Requirements for essential micronutrients during caloric restriction and fasting.

Caloric restriction (CR) or energy restriction, when carefully designed, monitored, and implemented in self-motivated and compliant individuals, proves to be a viable non-pharmacologic strategy for human weight control and obesity management. Beyond its role in weight management, CR has the potential to impede responses involved not only in the pathogenesis of various diseases but also in the aging process in adults, thereby being proposed to promote a healthier and longer life. The core objective of implementing caloric restriction is to establish a balance between energy intake and expenditure, typically involving a reduction in intake and an increase in expenditure-a negative balance at least initially. It may transition toward and maintain a more desired equilibrium over time. However, it is essential to note that CR may lead to a proportional reduction in micronutrient intake unless corresponding supplementation is provided. Historical human case reports on CR have consistently maintained adequate intakes (AI) or recommended dietary allowances (RDA) for essential micronutrients, including vitamins and minerals. Similarly, longevity studies involving non-human primates have upheld micronutrient consumption levels comparable to control groups or baseline measures. Recent randomized controlled trials (RCTs) have also endorsed daily supplementation of multivitamins and minerals to meet micronutrient needs. However, aside from these human case reports, limited human trials, and primate experiments, there remains a notable gap in human research specifically addressing precise micronutrient requirements during CR. While adhering to AI or RDA for minerals and vitamins appears sensible in the current practice, it's important to recognize that these guidelines are formulated for generally healthy populations under standard circumstances. The adequacy of these guidelines in the setting of prolonged and profound negative energy balance remains unclear. From perspectives of evidence-based medicine and precision nutrition, this field necessitates comprehensive exploration to uncover the intricacies of absorption, utilization, and metabolism and the requirement of each hydrophilic and lipophilic vitamin and mineral during these special periods. Such investigations are crucial to determine whether existing daily dietary recommendations for micronutrients are quantitatively inadequate, excessive, or appropriate when energy balance remains negative over extended durations.

Total bile acid to platelet ratio: A noninvasive index for predicting liver fibrosis in primary biliary cholangitis.

The aim of the study was to develop a new early noninvasive diagnostic model for primary biliary cholangitis (PBC).A total of 118 PBC patients who had undergone a liver biopsy were enrolled in the study, and were randomized into a model group (78 patients) and a validation group (40 patients). The patients' histological stages were based on the classifications of the Scheuer's stage. All common parameters and liver pathological results were analyzed. And total bile acid to platelet ratio, aspartate aminotransferase to platelet ratio index, fibrosis index based on 4 factors and red cell distribution width to platelet ratio were calculated.There were 106 (89.8%) women and 12 men in this study, and the number of patients in Scheuer stage I, II, III, and IV hepatic fibrosis was 52 (44.1%), 36 (30.5%), 26 (22.0%), and 4 (3.4%), respectively. The areas under the receiver operating characteristic curves of the total bile acid to platelet ratio (TPR), the aspartate aminotransferase to platelet ratio index, the fibrosis index based on 4 factors , and the red cell distribution width to platelet ratio for predicting advanced liver fibrosis were 0.771, 0.715, 0.618, and 0.517 respectively. The areas under the receiver operating characteristic curves of the TPR was higher than other non-invasive serological models.As a simple, inexpensive and easily accessible non-invasive liver fibrosis diagnostic model, the TPR may be a new noninvasive marker for predicting histologic severity of PBC.

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase-induced cardiomyopathy: involvement of mitofusin-2.

BACKGROUND AND PURPOSE: The anthracycline doxorubicin (DOX), although successful as a first-line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase-derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX-stimulated Nox2 activation. EXPERIMENTAL APPROACH: Nox2-/- and wild-type (WT) littermate mice were administered DOX (12 mg·kg-1 over 3 weeks) prior to study at 4 weeks. Detailed mechanisms were investigated in murine HL-1 cardiomyocytes, employing a robust model of oxidative stress, gene silencing and pharmacological tools. KEY RESULTS: DOX-induced cardiac dysfunction, cardiomyocyte remodelling, superoxide production and apoptosis in WT mice were attenuated in Nox2-/- mice. Transcriptional analysis of left ventricular tissue identified 152 differentially regulated genes (using adjusted P < 0.1) in DOX-treated Nox2-/- versus WT mice, and network analysis highlighted 'Cell death and survival' as the biological function most significant to the dataset. The mitochondrial membrane protein, mitofusin-2 (Mfn2), appeared as a strong candidate, with increased expression (1.5-fold), confirmed by qPCR (1.3-fold), matching clear published evidence of promotion of cardiomyocyte cell death. In HL-1 cardiomyocytes, targeted siRNA knockdown of Nox2 decreased Mfn2 protein expression, but not vice versa. While inhibition of Nox2 activity along with DOX treatment attenuated its apoptotic and cytotoxic effects, reduced apoptosis after Mfn2 silencing reflected a sustained cytotoxic response and reduced cell viability. CONCLUSIONS AND IMPLICATIONS: DOX-induced and Nox2-mediated up-regulation of Mfn2, rather than contributing to cardiomyocyte dysfunction through apoptotic pathways, appears to promote a protective mechanism. LINKED ARTICLES: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Effects of plant stanol or sterol-enriched diets on lipid profiles in patients treated with statins: systematic review and meta-analysis.

Efficacy and safety data from trials with suitable endpoints have shown that non-statin medication in combination with a statin is a potential strategy to further reduce cardiovascular events. We aimed to evaluate the overall effect of stanol- or sterol-enriched diets on serum lipid profiles in patients treated with statins by conducting a meta-analysis of randomized controlled trials (RCTs). We used the PubMed, Cochrane library and ClinicalTrials.gov databases to search for literature published up to December 2015. Trials were included in the analysis if they were RCTs evaluating the effect of plant stanols or sterols in patients under statin therapy that reported corresponding data on serum lipid profiles. We included 15 RCTs involving a total of 500 participants. Stanol- or sterol-enriched diets in combination with statins, compared with statins alone, produced significant reductions in total cholesterol of 0.30 mmol/L (95% CI -0.36 to -0.25) and low-density lipoprotein (LDL) cholesterol of 0.30 mmol/L (95% CI -0.35 to -0.25), but not in high-density lipoprotein cholesterol or triglycerides. These results persisted in the subgroup analysis. Our meta-analysis provides further evidence that stanol- or sterol-enriched diets additionally lower total cholesterol and LDL-cholesterol levels in patients treated with statins beyond that achieved by statins alone.

Plasma cholesterol-lowering activity of dietary dihydrocholesterol in hypercholesterolemia hamsters.

OBJECTIVE: Cholesterol analogs have been used to treat hypercholesterolemia. The present study was to examine the effect of dihydrocholesterol (DC) on plasma total cholesterol (TC) compared with that of ß-sitosterol (SI) in hamsters fed a high cholesterol diet. METHODS AND RESULTS: Forty-five male hamsters were randomly divided into 6 groups, fed either a non-cholesterol diet (NCD) or one of five high-cholesterol diets without addition of DC and SI (HCD) or with addition of 0.2% DC (DA), 0.3% DC (DB), 0.2% SI (SA), and 0.3% SI (SB), respectively, for 6 weeks. Results showed that DC added into diet at a dose of 0.2% could reduce plasma TC by 21%, comparable to that of SI (19%). At a higher dose of 0.3%, DC reduced plasma TC by 15%, less effective than SI (32%). Both DC and SI could increase the excretion of fecal sterols, however, DC was more effective in increasing the excretion of neutral sterols but it was less effective in increasing the excretion of acidic sterols compared with SI. Results on the incorporation of sterols in micellar solutions clearly demonstrated both DC and SI could displace the cholesterol from micelles with the former being more effective than the latter. CONCLUSION: DC was equally effective in reducing plasma cholesterol as SI at a low dose. Plasma TC-lowering activity of DC was mediated by inhibiting the cholesterol absorption and increasing the fecal sterol excretion.

Exendin-4 protects against post-myocardial infarction remodelling via specific actions on inflammation and the extracellular matrix.

Glucagon-like peptide-1 (GLP-1) is an insulin-releasing hormone clinically exploited for glycaemic control in diabetes, which also confers acute cardioprotection and benefits in experimental/clinical heart failure. We specifically investigated the role of the GLP-1 mimetic, exendin-4, in post-myocardial infarction (MI) remodelling, which is a key contributor to heart failure. Adult female normoglycaemic mice underwent coronary artery ligation/sham surgery prior to infusion with exendin-4/vehicle for 4 weeks. Metabolic parameters and infarct sizes were comparable between groups. Exendin-4 protected against cardiac dysfunction and chamber dilatation post-MI and improved survival. Furthermore, exendin-4 modestly decreased cardiomyocyte hypertrophy/apoptosis but markedly attenuated interstitial fibrosis and myocardial inflammation post-MI. This was associated with altered extracellular matrix (procollagen IαI/IIIαI, connective tissue growth factor, fibronectin, TGF-ß3) and inflammatory (IL-10, IL-1ß, IL-6) gene expression in exendin-4-treated mice, together with modulation of both Akt/GSK-3ß and Smad2/3 signalling. Exendin-4 also altered macrophage response gene expression in the absence of direct actions on cardiac fibroblast differentiation, suggesting cardioprotective effects occurring secondary to modulation of inflammation. Our findings indicate that exendin-4 protects against post-MI remodelling via preferential actions on inflammation and the extracellular matrix independently of its established actions on glycaemic control, thereby suggesting that selective targeting of GLP-1 signalling may be required to realise its clear therapeutic potential for post-MI heart failure.

Dairy consumption and risk of cardiovascular disease: an updated meta-analysis of prospective cohort studies.

BACKGROUND: Epidemiological studies to-date provided inconsistent findings on the effects of dairy consumption on the risk of cardiovascular disease (CVD). We aimed to examine the association of dairy consumption and its specific subtypes with CVD risk, including the risk of stroke and coronary heart disease (CHD) by a metaanalysis. METHODS: PubMed, EMBASE, and Cochrane Library databases were searched for articles published up to February 2014 to identify prospective cohort studies. Random-effects model or fix-effects model was used to compute the summary risk estimates. RESULTS: A total of 22 studies were eligible for analysis. An inverse association was found between dairy consumption and overall risk of CVD [9 studies; relative risk (RR)=0.88, 95% confidence interval (CI): 0.81, 0.96] and stroke (12 studies; RR=0.87, 95% CI: 0.77, 0.99). However, no association was established between dairy consumption and CHD risk (12 studies; RR=0.94, 95% CI: 0.82, 1.07). Stroke risk was significantly reduced by consumption of low-fat dairy (6 studies; RR=0.93, 95% CI: 0.88, 0.99) and cheese (4 studies; RR=0.91, 95% CI: 0.84, 0.98), and CHD risk was significantly lowered by cheese consumption (7 studies; RR=0.84, 95% CI: 0.71, 1.00). Restricting studies according to various inclusion criteria yielded similar results for CVD and CHD analyses, but showed attenuated results for stroke analysis. Heterogeneity across studies was found for stroke and CHD analyses, and publication bias was found for stroke analysis. CONCLUSION: This meta-analysis provided further evidence supporting the beneficial effect of dairy consumption on CVD. Low-fat dairy products and cheese may protect against stroke or CHD incidence.

Bioavailability of tramadol hydrochloride after administration via different routes in rats.

Tramadol is a synthetic non-opiate analgesic drug and effective for many kinds of chronic and acute pain. This study compared the bioavailability of tramadol after different administration routes in rats (oral, buccal and nasal). A simple HPLC analytical approach was used to determine the concentration of tramadol in plasma. The pharmacokinetic behavior and bioavailability of tramadol after administration via different routes in rats were investigated. Nasal and buccal administration of tramadol resulted in a fast increase followed by a rapid decrease in the plasma tramadol concentration. The Cmax values following buccal and nasal administration were 6 times and 20 times higher than that of oral administration, respectively, (6827.85 ± 7970.87 ng/ml, 22191.84 ± 5364.86 ng/ml, vs 1127.03 ± 778.34 ng/ml). The relative bioavailabilities of the nasal- and buccal-administered drug when compared with the oral route were 504.8% and 183.4%, respectively, which is much higher than that of oral administration. Nasal and buccal administration increased the bioavailability of tramadol, which may allow for a reduction in the dose of tramadol and a subsequent decrease in both side effects and toxicity. Therefore, this approach provides an effective choice for the delivery of tramadol, an analgesic drug.

Bitter gourd inhibits the development of obesity-associated fatty liver in C57BL/6 mice fed a high-fat diet.

Bitter gourd (BG) is a popular fruit in Asia with numerous well-known medicinal uses, including as an antidiabetic. In the current study, we aimed to explore the effects of BG on mitochondrial function during the development of obesity-associated fatty liver. C57BL/6 mice were divided into 4 experimental groups: mice fed a normal diet (control; included for reference only), mice fed a high-fat diet (HFD), and mice fed an HFD supplemented with freeze-dried BG powder through daily gavage at doses of 0.5 (HFD+0.5BG) and 5 (HFD+5BG) g/kg, respectively. After 16 wk, mice in the HFD+5BG group showed less body and tissue weight gain and less hyperglycemia and hyperlipidemia compared with those in the HFD group (P < 0.05). In both HFD+0.5BG and HFD+5BG groups, serum interleukin-6 concentration was lower than that in the HFD group (P < 0.02). The serum C-reactive protein concentration was lower in the HFD+5BG group compared with the HFD group (P < 0.04). An analysis of liver tissue revealed lower liver triglyceride and cholesterol concentrations in both HFD+0.5BG and HFD+5BG groups than in the HFD group (P < 0.01). The HFD+5BG group had less activation of the sterol regulatory element binding protein/fatty acid synthase (SREBP-1/FAS) pathway, greater superoxide dismutase activity, and less total protein and mitochondrial protein oxidation than did the HFD group (P < 0.05). Mitochondrial complex I, II, III, and V activity was greater in the HFD+0.5BG group than in the HFD group (P < 0.03). The HFD+5BG group only had greater complex V activity compared with the HFD group (P < 0.05). Mitochondrial dynamics regulators, including dynamin related protein 1 (DRP1) and mitofusin 1 (MFN1), as well as proapoptotic protein expression levels were restored by BG treatment (P < 0.02). Taken together, our results suggest that BG prevents inflammation and oxidative stress, modulates mitochondrial activity, suppresses apoptosis activation, and inhibits lipid accumulation during the development of fatty liver.

The wax glands and wax secretion of Matsucoccus matsumurae at different development stages.

In this paper, the wax secretions and wax glands of Matsucoccus matsumurae (Kuwana) at different instars were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The first and second instar nymphs were found to secrete wax filaments via the wax glands located in the atrium of the abdominal spiracles, which have a center open and a series of outer ring pores. The wax gland of the abdominal spiracle possesses a large central wax reservoir and several wax-secreting cells. Third-instar male nymphs secreted long and translucent wax filaments from monolocular, biolocular, trilocular and quadrilocular pores to form twine into cocoons. The adult male secreted long and straight wax filaments in bundles from a group of 18-19 wax-secreting tubular ducts on the abdominal segment VII. Each tube duct contained five or six wax pores. The adult female has dorsal cicatrices distributed in rows, many biolocular tubular ducts and multilocular disc pores with 8-12 loculi secreting wax filaments that form the egg sac, and a rare type wax pores with 10 loculi secreting 10 straight, hollow wax filaments. The ultrastructure and cytological characteristics of the wax glands include wax-secreting cells with a large nucleus, multiple mitochondria and several rough endoplasmic reticulum. The functions of the wax glands and wax secretions are discussed.

Mitochondrial dysfunction in obesity-associated nonalcoholic fatty liver disease: the protective effects of pomegranate with its active component punicalagin.

AIMS: Punicalagin (PU) is one of the major ellagitannins found in the pomegranate (Punica granatum), which is a popular fruit with several health benefits. So far, no studies have evaluated the effects of PU on nonalcoholic fatty liver disease (NAFLD). Our work aims at studying the effect of PU-enriched pomegranate extract (PE) on high fat diet (HFD)-induced NAFLD. RESULTS: PE administration at a dosage of 150 mg/kg/day significantly inhibited HFD-induced hyperlipidemia and hepatic lipid deposition. As major contributors to NAFLD, increased expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukins 1, 4, and 6 as well as augmented oxidative stress in hepatocytes followed by nuclear factor (erythroid-derived-2)-like 2 (Nrf2) activation were normalized through PE supplementation. In addition, PE treatment reduced uncoupling protein 2 (UCP2) expression, restored ATP content, suppressed mitochondrial protein oxidation, and improved mitochondrial complex activity in the liver. In contrast, mitochondrial content was not affected despite increased peroxisomal proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and elevated expression of genes related to mitochondrial beta-oxidation after PE treatment. Finally, PU was identified as the predominant active component of PE with regard to the lowering of triglyceride and cholesterol content in HepG2 cells, and both PU- and PE-protected cells from palmitate induced mitochondrial dysfunction and insulin resistance. INNOVATION: Our work presents the beneficial effects of PE on obesity-associated NAFLD and multiple risk factors. PU was proposed to be the major active component. CONCLUSIONS: By promoting mitochondrial function, eliminating oxidative stress and inflammation, PU may be a useful nutrient for the treatment of NAFLD.

Effect of probiotic fermented milk on blood pressure: a meta-analysis of randomised controlled trials.

Previous studies have suggested that probiotic fermented milk may possess blood pressure (BP)-lowering properties. In the present study, we aimed to systematically examine the effect of probiotic fermented milk on BP by conducting a meta-analysis of randomised controlled trials. PubMed, Cochrane library and the ClinicalTrials.gov databases were searched up to March 2012 to identify eligible studies.The reference lists of the obtained articles were also reviewed. Either a fixed-effects or a random-effects model was used to calculate the combined treatment effect. Meta-analysis of fourteen randomised placebo-controlled trials involving 702 participants showed that probiotic fermented milk, compared with placebo, produced a significant reduction of 3·10 mmHg (95% CI 24·64, 21·56) in systolic BP and 1·09 mmHg (95% CI 22·11, 20·06) in diastolic BP. Subgroup analyses suggested a slightly greater effect on systolic BP in hypertensive participants than in normotensive ones (23·98 v. 22·09 mmHg). Analysis of trials conducted in Japan showed a greater reduction than those conducted in European countries for both systolic BP (26·12 v. 22·08 mmHg) and diastolic BP (23·45 v. 20·52 mmHg). Some evidence of publication bias was present, but sensitivity analysis excluding small trials that reported extreme results only affected the pooled effect size minimally. In summary, the present meta-analysis suggested that probiotic fermented milk has BP-lowering effects in pre-hypertensive and hypertensive subjects.

[Determination of thiocyanate in dairy products by headspace gas chromatography].

A method for the determination of thiocyanate in dairy products by headspace gas chromatography was established. At first, the thiocyanate in dairy products was extracted by water. Then, the zinc acetate solution was added to the crude product for protein precipitation. The extract obtained above was centrifuged and the supernatant was added with chloramine T, which derivatized the thiocyanate ions to cyanogen chloride. The head-space vapor of the final extract was injected into a BP10 (14% cyanopropyl phenyl polysiloxane) gas chromatographic column, and detected by an electron capture detector (ECD). The target compound was quantified by external standard. The results showed that there was a good linearity between 0.005 mg/L and 0.1 mg/L with the correlation coefficient (r) of 0.997, and the limit of detection (signal-to-noise ratio (S/N) > or = 10) was 0.1 mg/kg. The recoveries were 90.0%-110.0% with the relative standard deviations (RSDs) (n = 10) of 4.98%-7.89% at the three spiked levels of 1.0, 2.0, 10.0 mg/kg. In conclusion, this method is simple, rapid and accurate. It can be applied in the determination of thiocyanate in dairy products, and meets the requirements of the daily testing. The method has been successfully used to test 18 kinds of commercially available dairy products and it was found that all the dairy products tested contained thiocyanate about 0.5-10 mg/kg

Antihypertensive effects and mechanisms of chlorogenic acids.

Chlorogenic acids (CGAs) are potent antioxidants found in certain foods and drinks, most notably in coffee. In recent years, basic and clinical investigations have implied that the consumption of chlorogenic acid can have an anti-hypertension effect. Mechanistically, the metabolites of CGAs attenuate oxidative stress (reactive oxygen species), which leads to the benefit of blood-pressure reduction through improved endothelial function and nitric oxide bioavailability in the arterial vasculature. This review article highlights the physiological and biochemical findings on this subject and highlights some remaining issues that merit further scientific and clinical exploration. In the framework of lifestyle modification for the management of cardiovascular risk factors, the dietary consumption of CGAs may hold promise for providing a non-pharmacological approach for the prevention and treatment of high blood pressure.

Effect of oral L-arginine supplementation on blood pressure: a meta-analysis of randomized, double-blind, placebo-controlled trials.

BACKGROUND: Previous studies suggest that L-arginine, an amino acid and a substrate of nitric oxide synthase, may have blood pressure (BP)-lowering effect. Because some studies were performed with limited number of patients with hypertension and therefore limited statistical power with sometimes inconsistent results, we aimed to examine the effect of oral L-arginine supplementation on BP by conducting a meta-analysis of randomized, double-blind, placebo-controlled trials. METHODS: PubMed, Cochrane Central Register of Controlled Trials, and the ClinicalTrials.gov databases were searched through June 2011 to identify randomized, double-blind, placebo-controlled trials of oral L-arginine supplementation on BP in humans. We also reviewed reference lists of obtained articles. Either a fixed-effects or, in the presence of heterogeneity, a random-effects model was used to calculate the combined treatment effect. RESULTS: We included 11 randomized, double-blind, placebo-controlled trials involving 387 participants with oral L-arginine intervention ranging from 4 to 24 g/d. Compared with placebo, L-arginine intervention significantly lowered systolic BP by 5.39 mm Hg (95% CI -8.54 to -2.25, P = .001) and diastolic BP by 2.66 mm Hg (95% CI -3.77 to -1.54, P < .001). Sensitivity analyses restricted to trials with a duration of 4 weeks or longer and to trials in which participants did not use antihypertensive medications yielded similar results. Meta-regression analysis suggested an inverse, though insignificant (P = .13), relation between baseline systolic BP and net change in systolic BP. CONCLUSIONS: This meta-analysis provides further evidence that oral L-arginine supplementation significantly lowers both systolic and diastolic BP.

Nox2 NADPH oxidase promotes pathologic cardiac remodeling associated with Doxorubicin chemotherapy.

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2-/-) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2-/- versus WT mice (fractional shortening: 29.5±1.4 versus 25.7±1.0%; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2-/- versus WT mice (ejection fraction: 52.6±2.5 versus 28.5±2.3%, LVdP/dtmin: -8,379±416 versus -5,198±527 mmHg s(-1); end-diastolic pressure-volume relation: 0.051±0.009 versus 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2-/- mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-ß3, and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15 µM at -150 mV to 148 µM at -75 mV in 120 mM external K(+). This current was insensitive to 10 µM glybenclamide. A component of whole-cell current was sensitive to 150 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype.

SRIF receptor subtype expression and involvement in positive and negative contractile effects of somatostatin-14 (SRIF-14) in ventricular cardiomyocytes.

BACKGROUND/AIMS: Somatostatin-14 (SRIF-14), a neuropeptide co-stored with acetylcholine in the cardiac parasympathetic innervation, exerts both positive and negative influences directly on contraction of ventricular cardiomyocytes, indicative of involvement of more than one of five known SRIF (SSTR) receptor subtypes. The aim was to characterize receptor subtype expression in adult rat ventricular cardiomyocytes and to investigate the influence of a series of SRIF (SSTR) subtype-selective agonists on contractile parameters. METHODS: mRNA and protein expression of each receptor subtype were quantified by RT-PCR and immunoblotting respectively; for contraction studies, cells were stimulated at 0.5 Hz under basal conditions and in the presence of isoprenaline (ISO, 10(-8)M). RESULTS: all five SRIF (SSTR) receptor subtypes were expressed in cardiomyocytes although SRIF1A (SSTR2) and SRIF2A (SSTR1) were less abundant than the other subtypes. L803087 (10(-8)M), a SRIF2B (SSTR4) agonist, attenuated ISO-stimulated peak contractile amplitude and prolonged relaxation time (T(50)). L796778 (10(-7)M), a SRIF1C (SSTR3) agonist, augmented basal and ISO-stimulated peak contractile amplitude; L779976 (10(-8)M) and L817818 (10(-9)M), agonists at SRIF1A (SSTR2) and SRIF1B (SSTR5) receptors, respectively, also augmented ISO-stimulated peak amplitude. CONCLUSION: These data support involvement of SRIF2B (SSTR4) receptors in the negative contractile effects of SRIF-14, while one or more of the three SRIF1 receptor subtypes (SSTR2, 3 or 5) may contribute to the positive contractile effects of SRIF-14.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

BACKGROUND: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD's actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). AIM/METHOD: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. RESULTS: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O(2-) anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. CONCLUSION: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency.

BACKGROUND: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. AIMS: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. METHODS: NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). RESULTS: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-alpha-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. CONCLUSION: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.