P21-activated kinase-1 signaling is required to preserve adipose tissue homeostasis and cardiac function.

While P21-activated kinase-1 (PAK1) has been extensively studied in relation to cardiovascular health and glucose metabolism, its roles within adipose tissue and cardiometabolic diseases are less understood. In this study, we explored the effects of PAK1 deletion on energy balance, adipose tissue homeostasis, and cardiac function utilizing a whole-body PAK1 knockout (PAK1-/-) mouse model. Our findings revealed that body weight differences between PAK1-/- and WT mice emerged at 9 weeks of age, with further increases observed at 12 weeks. Furthermore, PAK1-/- mice displayed increased fat mass and decreased lean mass at 12 weeks, indicating a shift towards adiposity. In conjunction with the increased body weight, PAK1-/- mice had increased food intake and reduced energy expenditure. At a mechanistic level, PAK1 deletion boosted the expression of lipogenic markers while diminishing thermogenic markers expression in adipose tissues, contributing to reduced energy expenditure and the overall obesogenic phenotype. Moreover, our findings highlighted a significant impact on cardiac function following PAK1 deletion, including alterations in calcium kinetics and compromised systolic and lusitropy functions. In summary, our study emphasizes the significant role of PAK1 in weight regulation and cardiac function, enriching our comprehension of heart health and metabolism. These findings could potentially facilitate the identification of novel therapeutic targets in cardiometabolic diseases.

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups' limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

B-arrestin-2 Signaling Is Important to Preserve Cardiac Function During Aging.

Experiments reported here tested the hypothesis that ß-arrestin-2 is an important element in the preservation of cardiac function during aging. We tested this hypothesis by aging ß-arrestin-2 knock-out (KO) mice, and wild-type equivalent (WT) to 12-16months. We developed the rationale for these experiments on the basis that angiotensin II (ang II) signaling at ang II receptor type 1 (AT1R), which is a G-protein coupled receptor (GPCR) promotes both G-protein signaling as well as ß-arrestin-2 signaling. ß-arrestin-2 participates in GPCR desensitization, internalization, but also acts as a scaffold for adaptive signal transduction that may occur independently or in parallel to G-protein signaling. We have previously reported that biased ligands acting at the AT1R promote ß-arrestin-2 signaling increasing cardiac contractility and reducing maladaptations in a mouse model of dilated cardiomyopathy. Although there is evidence that ang II induces maladaptive senescence in the cardiovascular system, a role for ß-arrestin-2 signaling has not been studied in aging. By echocardiography, we found that compared to controls aged KO mice exhibited enlarged left atria and left ventricular diameters as well as depressed contractility parameters with preserved ejection fraction. Aged KO also exhibited depressed relaxation parameters when compared to WT controls at the same age. Moreover, cardiac dysfunction in aged KO mice was correlated with alterations in the phosphorylation of myofilament proteins, such as cardiac myosin binding protein-C, and myosin regulatory light chain. Our evidence provides novel insights into a role for ß-arrestin-2 as an important signaling mechanism that preserves cardiac function during aging.

Modifications of Sarcoplasmic Reticulum Function Prevent Progression of Sarcomere-Linked Hypertrophic Cardiomyopathy Despite a Persistent Increase in Myofilament Calcium Response.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in different genes mainly encoding myofilament proteins and therefore called a "disease of the sarcomere." Despite the discovery of sarcomere protein mutations linked to HCM almost 30 years ago, the cellular mechanisms responsible for the development of this disease are not completely understood and likely vary among different mutations. Moreover, despite many efforts to develop effective treatments for HCM, these have largely been unsuccessful, and more studies are needed to better understand the cellular mechanisms of the disease. In experiments reported here, we investigated a mouse model expressing the mutant cTnT-R92Q, which is linked to HCM and induces an increase in myofilament Ca2+ sensitivity and diastolic dysfunction. We found that early correction of the diastolic dysfunction by phospholamban knockout (PLNKO) was able to prevent the development of the HCM phenotype in troponin T (TnT)-R92Q transgenic (TG) mice. Four groups of mice in FVB/N background were generated and used for the experiments: (1) non-transgenic (NTG)/PLN mice, which express wild-type TnT and normal level of PLN; (2) NTG/PLNKO mice, which express wild-type TnT and no PLN; (3) TG/PLN mice, which express TnT-R92Q and normal level of PLN; (4) TG/PLNKO mice, which express TnT-R92Q and no PLN. Cardiac function was determined using both standard echocardiographic parameters and speckle tracking strain measurements. We found that both atrial morphology and diastolic function were altered in TG/PLN mice but normal in TG/PLNKO mice. Histological analysis showed a disarray of myocytes and increased collagen deposition only in TG/PLN hearts. We also observed increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation only in TG/PLN hearts but not in TG/PLNKO hearts. The rescue of the HCM phenotype was not associated with differences in myofilament Ca2+ sensitivity between TG/PLN and TG/PLNKO mice. Moreover, compared to standard systolic echo parameters, such as ejection fraction (EF), speckle strain measurements provided a more sensitive approach to detect early systolic dysfunction in TG/PLN mice. In summary, our results indicate that targeting diastolic dysfunction through altering Ca2+ fluxes with no change in myofilament response to Ca2+ was able to prevent the development of the HCM phenotype and should be considered as a potential additional treatment for HCM patients.

Sphingosine-1-Phosphate Receptor Modulator, FTY720, Improves Diastolic Dysfunction and Partially Reverses Atrial Remodeling in a Tm-E180G Mouse Model Linked to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic cardiovascular disorder, primarily involving mutations in sarcomeric proteins. HCM patients present with hypertrophy, diastolic dysfunction, and fibrosis, but there is no specific treatment. The sphingosine-1-phosphate receptor modulator, FTY720/fingolimod, is approved for treatment of multiple sclerosis. We hypothesize that modulation of the sphingosine-1-phosphate receptor by FTY720 would be of therapeutic benefit in sarcomere-linked HCM. METHODS: We treated mice with an HCM-linked mutation in tropomyosin (Tm-E180G) and nontransgenic littermates with FTY720 or vehicle for 6 weeks. Compared with vehicle-treated, FTY720-treated Tm-E180G mice had a significant reduction in left atrial size (1.99±0.19 [n=7] versus 2.70±0.44 [n=6] mm; P<0.001) and improvement in diastolic function (E/A ratio: 2.69±0.38 [n=7] versus 5.34±1.19 [n=6]; P=0.004) as assessed by echocardiography. RESULTS: Pressure-volume relations revealed significant improvements in the end-diastolic pressure volume relationship, relaxation kinetics, preload recruitable stroke work, and ejection fraction. Detergent-extracted fiber bundles revealed a significant decrease in myofilament Ca2+-responsiveness (pCa50=6.15±0.11 [n=13] versus 6.24±0.06 [n=14]; P=0.041). We attributed these improvements to a downregulation of S-glutathionylation of cardiac myosin binding protein-C in FTY720-treated Tm-E180G mice and reduction in oxidative stress by downregulation of NADPH oxidases with no changes in fibrosis. CONCLUSIONS: This is the first demonstration that modulation of S1PR results in decreased myofilament-Ca2+-responsiveness and improved diastolic function in HCM. We associated these changes with decreased oxidative modification of myofilament proteins via downregulation of NOX2. Our data support the hypothesis that modification of sphingolipid signaling may be a novel therapeutic approach in HCM.

Knockout of p21-activated kinase-1 attenuates exercise-induced cardiac remodelling through altered calcineurin signalling.

AIMS: Despite its known cardiovascular benefits, the intracellular signalling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy. METHODS AND RESULTS: Wild-type (WT) and Pak1 KO mice were subjected to 6 weeks of treadmill endurance exercise training (ex-training). Cardiac function was assessed via echocardiography, in situ haemodynamics, and the pCa-force relations in skinned fibre preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. Heart weight/tibia length and echocardiography data revealed that there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, and phosphorylation of cardiac myosin-binding protein C, cardiac TnT, and tropomyosin compared with KO mice. With ex-training in WT mice, there were also increased protein levels of calcineurin and increased phosphorylation of phospholamban. CONCLUSIONS: Our data suggest that Pak1 is essential for adaptive physiological cardiac remodelling and support previous evidence that demonstrates Pak1 signalling is important for cardiac growth and survival.

Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation.

Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia-reperfusion injury, little is known about ceramide's role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field-stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca(2+)]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2-dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC-dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present.

Decreasing tropomyosin phosphorylation rescues tropomyosin-induced familial hypertrophic cardiomyopathy.

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.

Conserved Asp-137 is important for both structure and regulatory functions of cardiac α-tropomyosin (α-TM) in a novel transgenic mouse model expressing α-TM-D137L.

α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca(2+) sensitivity and cardiomyocyte contractility without changes in intracellular Ca(2+) transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.

Cardiovascular responses and differential changes in mitogen-activated protein kinases following repeated episodes of binge drinking.

AIMS: Excessive alcohol use in the form of binge drinking is associated with many adverse medical outcomes. Using an animal model, the primary objective of this study was to determine the effects of repeated episodes of binge drinking on myocardial structure, blood pressure (BP) and activation of mitogen-activated protein kinases (MAPKs). The effects of carvedilol, a beta-adrenergic blocker, were also examined in this animal model of binge drinking. METHODS: Rats were randomized into three groups: control, binge and binge + carvedilol (20 mg/kg). Animals received intragastric administration of 5 g ethanol/kg in the morning × 4 days (Monday-Thursday) followed by no ethanol on Friday-Sunday. Animals were maintained on the protocol for 5 weeks. BP was measured using radiotelemetry methods. Animals underwent echocardiography at baseline, 2.5 and 5 weeks. Myocardial MAPKs were analyzed at 5 weeks using western blot techniques. RESULTS: Over the course of 5 weeks, binge drinking was associated with significant transient increases in BP that were greater at 4 and 5 weeks compared with earlier time points. Carvedilol treatment significantly attenuated the binge-induced transient increases in BP at 4 and 5 weeks. No significant changes were found in echocardiographic parameters at any time period; however, binge drinking was associated with increased phosphorylation of p38 MAPK, which was blocked by carvedilol treatment. CONCLUSION: Repeated episodes of binge drinking result in progressive and transient increases in BP, no change in myocardial structure and differential regulation of MAPK activation.

Maintenance of adult cardiac function requires the chromatin factor Asxl2.

During development and differentiation, cell type-specific chromatin configurations are set up to facilitate cell type-specific gene expression. Defects in the establishment or the maintenance of the correct chromatin configuration have been associated with diseases ranging from leukemia to muscular dystrophy. The heart expresses many chromatin factors, and we are only beginning to understand their roles in heart development and function. We have previously shown that the chromatin regulator Asxl2 is highly expressed in the murine heart both during development and adulthood. In the absence of Asxl2, there is a significant reduction in trimethylation of histone H3 lysine 27 (H3K27), a histone mark associated with lineage-specific silencing of developmental genes. Here we present evidence that Asxl2 is required for the long-term maintenance of ventricular function and for the maintenance of normal cardiac gene expression. Asxl2(-/-) hearts displayed progressive deterioration of ventricular function. By 10 months of age, there was ~37% reduction in fractional shortening in Asxl2(-/-) hearts compared to wild-type. Analysis of the expression of myofibril proteins suggests that Asxl2 is required for the repression of ß-MHC. Asxl2(-/-) hearts did not exhibit hypertrophy, suggesting that the de-repression of ß-MHC was not the result of hypertrophic response. Instead, Asxl2 and the histone methyltansferase Ezh2 co-localize to ß-MHC promoter, suggesting that Asxl2 directly represses ß-MHC. Interrogation of the CardioGenomics database revealed that ASXL2 is down-regulated in the hearts of patients with ischemic or idiopathic dilated cardiomyopathy. We propose that chromatin factors like Asxl2 function in the adult heart to regulate cell type- and stage-specific patterns of gene expression, and the disruption of such regulation may be involved in the etiology and/or development of certain forms of human heart disease.

Long-term rescue of a familial hypertrophic cardiomyopathy caused by a mutation in the thin filament protein, tropomyosin, via modulation of a calcium cycling protein.

We have recently shown that a temporary increase in sarcoplasmic reticulum (SR) cycling via adenovirus-mediated overexpression of sarcoplasmic reticulum ATPase (SERCA2) transiently improves relaxation and delays hypertrophic remodeling in a familial hypertrophic cardiomyopathy (FHC) caused by a mutation in the thin filament protein, tropomyosin (i.e., α-TmE180G or Tm180). In this study, we sought to permanently alter calcium fluxes via phospholamban (PLN) gene deletion in Tm180 mice in order to sustain long-term improvements in cardiac function and adverse cardiac remodeling/hypertrophy. While similar work has been done in FHCs resulting from mutations in thick myofilament proteins, no one has studied these effects in an FHC resulting from a thin filament protein mutation. Tm180 transgenic (TG) mice were crossbred with PLN knockout (KO) mice and four groups were studied in parallel: 1) non-TG (NTG), 2) Tm180, 3) PLNKO/NTG and 4) PLNKO/Tm180. Tm180 mice exhibit increased heart weight/body weight and hypertrophic gene markers compared to NTG mice, but levels in PLNKO/Tm180 mice were similar to NTG. Tm180 mice also displayed altered function as assessed via in situ pressure-volume analysis and echocardiography at 3-6 months and one year; however, altered function in Tm180 mice was rescued back to NTG levels in PLNKO/Tm180 mice. Collagen deposition, as assessed by Picrosirius Red staining, was increased in Tm180 mice but was similar in NTG and in PLNKO/Tm180 mice. Extracellular signal-regulated kinase (ERK1/2) phosphorylation increased in Tm180 mice while levels in PLNKO/Tm180 mice were similar to NTGs. The present study shows that by modulating SR calcium cycling, we were able to rescue many of the deleterious aspects of FHC caused by a mutation in the thin filament protein, Tm.

Effects of nicotine administration in a mouse model of familial hypertrophic cardiomyopathy, α-tropomyosin D175N.

The effects of nicotine (NIC) on normal hearts are fairly well established, yet its effects on hearts displaying familial hypertrophic cardiomyopathy have not been tested. We studied both the acute and chronic effects of NIC on a transgenic (TG) mouse model of FHC caused by a mutation in α-tropomyosin (Tm; i.e., α-Tm D175N TG, or Tm175). For acute effects, intravenously injected NIC increased heart rate, left ventricular (LV) pressure, and the maximal rate of LV pressure increase (+dP/dt) in non-TG (NTG) and Tm175 mice; however, Tm175 showed a significantly smaller increase in the maximal rate of LV pressure decrease (-dP/dt) compared with NTGs. Western blots revealed phosphorylation of phospholamban Ser16 and Thr17 residue increased in NTG mice following NIC injection but not in Tm175 mice. In contrast, phosphorylation of troponin I at serine residues 23 and 24 increased equally in both NTG and Tm175. Thus the attenuated increase in relaxation in Tm175 mice following acute NIC appears to result primarily from attenuated phospholamban phosphorylation. Chronic NIC administration (equivalent to smoking 2 packs of cigarettes/day for 4 mo) also increased +dP/dt in NTG and Tm175 mice compared with chronic saline. However, chronic NIC had little effect on heart rate, LV pressure, -dP/dt, LV wall and chamber dimensions, or collagen content for either group of mice.

Limited functional and metabolic improvements in hypertrophic and healthy rat heart overexpressing the skeletal muscle isoform of SERCA1 by adenoviral gene transfer in vivo.

Adenoviral gene transfer of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a to the hypertrophic heart in vivo has been consistently reported to lead to enhanced myocardial contractility. It is unknown if the faster skeletal muscle isoform, SERCA1, expressed in the whole heart in early failure, leads to similar improvements and whether metabolic requirements are maintained during an adrenergic challenge. In this study, Ad.cmv.SERCA1 was delivered in vivo to aortic banded and sham-operated Sprague-Dawley rat hearts. The total SERCA content increased 34%. At 48-72 h posttransfer, echocardiograms were acquired, hearts were excised and retrograded perfused, and hemodynamics were measured parallel to NMR measures of the phosphocreatine (PCr)-to-ATP ratio (PCr/ATP) and energy substrate selection at basal and high workloads (isoproterenol). In the Langendorff mode, the rate-pressure product was enhanced 27% with SERCA1 in hypertrophic hearts and 10% in shams. The adrenergic response to isoproterenol was significantly potentiated in both groups with SERCA1. 31P NMR analysis of PCr/ATP revealed that the ratio remained low in the hypertrophic group with SERCA1 overexpression and was not further compromised with adrenergic challenge. 13C NMR analysis revealed fat and carbohydrate oxidation were unaffected at basal with SERCA1 expression; however, there was a shift from fats to carbohydrates at higher workloads with SERCA1 in both groups. Transport of NADH-reducing equivalents into the mitochondria via the alpha-ketoglutamate-malate transporter was not affected by either SERCA1 overexpression or adrenergic challenge in both groups. Echocardiograms revealed an important distinction between in vivo versus ex vivo data. In contrast to previous SERCA2a studies, the echocardiogram data revealed that SERCA1 expression compromised function (fractional shortening) in the hypertrophic group. Shams were unaffected. While our ex vivo findings support much of the earlier cardiomyocyte and transgenic data, the in vivo data challenge previous reports of improved cardiac function in heart failure models after SERCA intervention.

Cigarette smoke-induced left ventricular remodelling is associated with activation of mitogen-activated protein kinases.

AIM: To determine the effects of cigarette smoke (CS) exposure on the expression/activation of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase [ERK1/2], p38-kinase [p38] and c-Jun NH2-terminal protein kinase [JNK]), norepinephrine (NE) levels and myocardial structure and function. METHODS: Rats were randomised to two groups: CS-exposed (n=12) or room air (CON) (n=10). After 5 weeks, the animals underwent echocardiography with pulse-wave Doppler flow measurements. Hearts were removed for microscopy and Western blot analysis. RESULTS: CS exposure was associated with significant increases in NE urinary levels and larger ventricular dimensions (mm) (CON=left ventricular end diastolic dimension [LVEDD] 7.99+/-0.10, LV end systolic dimension [LVESD] 4.55+/-0.20, CS=LVEDD 8.3+/-0.10, LVESD 5.3+/-0.09, p=0.026, p=0.003). There was also evidence of systolic dysfunction in the CS-exposed group compared to the CON group (fractional shortening %, CON=43+/-2, CS=36+/-.09, p=0.010). In CS-exposed hearts, significant increases in phosphorylated p38/total p38 (0.975+/-0.05) and phosphorylated ERK1/2/totalERK1/2 (1.919+/-0.050) were found compared to CON hearts (0.464+/-0.008, 0.459+/-0.050, respectively). No significant differences were found in JNK levels between the groups. CONCLUSIONS: Increased NE levels and MAPK activation are associated with CS-related left ventricular remodelling.

Long-term effects of alcohol consumption in male and female rats.

Discrepancies exist regarding potential sex differences in the effects of ethanol on the myocardium. Therefore, the aims of this study were to determine if long-term ethanol consumption was associated with the development of a dilated cardiomyopathy (CM) in female rats and, second, to determine if the absence of ovarian hormones modulated this effect. Adult male and female (n=6-8/group) sham-operated and ovariectomized (OVX) Sprague-Dawley rats received the Lieber DeCarli ethanol-containing (8% vol/vol) or control liquid diet for 8 months. All ethanol groups showed echocardiographic evidence of a cardiomyopathy; however, more significant ethanol-elicited differences were found in the male ethanol group than in either the female or female OVX groups. In addition, the male ethanol group had significant reductions in in vivo measures of contractility, such as the maximum derivative of change in systolic pressure and preload recruitable stroke work. Sex differences were also apparent in the pattern and degree of posterior and septal wall thickness changes, in that the male ethanol group had more posterior and septal wall thinning. In conclusion, similar to male rats long-term ethanol consumption in gonad-intact and OVX female rats is associated with the development of a dilated cardiomyopathy.

Epidermal growth factor stimulates chloride transport in primary cultures of weanling and adult rabbit colonocytes.

OBJECTIVES: We have shown that Ca2+-dependent regulation of Cl- secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca2+]i, it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl- transport in the developing rabbit distal colon. MATERIALS AND METHODS: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca2+]i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. RESULTS: Serum depletion had no effect on cell number, DNA content, or basal Cl- transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl- transport to a similar extent. EGF maximally stimulated Cl- transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca2+]i. EGF did not alter bethanechol-stimulated Cl- transport or [Ca2+]i. EGF acts via an EGF-receptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl- transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl- transport. CONCLUSIONS: EGF stimulates rabbit colonic Cl- transport via a Ca2+-independent, tyrosine kinase- and MAPK-dependent pathway, and its effects are not age dependent.

From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.