J. Jason Sims

Molecular Identification and Functional Characterization of a Mitochondrial Sulfonylurea Receptor 2 Splice Variant Generated by Intraexonic Splicing

Rationale: Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoKATP) channel but its composition is not fully understood. Objective: We hypothesized that the mitoKATP channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure. Methods and Results: Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (Rapid Amplification of cDNA Ends) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing (IES) event within the 4th and 29th exons of the SUR2 mRNA. The translated product matched the predicted size of the 55-kDa short form. In a knockout mouse (SUR2KO), in which the SUR2 gene was disrupted, the 130-kDa mitoSUR2 was absent, but the short forms remained expressed. Diazoxide failed to induce increased fluorescence of flavoprotein oxidation in SUR2KO cells, indicating that the diazoxide-sensitive mitoKATP channel activity was associated with 130-kDa–based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned wild type, suggesting a protective role for the remaining short form-based channels. Heterologous coexpression of the SUR2 IES variant and Kir6.2 in a K+ transport mutant Escherichia coli strain permitted improved cell growth under acidic pH conditions. The SUR2 IES variant was localized to mitochondria, and removal of a predicted mitochondrial targeting sequence allowed surface expression and detection of an ATP-sensitive current when coexpressed with Kir6.2. Conclusions: We identify a novel SUR2 IES variant in cardiac mitochondria and provide evidence that the variant-based channel can form an ATP-sensitive conductance and may contribute to cardioprotection.

Impaired vagal reflex activity in insulin-resistant rats.

Insulin resistance, without frank diabetes, is associated with sudden cardiac death. We postulated that a potential mechanism for this association is autonomic dysfunction. Male Sprague-Dawley rats were randomized into one of two groups: (a) insulin resistant (IR; n = 15), or (b) control (n = 11). Animals were made insulin resistant with a fructose-rich diet, whereas control animals received standard rat chow. Four weeks after randomization, arterial pressure and baroreceptor reflex were assessed. Baroreflex sensitivity was defined as the heart-rate response to acute blood pressure changes caused by nitroprusside (0.5-18 micrograms) or phenylephrine (0.2-3 micrograms). To determine the role of vagal stimulation specifically, each animal was randomized to receive atropine sulfate (1 mg/kg) or vehicle (normal saline) before administration of phenylephrine. Mean arterial pressure and fasting insulin concentrations were increased in the insulin-resistant group, whereas there were no differences in body weight, fasting glucose concentrations, or resting heart rate. Phenylephrine increased arterial blood pressure to a maximum of 54 +/- 2 mm Hg for control and 45 +/- 6 mm Hg for IR, p = 0.7. The maximal heart-rate change response to the increased blood pressure was markedly blunted in IR as compared with control (-88 +/- 12 beats/min for IR vs. -238 +/- 18 beats/min for control; p < 0.001). Thus the baroreflex sensitivity (BRS) was threefold less in IR versus the control group (-1.8 +/- 0.2 vs. -4.6 +/- 0.7 beats/min/mm Hg; p = 0.001). Pretreatment with atropine sulfate decreased the BRS in both groups, eliminating the difference between groups (-0.96 +/- 0.5 beats/min/mm Hg for control and -0.56 +/- 0.3 beats/min/mm Hg for IR; p = 0.2). Thus atropine sulfate caused the phenylephrine-induced heart rate and arterial blood pressure response to be equal between groups. On the other hand, BRS to nitroprusside-induced blood pressure changes were similar between groups. Insulin resistance, without the confounding factors of obesity, diabetes, and significant hypertension, is associated with a large reduction in vagal activity, which occurs via attenuation in reflex activity. In contrast, the insulin-resistant syndrome does not affect baroreflex sensitivity via sympathetic reflex.

Allopurinol as a Cardioprotectant during Coronary Artery Bypass Graft Surgery

OBJECTIVE: To examine the role of allopurinol as a cardioprotectant during coronary artery bypass graft (CABG) surgery. DATA SOURCES: A search of MEDLINE (1966–October 2002) was performed using the following terms: allopurinol, xanthine oxidase, oxygen free radical, and coronary artery bypass. References evaluated were limited to English-language and human studies, yielding 41 citations, 13 of which were found suitable. The 5 largest studies are discussed. DATA SYNTHESIS: Multiple studies with various doses have evaluated the effects of allopurinol on outcomes in CABG patients. These studies found that allopurinol can reduce in hospital mortality, improve cardiac performance, reduce incidence of arrhythmias, reduce markers of ischemia and free-radical generation, and reduce the need for inotropic support. However, these findings were not consistent between all studies. CONCLUSIONS: Allopurinol may reduce the incidence of CABG complications. Although the optimal dose has not been determined, reviewed literature suggests that patients should receive at least 600 mg one day prior to surgery, as well as at least 600 mg on the day of surgery.

High-dose lidocaine does not affect defibrillation efficacy: implications for defibrillation mechanisms.

This study assessed the effect of low (10 mg ⋅ kg-1 ⋅ h-1) and very high (18 mg ⋅ kg-1 ⋅ h-1) doses of lidocaine on defibrillation energy requirements (DER) to relate changes in indexes of sodium-channel blockade with changes in DER values using a dose-response study design. In group 1 (control; n = 6 pigs), DER values were determined at baseline and during treatment with 5% dextrose in water (D5W) and with D5W added to D5W. In group 2 ( n = 7), DER values were determined at baseline and during treatment with low-dose lidocaine followed by high-dose lidocaine. In group 3 ( n = 3), DER values were determined at baseline and high-dose lidocaine. Group 3 controlled for the order of lidocaine treatment with the addition of high-dose lidocaine after baseline. DER values in group 1 did not change during D5W. In group 2, low-dose lidocaine increased DER values by 51% ( P = 0.01), whereas high-dose lidocaine added to low-dose lidocaine reduced DER values back to within 6% of baseline values ( P = 0.02, low dose vs. high dose). DER values during high-dose lidocaine in group 3 also remained near baseline values (16.2 ± 2.7 to 12.9 ± 2.7 J), demonstrating that treatment order had no impact on group 2. Progressive sodium-channel blockade was evident as incremental reduction in ventricular conduction velocity as the lidocaine dose increased. Lidocaine also significantly increased ventricular fibrillation cycle length as the lidocaine dose increased. However, the greatest increase in DER occurred when ventricular fibrillation cycle length was minimally affected, demonstrating a negative correlation ( P = 0.04). In summary, lidocaine has an inverted U-shaped DER dose-response curve. At very high lidocaine doses, DER values are similar to baseline and tend to decrease rather than increase. Increased refractoriness during ventricular fibrillation may be the electrophysiological mechanism by which high-dose lidocaine limits the adverse effects that low-dose lidocaine has on DER values. However, there is a possibility that an unidentified action of lidocaine is responsible for these effects.This study assessed the effect of low (10 mg.kg-1.h-1) and very high (18 mg.kg-1.h-1) doses of lidocaine on defibrillation energy requirements (DER) to relate changes in indexes of sodium-channel blockade with changes in DER values using a dose-response study design. In group 1 (control; n = 6 pigs), DER values were determined at baseline and during treatment with 5% dextrose in water (D5W) and with D5W added to D5W. In group 2 (n = 7), DER values were determined at baseline and during treatment with low-dose lidocaine followed by high-dose lidocaine. In group 3 (n = 3), DER values were determined at baseline and high-dose lidocaine. Group 3 controlled for the order of lidocaine treatment with the addition of high-dose lidocaine after baseline. DER values in group 1 did not change during D5W. In group 2, low-dose lidocaine increased DER values by 51% (P = 0.01), whereas high-dose lidocaine added to low-dose lidocaine reduced DER values back to within 6% of baseline values (P = 0.02, low dose vs. high dose). DER values during high-dose lidocaine in group 3 also remained near baseline values (16.2 +/- 2.7 to 12.9 +/- 2.7 J), demonstrating that treatment order had no impact on group 2. Progressive sodium-channel blockade was evident as incremental reduction in ventricular conduction velocity as the lidocaine dose increased. Lidocaine also significantly increased ventricular fibrillation cycle length as the lidocaine dose increased. However, the greatest increase in DER occurred when ventricular fibrillation cycle length was minimally affected, demonstrating a negative correlation (P = 0.04). In summary, lidocaine has an inverted U-shaped DER dose-response curve. At very high lidocaine doses, DER values are similar to baseline and tend to decrease rather than increase. Increased refractoriness during ventricular fibrillation may be the electrophysiological mechanism by which high-dose lidocaine limits the adverse effects that low-dose lidocaine has on DER values. However, there is a possibility that an unidentified action of lidocaine is responsible for these effects.

Influence of Intermittent Hypoxia on Myocardial and Hepatic P‐glycoprotein Expression in a Rodent Model

Study Objective. Patients with obstructive sleep apnea who receive drug therapy for cardiovascular disease may experience resistant hypertension, arrhythmias, or more severe heart failure, and many of the drugs used to treat these conditions are substrates for P‐glycoprotein (P‐gp) transporters. Therefore, we sought to determine if intermittent hypoxia, which mimics obstructive sleep apnea, would upregulate myocardial and hepatic P‐gp expression and Abcb1a and Abcb1b messenger RNA (mRNA) expression (genes that encode for P‐gp) in an animal model.

Disparate effects of biphasic and monophasic shocks on postshock refractory period dispersion

The magnitude by which a defibrillation shock extends the refractory period immediately postshock (refractory period extension, RPE) does not explain why biphasic shocks defibrillate with greater efficacy than monophasic shocks. It may be that spatial heterogeneity of RPE is a more important regulator of defibrillation efficacy. We measured RPE in 15 pentobarbital-anesthetized swine using 400-V biphasic and monophasic shocks of equal pulse duration at three discrete myocardial sites. Spatial heterogeneity of RPE was calculated as the difference between the maximum and minimum values of the three recording sites. Monophasic shocks produced greater magnitude of RPE than biphasic shocks at all sites tested (82 +/- 6 to 99 +/- 13 and 64 +/- 6 to 68 +/- 5 ms, respectively; P < 0.05). However, RPE dispersion was significantly less with biphasic shocks versus monophasic shocks (29 +/- 4 and 48 +/- 7 ms, respectively; P < 0.05). This suggests that one potential mechanism by which biphasic shocks defibrillate with greater efficacy is limiting postshock spatial heterogeneity of refractoriness. Thus these data support our hypothesis that RPE heterogeneity is a more likely predictor of defibrillation efficacy than magnitude of RPE.The magnitude by which a defibrillation shock extends the refractory period immediately postshock (refractory period extension, RPE) does not explain why biphasic shocks defibrillate with greater efficacy than monophasic shocks. It may be that spatial heterogeneity of RPE is a more important regulator of defibrillation efficacy. We measured RPE in 15 pentobarbital-anesthetized swine using 400-V biphasic and monophasic shocks of equal pulse duration at three discrete myocardial sites. Spatial heterogeneity of RPE was calculated as the difference between the maximum and minimum values of the three recording sites. Monophasic shocks produced greater magnitude of RPE than biphasic shocks at all sites tested (82 ± 6 to 99 ± 13 and 64 ± 6 to 68 ± 5 ms, respectively; P < 0.05). However, RPE dispersion was significantly less with biphasic shocks versus monophasic shocks (29 ± 4 and 48 ± 7 ms, respectively; P < 0.05). This suggests that one potential mechanism by which biphasic shocks defibrillate with greater efficacy is limiting postshock spatial heterogeneity of refractoriness. Thus these data support our hypothesis that RPE heterogeneity is a more likely predictor of defibrillation efficacy than magnitude of RPE.

Lidocaine does not affect myocardial electrical heterogeneity : Implications for low proarrhythmic actions

An area of unidirectional conduction block is one requirement for reentrant arrhythmias to occur. Functional block caused by dispersion of repolarization and refractoriness is the most probable mechanism of drug‐induced unidirectional conduction block. We assessed the effects of lidocaine on spatial dispersion of myocardial repolarization and refractoriness in the intact porcine heart. Monophasic action potential duration at 90% repolarization, effective refractory period (ERP), and ventricular fibrillation cycle length (VFCL) were measured at two endocardial and one epicardial sites at baseline and during a treatment phase with D5W (n=11) or lidocaine 10 mg/kg/hour (n=12). Dispersion was calculated as the difference between the maximum and minimum values of the three recording sites. Lidocaine produced significant changes in ERP, VFCL, paced QRS duration, and intraventricular conduction time. It did not change basal levels of dispersion in repolarization and refractoriness. Lidocaine produced changes in myocardial electrophysiology that are uniform across the myocardium and thus did not change myocardial electrical heterogeneity. This may be a mechanism of the agents lower proarrhythmic effects compared with other sodium channel blockers that increase myocardial electrical heterogeneity.

Electrical heterogeneity and arrhythmogenesis: importance of conduction velocity dispersion.

An experimental model of conduction velocity (CV) and refractory period dispersion was established to determine which variable is a determinant of myocardial vulnerability. Anesthetized swine were instrumented with a left anterior descending coronary artery catheter for regional infusion of lidocaine (n = 6), low-dose d-sotalol (n = 4), high-dose d-sotalol (n = 6), or saline (n = 6), to create dispersion in CV (lidocaine), refractoriness (d-sotalol), or neither (saline). Ventricular fibrillation thresholds (VFTs) and refractory periods were determined at five sites (one drug perfused, four non–drug perfused). CV was determined in one drug-perfused area (left ventricular epicardial apex) and one non–drug perfused area (right ventricular epicardial base). Lidocaine and low- and high-dose d-sotalol increased VFT when stimuli were delivered in the drug-perfused area. However, lidocaine decreased VFT when stimuli were delivered at non–drug perfused areas by an average of 52%. Neither d-sotalol dose affected VFT when stimuli were delivered in non–drug perfused areas. Lidocaine increased CV dispersion by 18–26 cm/s but did not alter refractoriness. Both d-sotalol doses increased dispersion in refractoriness by 15–27 ms but did not alter CV. Saline did not affect either variable. Regional lidocaine had profibrillatory effects when stimuli were delivered in non–drug perfused areas, whereas regional d-sotalol did not. Hence, CV dispersion is a more likely determinant of myocardial vulnerability than refractoriness.

Endotoxemia alters splanchnic capacitance

The splanchnic circulation constitutes a major portion of the total capacitance vasculature and may affect venous return and subsequently cardiac output during low output states. This study assessed the effects of rapid (10 microg/kg over 5 min) and slow (10 microg/kg over 60 min) induction of endotoxin (Escherichia coli) shock on splanchnic blood volume in 8 farm swine. Blood volume was measured by using Tc99m-labeled erythrocytes and radionuclide imaging. Baseline arterial pressure (MAP), central venous pressure (CVP), and liver, splenic, mesenteric and total splanchnic volumes were stable during the 30-min baseline. Approximately 30 min after the rapid endotoxin infusion, splenic volume decreased by 45%, whereas liver volume increased by 40% and MAP decreased by 60% (P < 0.01). The reduction in splenic volume occurred within 10 min of the endotoxin infusion, whereas liver volume changes occurred after MAP reduction. The slow endotoxin infusion also reduced splenic volume by approximately 50% (P = 0.05), whereas MAP declined by 30% (P < 0.05). However, the slow endotoxin infusion lowered liver volume (P < 0.05). Mesenteric volume was unaffected by the fast or slow endotoxin infusion. Total splanchnic volume was unaffected by the fast infusion but decreased by 37% in the slow infusion group (P < 0.05). In summary, E. coli endotoxin reduces splenic blood volume and increases liver blood volume after acute hypotension ensues. Endotoxin does not increase total splanchnic blood volume and may actually decrease total splanchnic volume in the absence of circulatory collapse. This endotoxin shock model is not associated with blood volume pooling in the splanchnic capacitance circulation.

Tachycardia-induced heart failure does not alter myocardial P-glycoprotein expression.

Study Objective. To determine the effects of tachycardia‐induced heart failure on myocardial P‐glycoprotein (P‐gp) expression.