, and video and confocal microscopy were used to measure mechanical properties and Ca(2+) transients, respectively. Spectrophotometric assays of tissue extracts were also done to measure the activities of enzymes that control GSH levels. Four weeks after injection of streptozotocin, mean GSH concentration ([GSH]) in isolated diabetic rat myocytes was approximately 36% less than in control, correlating with decreased activities of two major enzymes regulating GSH levels: glutathione reductase and gamma-glutamylcysteine synthetase. Treatment of diabetic rat myocytes with insulin normalized [GSH] after a delay of 3-4 h. A more rapid but transient upregulation of [GSH] occurred in myocytes treated with dichloroacetate, an activator of pyruvate dehydrogenase. Inhibitor experiments indicated that insulin normalized [GSH] via the pentose pathway and gamma-glutamylcysteine synthetase, although the basal activity of glucose-6-phosphate dehydrogenase was not different between diabetic and control hearts. Diabetic rat myocytes were characterized by significant mechanical dysfunction that correlated with diminished and prolonged Ca(2+) transients. This phenotype was reversed by in vitro treatment with insulin and also by exogenous GSH or N-acetylcysteine, a precursor of GSH. Our data suggest that insulin regulates GSH through pathways involving de novo GSH synthesis and reduction of its oxidized form. It is proposed that a key function of glucose metabolism in heart is to supply reducing equivalents required to maintain adequate GSH levels for the redox control of Ca(2+) handling proteins and contraction.