Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na/HCO3 cotransport

The sarcolemmal Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH_i was measured in single cells in a medium with HCO₃⁻ using the fluorescent pH indicator BCECF. The NH₄⁺ pulse method was used to induce an intracellular acid load and the acid efflux (J_H) in the presence of the Na⁺/H⁺ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following J_H data are presented at pH_i of 6.8 ( and ^# indicate p < 0.05 after ANOVA vs. control and Ang II, respectively). The basal J_H (1.03 ± 0.12 mM/min, n = 11) was significantly increased in the presence of 100 nM Ang II (1.70 ± 0.15 mM/min, n = 8). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80 ± 0.08 mM/min, n = 11^#), 300 μM apocynin (NADPH oxidase blocker; 0.80 ± 0.13 mM/min, n = 6^#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K⁺ channel, mK_ATP, blocker; 0.97 ± 0.21 mM/min, n = 9^#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56 ± 0.18 mM/min, n = 6^#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK_ATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.     

Wide long lasting perinuclear Ca2+ release events generated by an interaction between ryanodine and IP3 receptors in canine Purkinje cells

The purpose of this study was to determine whether IP₃Rs contribute to the generation of wide long lasting perinuclear Ca²⁺ release events in canine Purkinje cells. Spontaneous Ca²⁺ release events (elevations of basal [Ca²⁺] equivalent to F/F₀ 3.4SD over F₀) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca²⁺ waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 μm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035 ± 0.0007 events (ev)/μm²/s, N = 34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022 ± 0.00005 ev/μm²/s, N = 41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0 ± 3.9 vs. TE 9.0 ± 0.3 μm², P < 0.01) and higher amplitude (F/F₀ 1.38 ± 0.02 vs. TE 1.20 ± 0.003, P < 0.01). WLE event rate was increased by phenylephrine (10 μM, P < 0.01), inhibited by 2APB and U73122 (P < 0.05), and abolished by tetracaine (1 mM) and ryanodine (100 μM). While SSL WLEs were scattered randomly, Core WLEs (n = 69 events) were predominantly distributed longitudinally 18.2 ± 1.6 μm from the center of nuclei. Immunocytochemistry showed that IP₃R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca²⁺ release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP₃R1s evoked Ca²⁺ release and may play a role in Ca²⁺ dependent nuclear processes.     

Serum bilirubin levels, UGT1A1 polymorphisms and risk for coronary artery disease

Low levels of the antioxidative serum bilirubin are associated with vascular aging and an increased risk for coronary artery disease (CAD). UGT1A1 is the major gene influencing bilirubin concentrations. Therefore, we investigated an association of bilirubin levels and two polymorphisms in the promoter of UGT1A1 (-53(TA-repeat) polymorphism and T-3279G) in 477 patients with premature, familial CAD and 619 age- and sex-matched controls. Bilirubin concentrations were significantly lower in cases than in controls (0.62 ± 0.36 vs. 0.76 ± 0.41 mg/dl for men, p = 1.2 × 10⁻¹⁰; and 0.42 ± 0.29 vs. 0.55 ± 0.23 mg/dl, p = 1.9 × 10⁻⁹ for women). Both polymorphisms showed a strong association with bilirubin levels with higher levels for homozygote carriers of the minor allele. These associations were most pronounced in male controls and patients (p = 5.9 × 10⁻²⁶ and p = 3.4 × 10⁻¹⁶, respectively, for the -53(TA-repeat) polymorphism). Logistic regression analysis revealed low bilirubin levels but not the UGT1A1 polymorphisms to be significantly associated with CAD: OR (95% CI) 0.90 (0.86–0.94), p = 2.6 × 10⁻⁶ for men and 0.77 (0.68–0.87), p = 3.2 × 10⁻⁵ for women, respectively for each 0.1 mg/dl increase of bilirubin. These results indicate that it is rather decreased bilirubin levels in general than the changes in the genetic variation of this gene that increase the risk for CAD.     

Morphological and functional platelet abnormalities in Berkeley sickle cell mice

Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37 ± 3.2 vs. 27 ± 1.4, mean ± SD; p < 0.001), in association with moderate thrombocytopenia (505 ± 49 × 10³/μl vs. 1151 ± 162 × 10³/μl; p < 0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell–Jolly bodies and “pocked” erythrocytes (p < 0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5 ± 1% vs. 1 ± 1%; p < 0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.     

NO-mediated vasodilation in the rat liver: Role of hepatocytes and liver endothelial cells

Background/Aims: Nitric oxide (NO) is a potent vasodilator. We investigated the mechanisms responsible for this effect in the liver.Methods: Isolated perfused rat liver and cultures of endothelial sinusoidal cells and hepatocytes were used.Results: L-arginine (10⁻³ M) and NO donor Sin-1 (10⁻⁵ M) respectively increased the liver flow by 52% (p<0.01) and 93% (p<0.01) vs controls. The NO synthase inhibitor N^w-nitro-L-arginine (10⁻³ M) and the guanylate cyclase inhibitor methylene blue (10⁻⁵ M) respectively decreased the basal liver flow by 26% and 16% (p<0.05) and inhibited the vasodilating effects of L-arginine. L-arginine (10⁻³ M) increased nitrite concentration in hepatocyte culture (77.25±7.40 μmol · 1⁻¹ vs 14.70±3.55 μmol · 1⁻¹ in controls; p<0.01) and in liver endothelial cell culture (0.36±0.09 μmol · 1⁻¹ vs 0.12±0.05 μmol · 1⁻¹ in controls; p<0.05). N^w-nitro-L-arginine inhibited the basal production and abolished the L-arginine-induced production of nitrites both in hepatocyte and in liver endothelial cell cultures. The concentration of nitrites in the hepatocyte supernatant rose from 14.70±3.55 μmol⁻¹ · 1⁻ to 150.50±45.55 μmol · 1⁻¹ in the presence of a combination of interleukin-1β, TNF a and interferon γ.Conclusions: Under basal conditions, NO regulates the vascular tone of liver circulation. Both liver endothelial cells and hepatocytes can be implicated. NO production by hepatocytes may increase during inflammation.     

The effect of norepinephrine on insulin secretion and glucose effectiveness in non-insulin-dependent diabetes

It has previously been shown that in normal subjects, physiological elevation of norepinephrine (NE) impairs insulin sensitivity (Si) but does not influence insulin secretion. The aim of this study was to determine the effect of short-term physiological elevation of NE on insulin secretion, Si, and glucose-mediated glucose disposal, or the glucose effectiveness index (Sg), in non-insulin-dependent diabetes mellitus (NIDDM). Two intravenous glucose tolerance tests (IVGTTs) were performed in eight well-controlled NIDDM patients, using a supplemental exogenous insulin infusion to achieve an approximation of normal endogenous insulin secretion. The IVGTTs were performed in random order after 30 minutes of either the saline (SAL) or NE (25 ng/kg/min) infusions, which were continued throughout the 3-hour IVGTT. Sg and Si were estimated by minimal model analysis of the IVGTT data as previously described. Plasma C-peptide was used to estimate insulin secretion rate using the ISEC program. NE infusion produced approximately a threefold increase in plasma NE, associated with (1) a significant reduction in glucose disposal ([K_G] SAL v NE, 0.73 ± 0.06 v 0.61 ± 0.06 × 10⁻² · min⁻², P < .05), (2) no reduction in Si (2.33 ± 0.8 v 2.62 ± 0.9 × 10⁻⁴ · min⁻¹/mU/L, NS), (3) a reduced mean second-phase insulin secretion rate (1.21 ± 0.19 v 1.01 ± 0.16 × 10⁻³ pmol/kg/min per mmol/L glucose, P < .05), (4) a significant increase in Sg (0.89 ± 0.08 v 1.63 ± 0.2 × 10⁻² · min⁻¹ P < .05), and (5) a corresponding increase in glucose effectiveness at zero insulin ([GEZI] 0.55 ± 0.13 v 1.30 ± 0.33 × 10⁻² · min⁻¹, P < .05). These results show that in contrast to normal subjects, physiological elevation of NE in NIDDM does not result in a reduction in Si, but causes a reduction in glucose disposal related to inhibition of insulin secretion that is only partially compensated for by increased Sg.     

Ca2+ sparks and cellular distribution of ryanodine receptors in developing cardiomyocytes from rat

Although abundant ryanodine receptors (RyRs) exist in cardiomyocytes from newborn (NB) rat and despite the maturity of their single-channel properties, the RyR contribution to excitation–contraction (E-C) coupling is minimal. Immature arrangement of RyRs in the Ca²⁺ release site of the sarcoplasmic reticulum and/or distant RyRs location from the sarcolemmal Ca²⁺ signal could explain this quiescence. Consequently, Ca²⁺ sparks and their cellular distribution were studied in NB myocytes and correlated with the formation of dyads and transverse (T) tubules. Ca²⁺ sparks were recorded in fluo-4-loaded intact ventricular myocytes acutely dissociated from adult and NB rats (0–9 days old). Sparks were defined/compared in the center and periphery of the cell. Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyad formation, while the development of T tubules was studied using di-8-ANEPPS and diIC12. Our results indicate that in NB cells, Ca²⁺ sparks exhibited lower amplitude (1.7 ± 0.5 vs. 3.6 ± 1.7 F/F₀), shorter duration (47 ± 3.2 vs. 54.1 ± 3 ms), and larger width (1.7 ± 0.8 vs. 1.2 ± 0.4 μm) than in adult. Although no significant changes were observed in the overall frequency, central sparks increased from ~ 60% at 0–1 day to 82% at 7–9 days. While immunolocalization revealed many central release sites at 7–8 days, fluorescence labeling of the plasma membrane showed less abundant internal T tubules. This could imply that although during the first week, release sites emerge forming dyads with DHPR-containing T tubules; some of these T tubules may not be connected to the surface, explaining the RyR quiescence during E-C coupling in NB.     

Action potential clamp and chloroquine sensitivity of mutant Kir2.1 channels responsible for variant 3 short QT syndrome

Recently identified genetic forms of short QT syndrome (SQTS) are associated with an increased risk of arrhythmia and sudden death. The SQT3 variant is associated with an amino-acid substitution (D172N) in the KCNJ2-encoded Kir2.1 K⁺ channel. In this study, whole-cell action potential (AP) clamp recording from transiently transfected Chinese Hamster Ovary cells at 37 °C showed marked augmentation of outward Kir2.1 current through D172N channels, associated with right-ward voltage-shifts of peak repolarizing current during both ventricular and atrial AP commands. Peak outward current elicited by ventricular AP commands was inhibited by chloroquine with an IC₅₀ of 2.45 μM for wild-type (WT) Kir2.1, of 3.30 μM for D172N-Kir2.1 alone and of 3.11 μM for co-expressed WT and D172N (P > 0.05 for all). These findings establish chloroquine as an effective inhibitor of SQT3 mutant Kir2.1 channels.     

Prolonged exposure to high dietary lipids is not associated with lipotoxicity in heart failure

Previous studies have reported that elevated myocardial lipids in a model of mild-to-moderate heart failure increased mitochondrial function, but did not alter left ventricular function. Whether more prolonged exposure to high dietary lipids would promote a lipotoxic phenotype in mitochondrial and myocardial contractile function has not been determined. We tested the hypothesis that prolonged exposure to high dietary lipids, following coronary artery ligation, would preserve myocardial and mitochondrial function in heart failure. Rats underwent ligation or sham surgery and were fed normal (10% kcal fat) (SHAM, HF) or high fat diet (60% kcal saturated fat) (SHAM+FAT, HF+FAT) for sixteen weeks. Although high dietary fat was accompanied by myocardial tissue triglyceride accumulation (SHAM 1.47 ± 0.14; SHAM+FAT 2.32 ± 0.14; HF 1.34 ± 0.14; HF+FAT 2.21 ± 0.20 μmol/gww), fractional shortening was increased 16% in SHAM+FAT and 28% in HF+FAT compared to SHAM and HF, respectively. Despite increased medium-chain acyl-CoA dehydrogenase (MCAD) activity in interfibrillar mitochondria (IFM) of both SHAM+FAT and HF+FAT, dietary lipids also were associated with decreased state 3 respiration using palmitoylcarnitine (SHAM 369 ± 14; SHAM+FAT 307 ± 23; HF 354 ± 13; HF+FAT 366 ± 18 nAO min^− 1 mg^− 1) in SHAM+FAT compared to SHAM and HF+FAT. State 3 respiration in IFM also was decreased in SHAM+FAT relative to SHAM using succinate and DHQ. In conclusion, high dietary lipids promoted myocardial lipid accumulation, but were not accompanied by alterations in myocardial contractile function typically associated with lipotoxicity. In normal animals, high dietary fat decreased mitochondrial respiration, but also increased MCAD activity. These studies support the concept that high fat feeding can modify multiple cellular pathways that differentially affect mitochondrial function under normal and pathological conditions.     

Electrical properties of cultured heart cell reaggregates from newborn rat ventricles: Comparison with intact non-cultured ventricles

The electrophysiological properties of cultured rat heart cells were investigated and compared with those of cells in intact hearts of the same age rats. Reaggregates (100 to 200 μm diameter) from newborn rat hearts (ventricles) were prepared from collagenase-hyaluronidase-dissociated cells and cultured for periods up to 7 days. Intracellular micro-electrode recordings were made using standard techniques. Ventricular reaggregates cultured under standard conditions had a low resting potential (−67 ± 1.5 mV) and the action potentials had a slow rate of rise (18 ± 1.4 V/s). Tetrodotoxin (5 × 10⁻⁶ ) decreased only slightly the rate of rise and verapamil (10⁻⁶ ) inhibited completely the action potentials. In contrast the ventricular action potentials recorded from isolated ventricles were characterized by a high resting potential (−77 ± 0.7 mV) and a fast rate of rise (80 to 193 V/s). Tetrodotoxin (5 × 10⁻⁶ ) decreased the rate of rise and verapamil (10⁻⁶ ) only depressed the plateau level of the action potentials. In cultured reaggregates hyperpolarizing current pulses increased the rate of rise. This effect was prevented by tetrodotoxin. Reducing the amount of fibroblasts in the reaggregates by using the differential attachment technique, caused the AP parameters to become similar to those found in non-cultured cells. We conclude that, under certain conditions (e.g. removal of fibroblasts) fully differentiated properties can be maintained in reaggregate cell cultures. Thus, as in the case of cultured chick heart cells, cultured rat heart cells can be maintained in vitro in a highly differentiated state.     

Consequences of seeded cell type on vascularization of tissue engineering constructs in vivo

Implantation of tissue engineering constructs is a promising technique to reconstruct injured tissue. However, after implantation the nutrition of the constructs is predominantly restricted to vascularization. Since cells possess distinct angiogenic potency, we herein assessed whether scaffold vitalization with different cell types improves scaffold vascularization. 32 male balb/c mice received a dorsal skinfold chamber. Angiogenesis, microhemodynamics, leukocyte–endothelial cell interaction and microvascular permeability induced in the host tissue after implantation of either collagen coated poly (l-lactide-co-glycolide) (PLGA) scaffolds (group 4), additionally seeded with osteoblast-like cells (OLCs, group 1), bone marrow mesenchymal stem cells (bmMSCs, group 2) or a combination of OLCs and bmMSCs (group 3) were analyzed repetitively over 14 days using intravital fluorescence microscopy. Apart from a weak inflammatory response in all groups, vascularization was found distinctly accelerated in vitalized scaffolds, indicated by a significantly increased microvascular density (day 6, group 1: 202 ± 15 cm/cm², group 2: 202 ± 12 cm/cm², group 3: 194 ± 8 cm/cm²), when compared with controls (group 4: 72 ± 5 cm/cm²). This acceleration was independent from the seeded cell type. Immunohistochemistry revealed in vivo VEGF expression in close vicinity to the seeded OLCs and bmMSCs. Therefore, the observed lack of cell type confined differences in the vascularization process suggests that the accelerated vascularization of vitalized scaffolds is VEGF-related rather than dependent on the potential of bmMSCs to differentiate into specific vascular cells.     

Effects of phenytoin and quinidine on digitalis-induced oscillatory afterpotentials, aftercontractions, and inotropy in canine ventricular tissues

The object of this study was to compare the electrical and mechanical effects of quinidine and phenytoin on cardiac tissue intoxicated with digitalis. Phenytoin was selected because it is relatively effective against digitalis arrhythmias; quinidine was selected because it is contraindicated in treatment of digitalis arrhythmias. Isolated canine false tendons were exposed to concentrations of acetylstrophanthidin (or actodigin) sufficient to cause oscillatory afterpotentials and aftercontractions. Both phenytoin (1.2 × 10⁻⁵ ) and quinidine (1.6 × 10⁻⁶ ) decreased the amplitude of oscillatory afterpotentials at various membrane potentials selected by extracellular current application. Quinidine, but not phenytoin, also increased threshold potential. The lowest concentrations of these agents that stopped continuous automatic activity induced in false tendons by trains of rapid stimulation was 7.9 × 10⁻⁶ phenytoin and 1.6 × 10⁻⁶ quinidine. In the absence of digitalis, these concentrations decreased strength of contraction 69.5 (± 13 ) % and 49.6 (± 10 ) %, respectively. Similar concentrations of phenytoin (1.2 × 10⁻⁵ ) and quinidine (1.6 × 10⁻⁶ ) reduced or abolished oscillatory afterpotentials and aftercontractions induced by digitalis in false tendons. Abolition of aftercontractions eliminated potentiating and depotentiating effects of aftercontractions on superimposed beats and reversed the effects of digitalis on the configuration of the force-frequency relation and course of restitution of contractility. In muscle, studied simultaneously with the false tendons, phenytoin, but not quinidine, decreased strength of contraction.This study demonstrates that phenytoin and quinidine have remarkably similar, but not identical effects on isolated cardiac tissues exposed to digitalis.     

The clinical course of untreated Gaucher disease in 22 patients over 10 years: Hematological and skeletal manifestations

Gaucher disease (GD) is a lysosomal storage disorder characterized by anemia and thrombocytopenia, hepatosplenomegaly, and skeletal involvement. The management of Gaucher disease was improved by the development of enzyme replacement therapy (ERT). However, the bone response to ERT is generally slower compared to other clinical manifestations. Some have recommended the early use of ERT to prevent the development of severe skeletal complications. Because we have access to over 30 untreated patients in Ontario, we questioned the extent to which complications progress in severity over a long period of time. We examined retrospectively the natural history of GD and the extent of skeletal manifestations in 22 untreated type 1 GD adult patients (mean age, 49 ± 3.3; range, 20–81 years). The patients were followed for a median of 9.5 years (range, 3–16 years). Hemoglobin (Hb) concentration did not significantly change over time (mean baseline concentration of 12.8 ± 0.27 g/dL vs. mean recent concentration of 12.6 ± 0.37 g/dL, p =   0.65). Mean platelet count also remained relatively stable over time (mean baseline count of 138 ± 13 × 10⁹/L vs. mean recent count of 138.5 ± 18 × 10⁹/L, p =   0.98). Mean ferritin and ACE concentrations were elevated and were stable over time. Liver volumes decreased over time (mean baseline liver volume of 1.2 × normal (N) vs. mean recent volume of 1.06 × N, p =   0.27) and 6 of 22 (27%) patients had moderate hepatomegaly (liver volume, 1.25–2.5 × N). Spleen volumes remained stable over time (mean baseline spleen volume of 6.6 × N vs. mean recent volume of 5.2 × N, p = 0.5). None of the changes was statistically significant. Four of 20 (20%) patients had moderate splenomegaly (spleen volume, 5–15 × N), 2 of 20 (10%) had marked splenomegaly (spleen volume,  ≥ 15 × N), and 2 of 22 (9%) had had splenectomy. The most common skeletal manifestations were infiltration of the bone marrow in 16 of 22 (73%) patients followed by osteopenia in 15 of 22 (68%), Erlenmeyer flask deformity in 13 of 22 (59%), and infarctions in 6 of 22 (27%) patients. We observed that bone disease remained relatively stable over time in most patients, although three patients developed new infarcts over time, one developed an avascular necrosis (AVN), and four had an increase in the degree of osteopenia. Although GD and its skeletal complications progress in severity in some patients, our results suggest that GD complications, including bony disease, may stabilize over time. Therefore, early use of ERT may not be necessary in all type 1 GD patients.     

Insulin inhibits leukocyte–endothelium adherence via an Akt-NO-dependent mechanism in myocardial ischemia/reperfusion

Clinical evidence indicates that intensive insulin therapy during critical illness protects the endothelium and contributes to prevention of organ failure and death but the mechanisms involved remain unclear. This study was designed to test the hypothesis that insulin inhibits adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells in myocardial ischemia/reperfusion (MI/R) and to investigate the underlying mechanisms. Anesthetized rabbits were subjected to MI/R (45 min/4 h) and randomly received saline, glucose-insulin-potassium (GIK) or GK respectively (2 mL/kg/h, i.v.). In vitro study was performed on cultured endothelial cells subjected to simulated ischemia/reperfusion. In vivo treatment with GIK but not GK attenuated myocardial injury as evidenced by reduced plasma creatine kinase activity, myocardial apoptosis and infarct size in MI/R rabbits compared with the saline group. Interestingly, GIK but not GK significantly decreased coronary endothelial expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1), inhibited adherence of PMNs to coronary endothelium (107.7 ± 7.4 vs. 155.0 ± 9.2 PMNs/mm² in saline group, n = 8, P < 0.01), and therefore decreased myocardial PMNs accumulation. In cultured endothelial cells subjected to simulated ischemia/reperfusion, insulin (10^− 7 M) increased Akt activity and eNOS phosphorylation with subsequent NO production, and concurrently exerted an anti-adhesive effect as manifested by reduced endothelial P-selectin and ICAM-1 surface expression and PMNs adherence (13.7 ± 1.3% vs. 22.2 ± 1.9% in vehicle, n = 9, P < 0.01), all of which are abolished by the specific Akt inhibitor. Furthermore, inhibition of insulin-stimulated NO production using either the selective eNOS inhibitor cavtratin or the NOS inhibitor L-NAME blocked the anti-adhesive effect of insulin. These results demonstrate that insulin reduces endothelial P-selectin and ICAM-1 expression, and thus inhibits leukocyte–endothelium adherence in MI/R rabbit hearts. The anti-adhesive property by insulin may be mediated by the Akt-mediated and NO-dependent pathway.     

Pioglitazone treatment stimulates circulating CD34-positive cells in type 2 diabetes patients

Circulating bone marrow derived immature cells, including CD34-positive (CD34⁺) cells, contribute to maintenance of the vasculature, not only as a pool of endothelial progenitor cells (EPCs), but also as a source of growth/angiogenesis factor. We hypothesized that the thiazolidineone compound pioglitazone could stimulate the circulating CD34⁺ cells in diabetic patients. Thirty-four patients with type 2 diabetes received 15–30 mg pioglitazone for 24 weeks. The number of circulating CD34⁺ cells significantly increased at 12 and continued this effect for 24 weeks (1.08 ± 0.39, 1.34 ± 0.34 and 1.32 ± 0.28 cells/μl at 0, 12 and 24 weeks, respectively). The change of CD34⁺ cell levels (ΔCD34⁺ cells) between 0 and 12 weeks was significantly correlated with the change of high sensitive C reactive protein levels (Δhs-CRP) and change in adiponectin levels (Δadiponectin) (r = −0.412, r = 0.359, respectively). Our study demonstrated that pioglitazone treatment increased circulating CD34⁺ cells, suggesting that this effect may at least partly contribute to the anti-atherosclerotic action of pioglitazone.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Effects of low-dose recombinant human insulin-like growth factor-I on insulin sensitivity, growth hormone and glucagon levels in young adults with insulin-dependent diabetes mellitus

Despite recent interest in the therapeutic potential of recombinant human insulin-like growth factor-I (rhIGF-I) in the treatment of diabetes mellitus, its mechanism of action is still not defined. We have studied the effects of low-dose bolus subcutaneous rhIGF-I (40 μg/kg and 20 μg/kg) on insulin sensitivity, growth hormone (GH) and glucagon levels in seven young adults with insulin-dependent diabetes mellitus (IDDM) using a randomized double-blind placebo-controlled crossover study design. Each was subjected to a euglycemic clamp (5 mmol/L) protocol consisting of a variable-rate insulin infusion clamp (6:00 to 8:00 ) followed by a two-dose hyperinsulinemic clamp (insulin infusion of 0.75 mU · kg⁻¹ · min⁻¹ from 8 to 10 and 1.5 mU · kg⁻¹ · min⁻¹ from 10 to 12 noon) incorporating [6,6 ²H₂]glucose tracer for determination of glucose production/utilization rates. Following rhIGF-I administration, the serum IGF-I level (mean ± SEM) increased (40 μg/kg, 655 ± 90 ng/mL, P < .001; 20 μg/kg, 472 ± 67 ng/mL, P < .001; placebo, 258 ± 51 ng/mL). Dose-related reductions in insulin were observed during the period of steady-state euglycemia (1 to 8 ) (40 μg/kg, 48 ± 5 pmol/L, P = .01; 20 μg/kg, 58 ± 8 pmol/L, P = .03; placebo, 72 ± 8 pmol/L). The mean overnight GH level (40 μg/kg, 9.1 ± 1.4 mU/L, P = .04; 20 μg/kg, 9.6 ± 2.0 mU/L, P = .12; placebo, 11.3 ± 1.7 mU/L) and GH pulse amplitude (40 μg/kg, 18.8 ± 2.9 mU/L, P = .04; 20 μg/kg, 17.0 ± 3.4 mU/L, P> .05; placebo, 23.0 ± 3.7 mU/L) were also reduced. No differences in glucagon, IGF binding protein-1 (IGFBP-1), acetoacetate, or β-hydroxybutyrate levels were found. During the hyperinsulinemic clamp conditions, no differences in glucose utilization were noted, whereas hepatic glucose production was reduced by rhIGF-I 40 μg/kg (P = .05). Our data demonstrate that in subjects with IDDM, low-dose subcutaneous rhIGF-I leads to a dose-dependent reduction in the insulin level for euglycemia overnight that parallels the decrease in overnight GH levels, but glucagon and IGFBP-1 levels remain unchanged. The decreases in hepatic glucose production during the hyperinsulinemic clamp study observed the following day are likely related to GH suppression, although a direct effect by rhIGF-I cannot be entirely discounted.     

Role of nitric oxide in regulation of coronary blood flow during myocardial ischemia in dogs

Objectives. This study was undertaken to examine whether nitric oxide released in ischemic myocardium decreases the coronary vascular resistance and attenuates the severity of contractile and metabolic dysfunction.Background. Endothelium-derived relaxing factor, recently identified as nitric oxide, is a potent relaxant of coronary smooth muscle.Methods. The left anrterior descending coronary artery was perfused through an extracorporeal bypass tube placed in the carotid artery in 56 open chest dogs. After hemodynamic stabilization, we occluded this bypass tube to decrease coronary blood flow to one third of the control flow. Thereafter, we maintained a constant coronary perfusion pressure(40.9 ± 3.1mm Hg).Results. Under ischemic conditions, the coronary arteriovenous differences in nitrate and nitrite (end products of nitric oxide) increased (from 3.5 ± 0.4 [mean ± SEM] to 12.9 ± 2.1 μmol/liter, p < 0.01). ⁰-Monomethyl -arginine (3 μg/kg body weight per min, intracoronary) decreased the coronary arteriovenous differences in nitrate and nitrite (5.0 ± 0.9 μmol/liter, p < 0.05) and coronary blood flow (from 29.8 ± 0.5 to 18.1 ± 1.1 ml/100 g per min, p < 0.001). Fractional shortening (from 3.7 ± 1.0 to −1.3 ± 0.7%, p < 0.001) and lactate extraction ration (from −44.0 ± 4.1 to −59.2 ± 4.9%, p < 0.005) of the perfused area also decreased. These values were restored by the concomitant administration of -arginine. Blood flow to the endomyocardium was decreased relative to the epimyocardium. A reduction in coronary blood flow and worsening of myocardial contractile and metabolic functions due to the administration of ^G-monomethyl -arginine during ischemia were observed in denervated hearts. A reduction in coronary blood flow in ischemic myocardium was observed with the administration of ^W-nitro- -arginene methyl ester as well, although neither ^W-nitro- -arginine methyl ester nor ^G-monomethyl -arginine changed coronary blood flow and myocardial contractile and metabolic functions in the nonischemic myocardium. The cyclic guanosine monophosphate content of epicardial coronary artery increased due to myocardial ischemia; this increased was attenuated with ^G-monomethyl -arginine treatment.Conclusions. We conclude that endogenous nitric oxide predominantly decreases the coronary vascular resistance of ischemic endomyocardium, thereby improving myocardial contractility and metabolic function.     

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

PYK2 is a Ca²⁺-dependent, nonreceptor protein tyrosine kinase that is involved in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. We and others have previously investigated PYK2's function in vitro using cultured neonatal and adult rat ventricular myocytes as model systems. However, the function of PYK2 in the in vivo adult heart remains unclear. Here we evaluate the effect of PYK2 inhibition following myocardial infarction (MI) using adenoviral (Adv) overexpression of the C-terminal domain of PYK2, known as CRNK. First we demonstrate that CRNK functions as a dominant-negative inhibitor of PYK2-dependent signaling, presumably by displacing PYK2 from focal adhesions and costameres. Then, male Sprague–Dawley rats (~ 300 g) underwent permanent left anterior descending coronary artery ligation. One wk post-MI, either Adv-GFP (n = 34) or Adv-CRNK (n = 28) was administered (10¹⁰ pfu, 0.1 ml) via catheter-based, Optison^®-mediated gene transfer. LV structure and function were evaluated by echocardiography 1 and 3 wk after gene transfer, and LV tissue was analyzed by real-time RT-PCR and Western blotting. CRNK overexpression was readily detected by Western blotting 1 wk following gene transfer. Adv-CRNK improved overall survival (P = 0.03; Logrank Test) and LV fractional shortening (23 ± 2% vs. 31 ± 2% for Adv-GFP vs. Adv-CRNK infected animals, respectively; P < 0.05). Whereas MI hearts exhibited increased β-, and decreased α-myosin heavy chain (MHC) mRNA expression characteristic of LVH, Adv-CRNK reversed the MHC isoenzyme switch (3.3 ± 1.4 fold increase in αMHC; 0.4 ± 0.1 fold decrease in βMHC; P < 0.05 for both). In summary, CRNK gene transfer improves survival, increases LV function, and alters MHC gene expression suggesting an attenuation of LV remodeling post-MI.     

Inflammatory pathways in patients with heart failure and preserved ejection fraction

Immune activation is well established in patients with chronic heart failure and reduced ejection fraction (HF and reduced EF) and is associated with an impaired prognosis. Patients with heart failure and preserved ejection fraction (HF and preserved EF) have an impaired prognosis as well. It is not known whether they have signs of immune activation.

Methods

We studied patients with HF and preserved EF (n = 17, NYHA II [n = 7]/III [n = 10]) and patients with HF and reduced EF (n = 17 NYHA II [n = 1]/III [n = 16]) and 20 controls. Echocardiography demonstrated preserved ejection fraction (LVEF 59 ± 9%), but LV hypertrophy in patients with preserved EF as compared with patients with reduced EF (LVEF 23 ± 5%). We evaluated levels of TNFα, its receptors (sTNFR-1 and 2), IL-6, IL-10 and NT-proBNP.

Results

TNFα, was highest in HF with reduced EF (2.87 ± 0.65 vs 1.67 ± 0.58 pg/mL, p < 0.001) compared to preserved EF and similar between HF with preserved EF and controls. However, sTNFR1 (1618 ± 384 vs 1017 ± 302 pg/mL, p < 0.001) and sTNFR2 levels (3554 ± 916 vs 2041 ± 586 pg/mL, p < 0.001) in HF with preserved EF were significantly higher compared with controls. The same was true for IL-6, IL-10 and NT-proBNP. The highest cytokine and NT-proBNP levels were present in HF with reduced EF. There was a negative correlation between TNFα, and LVEF (r = −  0.700; p < 0.0001) and positive correlations between sTNFR1 and 2 with NT-proBNP.

Conclusion

Patients with HF and preserved EF already show signs of systemic-immune activation which may contribute to the impaired prognosis and the progression to HF with reduced EF.