Analysis of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger (NCX) function and current in murine cardiac myocytes during heart failure

Na⁺/Ca²⁺ exchanger (NCX) plays important roles in cardiac electrical activity and calcium homeostasis. NCX current (I_NCX) shows transmural gradient across left ventricle in many species. Previous studies demonstrated that NCX expression was increased and transmural gradient of I_NCX was disrupted in failing heart, but the mechanisms underlying I_NCX remodeling still remain unknown. In present study, we used patch clamp technique to record I_NCX from subepicardial (EPI) myocytes and subendocardial (ENDO) myocytes isolated from sham operation (SO) mice and heart failure (HF) mice. Our results showed that I_NCX was higher in normal EPI cells compared with that in ENDO, whatever for forward mode or reverse mode. In HF group, I_NCX was significantly up-regulated, but EPI-ENDO difference was disrupted because of a more increase of I_NCX in ENDO myocytes. In order to explore the molecular mechanism underlying remodeling of I_NCX in failing heart, we detected the protein expression of NCX1 and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) by Western blot. We found that CaMKII activity was dramatically enhanced and parallel with the expression of NCX1 in failing heart. Our study demonstrated that transmural gradient of I_NCX existed in murine left ventricle, and increased activity of CaMKII should account for I_NCX remodeling in failing heart.     

Gastric emptying and Ca2+ and K+ channels of circular smooth muscle cells in diet‐induced obese prone and resistant rats

Objective:

Accelerated gastric emptying that precipitates hunger and frequent eating could be a potential factor in the development of obesity. The aim of this study was to study gastric emptying in diet‐induced obese‐prone (DIO‐P) and DIO‐resistant (DIO‐R) rats and explore possible differences in electrical properties of calcium (Ca²⁺) and potassium (K⁺) channels of antral circular smooth muscle cells (SMCs).

Design and Methods:

Whole‐cell patch‐clamp technique was used to measure Ca²⁺ and K⁺ currents in single SMCs. Gastric emptying was evaluated 90 min after the ingestion of a solid meal.

Results:

Solid gastric emptying in the DIO‐P rats was significantly faster compared with that in the DIO‐R rats. The peak amplitude of L‐type Ca²⁺ current (I_Ba,L) at 10 mV in DIO‐P rats was greater than that in DIO‐R rats without alternation of the current–voltage curve and voltage‐dependent activation and inactivation. The half‐maximal inactivation voltage of transient outward K⁺ current (I_Kto) was more depolarized (～4 mV) in DIO‐P rats compared with that in DIO‐R rats. No difference was found in the current density or recovery kinetics of I_Kto between two groups. The current density of delayed rectifier K⁺ current (I_Kdr), which was sensitive to tetraethylammonium chloride but not 4‐aminopyridine, was lower in DIO‐P rats than that in DIO‐R rats.

Conclusion:

The accelerated gastric emptying in DIO‐P rats might be attributed to a higher density of I_Ba,L, depolarizing shift of inactivation curve of I_Kto and lower density of I_Kdr observed in the antral SMCs of DIO‐P rats.     

Stimulatory Effect of Leptin on Nitric Oxide Production Is Impaired in Dietary‐Induced Obesity

Objective: We investigated the effect of leptin on nitric oxide production in lean and rats made obese by a high‐calorie diet. Research Methods and Procedures: The animals were placed in metabolic cages, and urine was collected in 2‐hour periods after leptin (1 mg/kg intraperintoneally) or vehicle administration. Blood was obtained 0.5, 1, 2, 4, or 6 hours after injection. Results: Leptin had no effect on systolic blood pressure in either lean or obese animals. Plasma concentration of NO metabolites (nitrites + nitrates, NO_x) increased in lean rats by 31.5%, 58.0%, and 27.9% at 1, 2, and 4 hours after leptin injection, respectively. In the obese group, plasma NO_x increased only at 2 hours (+36.5%). Leptin increased urinary NO_x excretion by 31.8% in the first 2‐hour period after injection in lean but not in obese rats. In lean animals, leptin elevated plasma cyclic 3′, 5′‐guanosine monophosphate (cGMP) at 1, 2, and 4 hours by 35.3%, 96.3%, and 57.3%, respectively. In the obese group, plasma cGMP was higher only at 2 and 4 hours (+44.6% and +32.1%, respectively). Urinary excretion of cGMP increased in lean animals by 67.1% in the first period and by 50.4% in the second period. In the obese group, leptin induced a 53.9% increase in urinary cGMP excretion only in the first 2‐hour period. Discussion: The stimulatory effect of leptin on NO production is impaired in dietary‐induced obesity; however, leptin does not increase blood pressure in obese animals, suggesting that other NO—independent depressor mechanisms are stimulated.     

Cardiac fas receptor-dependent apoptotic pathway in obese Zucker rats

Objective: Very limited information regarding the cardiac molecular mechanism in obesity is available. The purpose of this study was to evaluate the cardiac Fas receptor‐dependent (type I) apoptotic pathway in obese Zucker rats. Research Methods and Procedures: Sixteen obese Zucker rats were studied at 5 to 6 months of age, and 16 age‐matched lean Zucker rats served as controls. Heart weight index, myocardial architecture, key components of the Fas receptor‐dependent apoptotic pathway, apoptotic activity, and fibrosis in the excised left ventricle of rats were measured by weight scales, hematoxylin and eosin staining, Western blotting, TUNEL assay, and Masson trichrome staining. Results: Body weight, whole heart weight, left ventricular weight, ratio of whole heart weight to tibia length, percentage of TUNEL‐positive cardiac myocytes, and percentage of cardiac fibrosis were significantly increased in the obese group. Cardiomyocyte disarray and increased cardiac interstitial space were observed in obese rats. Protein levels of Fas ligand, Fas death receptors, and Fas‐associated Death Domain were all significantly increased in the obese group. In addition, pro‐caspase‐8 and pro‐caspase‐3 were significantly decreased, whereas activated caspase‐8 and activated caspase‐3 were significantly increased in the obese group, which implies that pro‐forms of caspase‐8 and caspase‐3 were cleaved into active‐forms caspase‐8 and caspase‐3. Conclusions: Cardiac Fas receptor‐dependent apoptotic pathways were more activated in obese rats’ hearts, which may provide one of the possible apoptotic mechanisms for developing cardiac abnormality in obesity.     

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

Adiponectin Levels during Low‐ and High‐Fat Eucaloric Diets in Lean and Obese Women

Objective: Adiponectin influences insulin sensitivity (S_I) and fat oxidation. Little is known about changes in adiponectin with changes in the fat content of eucaloric diets. We hypothesized that dietary fat content may influence adiponectin according to an individual's S_I. Research Methods and Procedures: We measured changes in adiponectin, insulin, glucose, and leptin in response to high‐fat (HF) and low‐fat (LF) eucaloric diets in lean (n = 10) and obese (n = 11) subjects. Obese subjects were further subdivided in relation to a priori S_I. Results: We found significantly higher insulin, glucose, and leptin and lower adiponectin in obese vs. lean subjects during both HF and LF. The mean group values of these measurements, including adiponectin (lean, HF 21.9 ± 9.8; LF, 20.8 ± 6.6; obese, HF 10.0 ± 3.3; LF, 9.5 ± 2.3 ng/mL; mean ± SD), did not significantly change between HF and LF diets. However, within the obese group, the insulin‐sensitive subjects had significantly higher adiponectin during HF than did the insulin‐resistant subjects. Additionally, the change in adiponectin from LF to HF diet correlated positively with the obese subjects’ baseline S_I. Discussion: Although in lean and obese women, group mean values for adiponectin did not change significantly with a change in fat content of a eucaloric diet, a priori measured S_I in obese subjects predicted an increase in adiponectin during the HF diet; this may be a mechanism that preserves S_I in an already obese group.     

Vitamin C Pretreatment Attenuates Hypoxia-Induced Disturbance of Sodium Currents in Guinea Pig Ventricular Myocytes

As an important in vivo antioxidant, vitamin C is commonly used clinically to alleviate hypoxia-induced heart symptoms. To approach the protective mechanisms of vitamin C on hearts during hypoxia, we investigated the electrophysiological effects of vitamin C (1 mM, pretreated before hypoxia) on Na⁺ currents (including transient and persistent Na⁺ currents) in guinea pig ventricular myocytes during hypoxia by the whole-cell and single-channel patch-clamp techniques. Whole-cell recordings showed that the mean current density of I _NaT in the hypoxia group decreased from the control value of 40.2142 ± 1.7735 to 27.1663 ± 1.8441 pA/pF and current density of I _NaP increased from 0.3987 ± 0.0474 to 1.1854 ± 01994 pA/pF (n = 9, P < 0.05 vs. control) at 15 min. However, when vitamin C was administered before hypoxia as pretreatment, I _NaT and I _NaP varied moderately (mean current density of I _NaT decreasing from 41.6038 ± 2.9762 to 34.6341 ± 1.9651 pA/pF and current density of I _NaP increasing from 0.3843 ± 0.0636 to 0.6734 ± 0.1057 pA/pF; n = 9, P < 0.05 vs. hypoxia group). Single-channel recordings (cell-patched) showed that the mean open probability and open time of I _NaP increased significantly in both groups at hypoxia 15 min. However, the increased current values of the hypoxia group were still marked at hypoxia 15 min (n = 9, P < 0.05 vs. vitamin C + hypoxia group). Our results indicate that vitamin C can attenuate the disturbed effects of hypoxia on Na⁺ currents (I _NaT and I _NaP) of cardiac myocytes in guinea pigs effectively.     

The Venom of Ornithoctonus huwena affect the electrophysiological stability of neonatal rat ventricular myocytes by inhibiting sodium,potassium and calcium current

Spider venoms are known to contain various toxins that are used as an effective means to capture their prey or to defend themselves against predators. An investigation of the properties of Ornithoctonus huwena (O.huwena) crude venom found that the venom can block neuromuscular transmission of isolated mouse phrenic nerve-diaphragm and sciatic nerve-sartorius preparations. However, little is known about its electrophysiological effects on cardiac myocytes. In this study, electrophysiological activities of ventricular myocytes were detected by 100 μg/mL venom of O.huwena, and whole cell patch-clamp technique was used to study the acute effects of the venom on action potential (AP), sodium current (I_Na), potassium currents (I_Kr, I_Ks, I_to1 and I_K1) and L-type calcium current (I_CaL). The results indicated that the venom prolongs APD₉₀ in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 μg/mL venom inhibited 72.3 ± 3.6% I_Na current, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of I_Kr, and 65 ± 3.3% I_CaL current, yet, didn't have obvious effect on I_Ks, I_to1 and I_K1 currents. In conclusion, the O.huwena venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.     

S 23521 Decreases Food Intake and Body Weight Gain in Diet‐Induced Obese Rats

Objective: To investigate the effect of S 23521, a new glucagon‐like peptide‐1‐(7‐36) amide analogue, on food intake and body weight gain in obese rats, as well as on gene expression of several proteins involved in energy homeostasis. Research Methods and Procedures: Lean and diet‐induced obese rats were treated with either S 23521 or vehicle. S 23521 was given either intraperitoneally (10 or 100 μg/kg) or subcutaneously (100 μg/kg) for 14 and 20 days, respectively. Because the low‐dose treatment did not affect food intake and body weight, the subcutaneous treatment at high dose was selected to test the effect on selected end‐points. Results: Treated obese rats significantly decreased their cumulative energy intake in relation to vehicle‐treated counterparts (3401 ± 65 vs. 3898 ± 72 kcal/kg per 20 days; p < 0.05). Moreover, their body weight gain was reduced by 110%, adiposity was reduced by 20%, and plasma triglyceride levels were reduced by 38%. The treatment also improved glucose tolerance and insulin sensitivity of obese rats. Regarding gene expression, no changes in uncoupling protein‐1, uncoupling protein‐3, leptin, resistin, and peroxisome proliferator‐activated receptor (PPAR)‐γ were observed. Discussion: S 23521 is an effective glucagon‐like peptide‐1‐(7‐36) amide analogue, which induced a decrease in energy intake, body weight, and adiposity in a rat model of diet‐induced obesity. In addition, the treatment also improved glucose tolerance and insulin sensitivity of obese rats. These results strongly support S 23521 as a putative molecule for the treatment of obesity.     

Normal Distribution of Body Weight Gain in Male Sprague‐Dawley Rats Fed a High‐Energy Diet

Objective: To investigate the effect of a high‐energy (HE) diet on caloric intake, body weight, and related parameters in outbred male Sprague‐Dawley (SD) rats. Research Methods and Procedures: Twenty‐eight SD rats were fed either chow (C) for 19 weeks or HE diet for 14 weeks and then C for 5 weeks. Blood hormones and metabolites were assayed, and expression of uncoupling protein‐1 and hypothalamic energy‐balance‐related genes were determined by Northern blotting and in situ hybridization, respectively. Results: HE rats gained body weight more rapidly than C animals with a range of weight gains, but there was no evidence that weight gain was bimodally distributed. Caloric intake was transiently elevated after introduction of the HE diet. Transfer of HE rats back to C resulted in a drop in caloric intake, but a stable body weight. In terminal analysis, two of four dissected adipose tissue depots were heavier in rats that had previously been fed HE diet. Blood leptin, insulin, glucose, and nonesterified fatty acids were not different between the groups. Uncoupling protein‐1 mRNA was elevated in interscapular brown adipose tissue from HE rats. There was a trend for agouti‐related peptide mRNA in the hypothalamic arcuate nucleus to be higher in HE rats. Discussion: Contrary to other studies of the SD rat on HE diet, body weight and other measured parameters were normally distributed. There was no segregation into two distinct populations on the basis of susceptibility to diet‐induced obesity. This characteristic may be dependent on the breeding colony from which animals were sourced.     

Differential secretion of satiety hormones with progression of obesity in JCR:LA-corpulent rats

Objective: To characterize the gastrointestinal tract at the onset and in well‐established obesity. Methods and Procedures: Lean (+/?) and obese (cp/cp) male JCR:LA‐cp rats lacking a functional leptin receptor were killed at 3.5 weeks and 9 months of age and plasma concentrations of satiety hormones determined. The small intestine, colon, and stomach were measured, weighed, and mRNA levels of satiety genes quantified. Results: At the onset of obesity, obese rats had greater intestine, colon, and liver mass when adjusted for body weight compared to lean rats. Conversely, adult rats with established obesity had lower intestine and colon mass and length after adjustment for body weight. Early changes in gene expression included decreased ghrelin mRNA levels in stomach and increased peptide YY (PYY) mRNA levels in duodenum of young obese rats. After massive accumulation of adipose tissue had occurred, adult obese rats had increased proglucagon and ghrelin mRNA expression in the proximal intestine. In the distal small intestine, obese rats had lower proglucagon, ghrelin, and PYY mRNA levels. Finally, at the onset and in well‐established obesity, obese rats had higher plasma insulin, amylin, glucagon like peptide‐1 (GLP‐1), and PYY, a finding, with the exception of insulin, unique to this model. Plasma total ghrelin levels were significantly lower at the onset of obesity and established obesity compared to the lean rats. Discussion: Several defects are manifested in the obese gut early on in the disease before the accumulation of large excesses of body fat and represent potential targets for early intervention in obesity.     

Regional Fat Pad Growth and Cellularity in Obese Zucker Rats: Modulation by Caloric Restriction

Objective : To investigate, in young obese male Zucker rats, the effects of chronic food restriction and subsequent refeeding on: 1) parameters of nonadipose and adipose growth, 2) regional adipose depot cellularity [fat cell volume (FCV) and number], and 3) circulating leptin levels. Research Methods and Procedures : Obese (fa/fa) and lean (Fa/?) male Zucker rats were studied from age 5 to 19 weeks. After baseline food intake monitoring, 10 obese rats were subjected to 58 days of marked caloric restriction from ad libitum levels [obese‐restricted (OR)], followed by a return to ad libitum feeding for 22 days. Ten lean control rats and 10 obese control rats were fed ad libitum for the entire experiment. All rats were fed using a computer‐driven automated feeding system designed to mimic natural eating patterns. Results : After food restriction, OR rats weighed significantly less than did lean and obese rats and showed a significant diminution in body and adipose growth as compared with obese rats. Relative adiposity was not different between obese and OR rats and was significantly higher than that of lean rats. The limitation in growth of the adipose tissue mass in OR rats was due mostly to suppression of fat cell proliferation because the mean FCV in each of the four depots was not affected. Serum leptin levels of OR and obese rats were not different from each other but were significantly higher than those of lean rats. Discussion : Marked caloric restriction affects obese male Zucker rats in a manner different from that of nongenetic rodent models (i.e., Wistar rats). In comparison with the response to caloric deprivation of Wistar rats, these calorically restricted obese male Zucker rats appeared to defend their relative adiposity and mean FCV at the expense of fat cell number. These findings indicate that genetic and/or tissue‐specific controls override the general consequences of food restriction in this genetic model of obesity.     

Association between Serum Leptin Levels and 24‐Hour Blood Pressure in Obese Women

Objective: To assess the relationship between serum leptin and 24‐hour blood pressure (BP) in obese women, according to body fat distribution. Research Methods and Procedures: A cross‐sectional study was carried out in a population of 70 nondiabetic, normotensive, obese women (40 with android and 30 with gynoid type of obesity) and 20 nonobese healthy women as a control group. All subjects underwent 24‐hour ambulatory BP monitoring. Blood samples were collected for serum leptin and plasma insulin measurements. Total cholesterol and high‐density lipoprotein cholesterol were also measured. Results: Serum leptin levels were significantly higher in obese subjects than in controls, and they were more elevated in android obese women than in gynoid ones. Leptin levels were positively related to body mass index (BMI), insulin, and waist and hip circumferences in the android group. Among gynoid subjects, leptin levels showed positive associations with BMI and insulin. In women with android obesity, strong positive correlations (p < 0.001) were found between leptin levels and 24‐hour systolic BP (SBP), daytime SBP, nighttime SBP, 24‐hour diastolic BP (DBP), and daytime DBP. Multiple regression analyses, including age, insulin and leptin concentrations, BMI, and waist and hip circumferences on 24‐hour and daytime SBP and DBP, showed that only leptin levels contributed to the variability of BP. Conclusions: Our study shows that serum leptin levels are directly related to 24‐hour BP levels in normotensive women with android fat distribution, independently of BMI.     

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes

Much less is known about the contributions of the Na⁺/Ca²⁺ exchanger (NCX) and sarcoplasmic reticulum (SR) Ca²⁺ pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca²⁺ decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I_NCX) and the Ca²⁺ transient. We found that the integral of I_NCX, an indicator of SR Ca²⁺ content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca²⁺ extrusion by NCX (V_NCX) was linear with [Ca²⁺] and did not indicate saturation kinetics until [Ca²⁺] reached 1–3 µM for each age group. There was a significantly greater time delay between the peaks of I_NCX and the Ca²⁺ transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content