Interaction between high-fat diet and alcohol dehydrogenase on ethanol-elicited cardiac depression in murine myocytes

Objective: Consumption of high‐fat diet and alcohol is associated with obesity, leading to enhanced morbidity and mortality. This study was designed to examine the interaction between high‐fat diet and the alcohol metabolizing enzyme alcohol dehydrogenase (ADH) on ethanol‐induced cardiac depression. Research Methods and Procedures: Mechanical and intracellular Ca²⁺ properties were measured in cardiomyocytes from ADH transgenic and Friend Virus‐B type (FVB) mice fed a low‐ or high‐fat diet for 16 weeks. Expression of protein kinase B (Akt) and Foxo3a, two proteins essential for cardiac survival, was evaluated by Western blot. Cardiac damage was determined by carbonyl formation. Results: High fat but not ADH induced obesity without hyperglycemia or hypertension, prolonged time‐to‐90% relengthening (TR₉₀), and depressed peak shortening (PS) and maximal velocity of shortening/relengthening (± dL/dt) without affecting intracellular Ca²⁺ properties. Ethanol suppressed PS and intracellular Ca²⁺ rise in low‐fat‐fed FVB mouse cardiomyocytes. ADH but not high‐fat diet shifted the threshold of ethanol‐induced inhibition of PS and ± dL/dt to lower levels. The amplitude of ethanol‐induced cardiac depression was greater in the high‐fat but not the ADH group without additive effects. Ethanol down‐ and up‐regulated Akt and Foxo3a expression, respectively, and depressed intracellular Ca²⁺ rise, the effects of which were exaggerated by ADH, high‐fat, or both. High‐fat diet, but not ADH, enhanced Foxo3a expression and carbonyl content in non‐ethanol‐treated mice. Ethanol challenge significantly enhanced protein carbonyl formation, with the response being augmented by ADH, high‐fat, or both. Discussion: Our data suggest that high‐fat diet and ADH transgene may exaggerate ethanol‐induced cardiac depression and protein damage in response to ethanol.     

Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism

High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca²⁺ handling), ultrastructural disarray, apoptosis, O₂⁻, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser⁷⁵⁷ and Ser⁷⁷⁷), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.     

Deficiency of insulin‐like growth factor 1 reduces vulnerability to chronic alcohol intake‐induced cardiomyocyte mechanical dysfunction: role of AMPK

Circulating insulin‐like growth factor I (IGF‐1) levels are closely associated with cardiac performance although the role of IGF‐1 in alcoholic cardiac dysfunction is unknown. This study was designed to evaluate the impact of severe liver IGF‐1 deficiency (LID) on chronic alcohol‐induced cardiomyocyte contractile and intracellular Ca²⁺ dysfunction. Adult male C57 and LID mice were placed on a 4% alcohol diet for 15 weeks. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time‐to‐relengthening (TR₉₀), change in fura‐fluorescence intensity (ΔFFI) and intracellular Ca²⁺ decay. Levels of apoptotic regulators caspase‐3, Bcl‐2 and c‐Jun NH2‐terminal kinase (JNK), the ethanol metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), as well as the cellular fuel gauge AMP‐activated protein kinase (AMPK) were evaluated. Chronic alcohol intake enlarged myocyte cross‐sectional area, reduced PS, ± dL/dt and ΔFFI as well as prolonged TR₉₀ and intracellular Ca²⁺ decay, the effect of which was greatly attenuated by IGF‐1 deficiency. The beneficial effect of LID against alcoholic cardiac mechanical defect was ablated by IGF‐1 replenishment. Alcohol intake increased caspase‐3 activity/expression although it down‐regulated Bcl‐2, ALDH2 and pAMPK without affecting JNK and AMPK. IGF‐1 deficiency attenuated alcoholism‐induced responses in all these proteins with the exception of Bcl‐2. In addition, the AMPK agonist 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside abrogated short‐term ethanol incubation‐elicited cardiac mechanical dysfunction. Taken together, these data suggested that IGF‐1 deficiency may reduce the sensitivity to ethanol‐induced myocardial mechanical dysfunction. Our data further depicted a likely role of Caspase‐3, ALDH2 and AMPK activation in IGF‐1 deficiency induced ‘desensitization’ of alcoholic cardiomyopathy.     

Chromium (D-phenylalanine)3 improves obesity-induced cardiac contractile defect in ob/ob mice

Objective: Low‐molecular weight chromium compounds, such as chromium picolinate [Cr(pic)₃], improve insulin sensitivity, although toxicity is a concern. We synthesized a novel chromium complex, chromium (d ‐phenylalanine)₃ [Cr(d ‐phe)₃], in an attempt to improve insulin sensitivity with reduced toxicity. The aim of this study was to compare the two chromium compounds on cardiac contractile function in ob/ob obese mice. Research Methods and Procedures: C57BL lean and ob/ob obese mice were randomly divided into three groups: H₂O, Cr(d ‐phe)₃, or Cr(pic)₃ (45 µg/kg per day orally for 6 months). Results: The glucose tolerance test displayed improved glucose clearance by Cr(d ‐phe)₃ but not Cr(pic)₃. Myocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening (±dL/dt), prolonged time‐to‐PS and time‐to‐90% relengthening (TR90), reduced electrically stimulated rise in intracellular Ca²⁺ (Δfura‐2 fluorescence intensity), and slowed intracellular Ca²⁺ decay. Although a 3‐month Cr(d ‐phe)₃ treatment for a separate group of ob/ob and lean 2‐month‐old mice only rectified reduced ±dL/dt in ob/ob mice, all mechanical and intracellular Ca²⁺ abnormalities were significantly attenuated or ablated by 6 months of Cr(d ‐phe)₃ but not Cr(pic)₃ treatment (except TR90). Sarco(endo)plasmic reticulum Ca²⁺ ATPase activity and Na⁺‐Ca²⁺ exchanger expression were depressed in ob/ob mice, which were reversed by both Cr(d ‐phe)₃ and Cr(pic)₃, with a more pronounced effect from Cr(d ‐phe)₃. Cr(d ‐phe)₃ corrected reduced insulin‐stimulated glucose uptake and improved basal phosphorylation of Akt and insulin receptor, as well as insulin‐stimulated phosphorylation of Akt and insulin receptor in ob/ob myocytes. Heart homogenates from ob/ob mice had enhanced oxidative stress and protein carbonyl formation compared with the lean group, which were attenuated by both Cr(d ‐phe)₃ and Cr(pic)₃. Discussion: Our data suggest that the new Cr(d ‐phe)₃ compound possesses better cardio‐protective and insulin‐sensitizing properties against obesity.     

Short‐Term Lenalidomide (Revlimid) Administration Ameliorates Cardiomyocyte Contractile Dysfunction in ob/ob Obese Mice

Lenalidomide is a potent immunomodulatory agent capable of downregulating proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and upregulating anti‐inflammatory cytokines. Lenalidomide has been shown to elicit cardiovascular effects, although its impact on cardiac function remains obscure. This study was designed to examine the effect of lenalidomide on cardiac contractile function in ob/ob obese mice. C57BL lean and ob/ob obese mice were given lenalidomide (50 mg/kg/day, p.o.) for 3 days. Body fat composition was assessed by dual‐energy X‐ray absorptiometry. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated. Expression of TNF‐α, interleukin‐6 (IL‐6), Fas, Fas ligand (FasL), the short‐chain fatty acid receptor GPR41, the NFκB regulator IκB, endoplasmic reticulum (ER) stress, the apoptotic protein markers Bax, Bcl‐2, caspase‐8, tBid, cytosolic cytochrome C, and caspase‐12; and the stress signaling molecules p38 and extracellular signal‐regulated kinase (ERK) were evaluated by western blot. ob/ob mice displayed elevated serum TNF‐α and IL‐6 levels, fat composition and glucose intolerance, the effects of which except glucose intolerance and fat composition were attenuated by lenalidomide. Cardiomyocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening, prolonged time‐to‐PS and time‐to‐90% relengthening as well as intracellular Ca²⁺ mishandling, which were ablated by lenalidomide. Western blot analysis revealed elevated levels of TNF‐α, IL‐6, Fas, Bip, Bax, caspase‐8, tBid, cleaved caspase‐3 caspase‐12, cytochrome C, phosphorylation of p38, and ERK in ob/ob mouse hearts, the effects of which with the exception of Bip, Bax, and caspase‐12 were alleviated by lenalidomide. Taken together, these data suggest that lenalidomide is protective against obesity‐induced cardiomyopathy possibly through antagonism of cytokine/Fas‐induced activation of stress signaling and apoptosis.     

Tauroursodeoxycholic Acid Mitigates High Fat Diet-Induced Cardiomyocyte Contractile and Intracellular Ca2+ Anomalies

ObjectivesThe endoplasmic reticulum (ER) chaperone tauroursodeoxycholic acid (TUDCA) has exhibited promises in the treatment of obesity, although its impact on obesity-induced cardiac dysfunction is unknown. This study examined the effect of TUDCA on cardiomyocyte function in high-fat diet-induced obesity.MethodsAdult mice were fed low or high fat diet for 5 months prior to treatment of TUDCA (300 mg/kg. i.p., for 15d). Intraperitoneal glucose tolerance test (IPGTT), cardiomyocyte mechanical and intracellular Ca²⁺ property, insulin signaling molecules including IRS-1, Akt, AMPK, ACC, GSK-3β, c-Jun, ERK and c-Jun N terminal kinase (JNK) as well as ER stress and intracellular Ca²⁺ regulatory proteins were examined. Myocardial ultrastructure was evaluated using transmission electron microscopy (TEM).ResultsHigh-fat diet depressed peak shortening (PS) and maximal velocity of shortening/relengthenin as well as prolonged relengthening duration. TUDCA reversed or overtly ameliorated high fat diet-induced cardiomyocyte dysfunction including prolongation in relengthening. TUDCA alleviated high-fat diet-induced decrease in SERCA2a and phosphorylation of phospholamban, increase in ER stress (GRP78/BiP, CHOP, phosphorylation of PERK, IRE1α and eIF2α), ultrastructural changes and mitochondrial permeation pore opening. High-fat diet feeding inhibited phosphorylation of AMPK and promoted phosphorylation of GSK-3β. TUDCA prevented high fat-induced dephosphorylation of AMPK but not GSK-3β. High fat diet promoted phosphorylation of IRS-1 (Ser³⁰⁷), JNK, and ERK without affecting c-Jun phosphorylation, the effect of which with the exception of ERK phosphorylation was attenuated by TUDCA.ConclusionsThese data depict that TUDCA may ameliorate high fat diet feeding-induced cardiomyocyte contractile and intracellular Ca²⁺ defects through mechanisms associated with mitochondrial integrity, AMPK, JNK and IRS-1 serine phosphorylation.     

Interaction between Age and Obesity on Cardiomyocyte Contractile Function: Role of Leptin and Stress Signaling

Objectives

This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca²⁺ properties.

Methods

Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ levels and decay. O₂ ⁻ levels were measured by dihydroethidium fluorescence.

Results

Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, ± dL/dt, intracellular Ca²⁺ rise, prolonged TR₉₀ and intracellular Ca²⁺ decay, enhanced O₂ ⁻ production and p ^47phox expression without an additive effect of the two, with the exception of intracellular Ca²⁺ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIκB. Low levels of leptin reconciled contractile, intracellular Ca²⁺ and cell signaling defects as well as O₂ ⁻ production and p ^47phox upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca²⁺ response as well as O₂ ⁻ production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.

Conclusions

Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O₂ ⁻ release.     

Cardiac‐specific overexpression of metallothionein attenuates L‐NAME‐induced myocardial contractile anomalies and apoptosis

Hypertension contributes to the high cardiac morbidity and mortality. Although oxidative stress plays an essential role in hypertensive heart diseases, the mechanism remains elusive. Transgenic mice with cardiac overexpression of metallothionein, a heavy metal‐binding scavenger, were challenged with N^G‐nitro‐L‐arginine methyl ester (L‐NAME) for 14 days prior to measurement of myocardial contractile and intracellular Ca²⁺ anomalies as well as cell signalling mechanisms using Western blot and immunofluorescence analysis. L‐NAME challenge elicited hypertension, macrophage infiltration, oxidative stress, inflammation and cardiac dysfunction manifested as increased proinflammatory macrophage marker F4/80, interleukin‐1β (IL‐1β), intracellular production, LV end systolic and diastolic diameters as well as depressed fractional shortening. L‐NAME treatment reduced mitochondrial membrane potential (MMP), impaired cardiomyocyte contractile and intracellular Ca²⁺ properties as evidenced by suppressed peak shortening, maximal velocity of shortening/relengthening, rise in intracellular Ca²⁺, along with elevated baseline and peak intracellular Ca²⁺. These unfavourable mechanical changes and decreased MMP (except blood pressure and macrophage infiltration) were alleviated by overexpression of metallothionein. Furthermore, the apoptosis markers including BAD, Bax, Caspase 9, Caspase 12 and cleaved Caspase 3 were up‐regulated while the anti‐apoptotic marker Bcl‐2 was decreased by L‐NAME treatment. Metallothionein transgene reversed L‐NAME‐induced changes in Bax, Bcl‐2, BAD phosphorylation, Caspase 9, Caspase 12 and cleaved Caspase 3. Our results suggest that metallothionein protects against L‐NAME‐induced myocardial contractile anomalies in part through inhibition of apoptosis.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

The effects of dietary additives on faecal levels of Lactobacillus spp., coliforms,and Escherichia coli,and faecal prevalence of Salmonella spp. and Campylobacter spp. in US production nursery swine1

Aims: In the United States, carbadox and copper sulfate are growth promoters commonly used in combination in nursery swine diets. Our aim was to determine how selected dietary additives affect selected bacterial populations and pathogens in nursery swine, and compare to larch extract, which contains potential antibacterial activities. Methods and Results: Piglets were weaned and sorted into one of the four treatments: (i) basal diet without antimicrobials; (ii) basal diet with carbadox + copper sulfate; (iii) basal diet + 1000 ppm larch extract; or (iv) basal diet + 2000 ppm larch extract. Diets were fed for a 4‐week period after weaning. In both trials, the carbadox + copper sulfate group consumed more feed over the 4‐week period relative to the other three diet groups (P < 0·05), but did not gain significantly more weight. Faecal shedding of Salmonella spp. was not affected by dietary supplement in either trial, but faecal shedding of Campylobacter spp. was the lowest for the carbadox + copper sulfate diet. In faecal samples collected at the end of each trial, Lactobacillus spp. cell counts for the basal and larch extract diets were nearly 1·0 log₁₀ g^?1 faeces greater (P < 0·05) than the carbadox + copper sulfate group, whereas the coliforms and Escherichia coli were nearly 1·0 log₁₀ g^?1 faeces lower (P < 0·05). Conclusions: Compared to basal fed animals, supplementation with carbadox + copper sulfate significantly altered faecal E. coli, coliform bacteria and Lactobacillus spp. Larch extract has no benefit up to 0·2% of diet in regard to pathogen shedding, whereas carbadox + copper sulfate decreased faecal shedding of Campylobacter spp. Significance and Impact of the Study: Current swine management practices in the United States may be beneficial to managing Campylobacter spp. shedding in nursery swine, but also result in significant changes in the resident gastrointestinal microflora.     

Adiponectin Improves Cardiomyocyte Contractile Function in db/db Diabetic Obese Mice

Low levels of adiponectin, a fat‐derived hormone, are found to be correlated with coronary heart disease, type 2 diabetes, obesity, and insulin resistance. Conversely, high adiponectin levels are predictive of reduced coronary risk in long‐term epidemiologic studies. However, the precise role of adiponectin in cardiomyocyte function is still not clear. This study was designed to examine the role of adiponectin in cardiac contractile function in the db/db model of diabetic obesity. Mechanical properties and intracellular Ca²⁺ transients were evaluated in cardiomyocytes from lean control and db/db mice with or without adiponectin (10 µg/ml) treatment. Expression and phosphorylation of IRS‐1, Akt, c‐Jun, and c‐Jun N terminal kinase (JNK) as well as markers of endoplasmic reticulum (ER) stress were evaluated using western blotting. Cardiomyocytes from db/db mice exhibited greater cross‐sectional area, depressed peak shortening (PS), and maximal velocity of shortening/re‐lengthening as well as prolonged duration of re‐lengthening. Consistently, myocytes from db/db mice displayed a reduced electrically stimulated rise in intracellular Ca²⁺ and prolonged intracellular Ca²⁺ decay, which were abrogated by adiponectin treatment. Ratios between phosphorylated c‐Jun and c‐Jun as well as phosphorylated IRS‐1 and IRS‐1 were increased in db/db mice, the effect of which was attenuated by adiponectin. Levels of the phosphorylated ER stress makers PERK (Thr980), IRE‐1, and eIF2α were significantly elevated in db/db mice compared with lean controls, although the effect was unaffected by adiponectin. Collectively, our data suggest that adiponectin improves cardiomyocyte dysfunction in db/db diabetic obese mice through a mechanism possibly related to c‐Jun and IRS‐1.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Mitochondrial ALDH2 protects against lipopolysaccharide-induced myocardial contractile dysfunction by suppression of ER stress and autophagy

Lipopolysaccharide (LPS), an essential component of outer membrane of the Gram-negative bacteria, plays a pivotal role in myocardial anomalies in sepsis. Recent evidence depicted an essential role for mitochondrial aldehyde dehydrogenase (ALDH2) in cardiac homeostasis. This study examined the effect of ALDH2 on endotoxemia-induced cardiac anomalies. Echocardiographic, cardiac contractile and intracellular Ca²⁺ properties were examined. Our results indicated that LPS impaired cardiac contractile function (reduced fractional shortening, LV end systolic diameter, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration, oxidation of SERCA, and intracellular Ca²⁺ mishandling), associated with ER stress, inflammation, O₂⁻ production, increased autophagy, CAMKKβ, phosphorylated AMPK and suppressed phosphorylation of mTOR, the effects of which were significantly attenuated or negated by ALDH2. LPS promoted early endosomal formation (as evidenced by RAB4 and RAB5a), apoptosis and necrosis (MTT and LDH) while decreasing late endosomal formation (RAB7 and RAB 9), the effects were reversed by ALDH2. In vitro study revealed that LPS-induced SERCA oxidation, autophagy and cardiac dysfunction were abrogated by ALDH2 activator Alda-1, the ER chaperone TUDCA, the autophagy inhibitor 3-MA, or the AMPK inhibitor Compound C. The beneficial effect of Alda-1 against LPS was nullified by AMPK activator AICAR or rapamycin. CAMKKβ inhibition failed to rescue LPS-induced ER stress. Tunicamycin–induced cardiomyocyte dysfunction was ameliorated by Alda-1 and autophagy inhibition, the effect of which was abolished by rapamycin. These data suggested that ALDH2 protected against LPS-induced cardiac anomalies via suppression of ER stress, autophagy in a CAMKKβ/AMPK/mTOR-dependent manner.     

The progressive effects of a fat enriched diet on ventricular myocyte contraction and intracellular Ca2+ in the C57BL/6J mouse

The C57BL/6J mouse has a genetic susceptibility to develop diabetes when fed with a high-fat, high-sucrose diet. The general characteristics of diet-induced diabetes in this model include progressive development of hyperinsulinaemia, hyperglycaemia, insulin resistance and obesity, features that are frequently observed in the clinical setting. This study investigated the progressive effects of a fat enriched (FE) diet on contraction and intracellular Ca²⁺ in ventricular myocytes from the C57BL/6J mouse. The characteristics of the mice fed with the FE diet compared to mice receiving control diet included progressive increase in the rate of body weight gain, increased fasting blood glucose and time-dependent differences in the disposal of blood glucose after a glucose challenge. The ultrastructure of cardiac myocytes and associated capillaries did not show any gross morphological alteration after 27 weeks of FE diet compared to controls.At 5 months the resting cell length (RCL) and the kinetics of shortening were not significantly altered in ventricular myocytes from mice receiving the FE diet compared to age-matched controls. At 5 and at 7 months the amplitude of shortening was increased in myocytes receiving the FED diet compared to controls. At 7 months the time to half (THALF) relaxation of myocyte contraction was shortened in myocytes from mice receiving the FE diet compared to controls. Mean THALF relaxation in myocytes from mice fed the FE diet was 32.0 ± 1.4 ms (n = 23) compared to 40.2 ± 2.0 ms (n = 27) in controls. Neither resting intracellular Ca²⁺ nor the kinetics or amplitude of the Ca²⁺ transient were altered by FE diet. Differences in myofilament sensitivity to Ca²⁺ might underlie the changes in contractility.     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction

Aims

Accumulation of advanced glycation endproduct (AGE) contributes to diabetic complication including diabetic cardiomyopathy although the precise underlying mechanism still remains elusive. Recent evidence depicted a pivotal role of protein kinase C (PKC) in diabetic complications. To this end, this study was designed to examine if PKCβII contributes to AGE-induced cardiomyocyte contractile and intracellular Ca^2 + aberrations.

Main methods

Adult rat cardiomyocytes were incubated with methylglyoxal-AGE (MG-AGE) in the absence or presence of the PKCβII inhibitor LY333531 for 12 h. Contractile and intracellular Ca^2 + properties were assessed using an IonOptix system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), rise in intracellular Ca^2 + Fura-2 fluorescence intensity and intracellular Ca^2 + decay. Oxidative stress, O₂⁻ production and mitochondrial integrity were examined using TBARS, fluorescence imaging, aconitase activity and Western blotting.

Key findings

MG-AGE compromised contractile and intracellular Ca^2 + properties including reduced PS, ± dL/dt, prolonged TPS and TR₉₀, decreased electrically stimulated rise in intracellular Ca^2 + and delayed intracellular Ca^2 + clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued MG-AGE-induced oxidative stress, O₂⁻ generation, cell death, apoptosis and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were replicated by the NADPH oxidase inhibitor apocynin and were mitigated by the mitochondrial uncoupler FCCP.

Significance

These findings implicated the therapeutic potential of specific inhibition of PKCβII isoform in the management of AGE accumulation-induced myopathic anomalies.     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Low ethanol intake prevents salt-induced hypertension in WKY rats

Low alcohol intake in humans lowers the risk of coronary heart disease and may lower blood pressure. In hypertension, insulin resistance with altered glucose metabolism leads to increased formation of aldehydes. We have shown that chronic low alcohol intake decreased systolic blood pressure (SBP) and tissue aldehyde conjugates in spontaneously hypertensive rats and demonstrated a strong link between elevated tissue aldehyde conjugates and hypertension in salt-induced hypertensive Wistar-Kyoto (WKY) rats. This study investigated the antihypertensive effect of chronic low alcohol consumption in high salt-treated WKY rats and its effect on tissue aldehyde conjugates, platelet cytosolic free calcium ([Ca^{2 +}] _i )_,and renal vascular changes. Animals, aged 7 weeks, were divided into three groups of six animals each. The control group was given normal salt diet (0.7% NaCl) and regular drinking water; the high salt group was given a high salt diet (8% NaCl) and regular drinking water; the high salt + ethanol group was given a high salt diet and 0.25% ethanol in drinking water. After 10 weeks, SBP, platelet [Ca^{2 +}] _i , and tissue aldehyde conjugates were significantly higher in rats in the high salt group as compared with controls. Animals on high salt diets also showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidney. Ethanol supplementation prevented the increase in SBP and platelet [Ca^{2 +}] _i and aldehyde conjugates in liver and aorta. Kidney aldehyde conjugates and renal vascular changes were attenuated. These results suggest that chronic low ethanol intake prevents salt-induced hypertension and attenuates renal vascular changes in WKY rats by preventing an increase in tissue aldehyde conjugates and cytosolic [Ca^{2 +}] _i .     

Cardiomyocyte Contractile Dysfunction in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease

Objectives

Ample clinical and experimental evidence indicated that patients with Alzheimer''s disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca²⁺ homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer''s disease.

Methods

Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (±dL/dt), intracellular Ca²⁺ transient rise and decay.

Results

Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced±dL/dt, normal TPS and TR compared with WT mice_. Rise in intracellular Ca²⁺ was lower accompanied by unchanged resting/peak intracellular Ca²⁺ levels and intracellular Ca²⁺ decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although β₁-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca²⁺ regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na⁺-Ca²⁺ exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.

Conclusions

Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer''s disease model, possibly due to oxidative stress.     

Atrial Myocyte Function and Ca2+ Handling Is Associated with Inborn Aerobic Capacity

Background

Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO_{2 max}) had impaired atrial myocyte contractile function when compared to rats with high inborn VO_{2 max}.

Methods and Results

Atrial myocyte function was depressed in Low Capacity Runners (LCR) relative to High Capacity Runners (HCR) which was associated with impaired Ca²⁺ handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca²⁺ amplitude and diastolic Ca²⁺ level were observed at 5 Hz stimulation where Ca²⁺ amplitude was 70% lower and diastolic Ca²⁺ level was 11% higher in LCR rats. Prolonged time to 50% Ca²⁺ decay was associated with reduced sarcoplasmic reticulum (SR) Ca²⁺ ATPase function in LCR (39%). Na⁺/Ca²⁺ exchanger activity was comparable between the groups. Diastolic SR Ca²⁺ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca²⁺-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca²⁺ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca²⁺-signal onset.

Conclusion

This data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats.