Roger J. Bick

Relation of tissue Doppler derived myocardial velocities to myocardial structure and beta-adrenergic receptor density in humans.

OBJECTIVES We sought to evaluate the relation of segmental tissue Doppler (TD) velocities to both the regional amount of interstitial fibrosis and the myocyte beta-adrenergic receptor density in humans. BACKGROUND The systolic myocardial velocity (Sm) and early diastolic myocardial velocity (Em) acquired by TD are promising new indexes of left ventricular function. However, their structural and functional correlates in humans are still unknown. METHODS Ten patients with coronary artery disease underwent echocardiographic examination including TD imaging, along with transmural endomyocardial biopsy at the time of coronary bypass surgery (two biopsies per patient for a total of 20 specimens). The specimens were analyzed for percent interstitial fibrosis and beta-adrenergic receptor density. RESULTS Normal segments (n = 8) had a higher beta-adrenoceptor density (2,280 +/- 738 vs. 1,373 +/- 460, p = 0.03) and a lower amount of interstitial fibrosis (13 +/- 3.3% vs. 28 +/- 11.5%, p = 0.002) than dysfunctional segments (n = 12). Myocardial systolic velocity and Em were also significantly higher (9.5 +/- 2.7 vs. 5.9 +/- 1.8 cm/s, p = 0.025 and 11.3 +/- 2.8 vs. 6.4 +/- 2.1 cm/s, p = 0.002, respectively) in normal segments. A significant relationship was present between Em and the beta-adrenergic receptor density (r = 0.78, p < 0.001) and percent interstitial fibrosis (r = -0.7, p = 0.0026), which together accounted for 81% of the variance observed in Em. Likewise, a significant relationship was present between Sm and the beta-adrenergic receptor density (r = 0.68, p < 0.001) and the percent interstitial fibrosis (r = -0.66, p = 0.004) and together accounted for 62% of the variance observed in Sm. CONCLUSIONS Systolic myocardial velocity and Em are strongly dependent on both the number of myocytes and the myocardial beta-adrenergic receptor density.

C/EBPα is required for differentiation of white, but not brown, adipose tissue

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is expressed at high levels in liver and adipose tissue. Cell culture studies show that C/EBPα is sufficient to trigger differentiation of preadipocytes into mature adipocytes, suggesting a central role for C/EBPα in the development of adipose tissue. C/EBPα knockout mice die within 7–12 h after birth. Defective gluconeogenesis of the liver and subsequent hypoglycemia contribute to the early death of these animals. This short life span impairs investigation of the development of adipose tissue in these mice. To improve the survival of C/EBPα−/− animals, we generated a transgenic line that expresses C/EBPα under the control of the albumin enhancer/promoter. This line was bred into the knockout strain to generate animals that express C/EBPα in the liver but in no other tissue. The presence of the transgene improved survival of C/EBPα−/− animals almost 3-fold. Transgenic C/EBPα−/− animals at 7 days of age show an absence of s.c., perirenal, and epididymal white fat despite excess lipid substrate in the serum, whereas brown adipose tissue is somewhat hypertrophied and shows minimal biochemical alterations. Interestingly, mammary gland fat tissue is present and exhibits normal morphology. The absence of white adipose tissue in many depots in the presence of high serum lipid levels shows that C/EBPα is required for the in vivo development of this tissue. In contrast, brown adipose tissue differentiation is independent of C/EBPα expression. The presence of lipid in brown adipose tissue serves as an internal nutritional control, indicating that neither nutritional intake nor lipoprotein composition is likely responsible for the absence of white fat.

Identification of a Gene That Reverses the Immortal Phenotype of a Subset of Cells and Is a Member of a Novel Family of Transcription Factor-Like Genes

ABSTRACT Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

The neuroprotective activities of melatonin against the Alzheimer β‐protein are not mediated by melatonin membrane receptors

Exposure of neuronal cells to the Alzheimers amyloid β protein (Aβ) results in extensive oxidative damage of bio‐molecules that are profoundly harmful to neuronal homeostasis. It has been demonstrated that melatonin protects neurons against Aβ‐mediated neurotoxicity, including cell death and a spectrum of oxidative lesions. We undertook the current study to determine whether melatonin membrane receptors are involved in the mechanism of neuroprotection against Aβ neurotoxicity. For this purpose, we characterized the free‐radical scavenging potency of several compounds exhibiting various affinities for melatonin membrane receptors (MLT 1a and 1b). Aβ‐mediated neurotoxicity was assessed in human neuroblastoma cells and in primary hippocampal neurons. In sharp contrast with melatonin, no neuroprotection against Aβ toxicity was observed when we used melatonin membrane receptor agonists that were devoid of antioxidant activity. In contrast, the cells were fully protected in parallel control experiments when either melatonin, or the structurally unrelated free‐radical scavenger phenyl‐N‐t‐butyl nitrone (PBN), were added to Aβ‐containing culture media. This study demonstrates that the neuroprotective properties of melatonin against Aβ‐mediated toxicity does not require binding of melatonin to a membrane receptor and is likely the result of the antioxidant and antiamyloidogenic features of the agent.

Altered Adrenergic Receptor Density in Myocardial Hibernation in Humans A Possible Mechanism of Depressed Myocardial Function

Background—Alterations in adrenergic receptor densities can potentially contribute to myocardial dysfunction. Their relevance to myocardial hibernation in humans is unknown. Methods and Results—Accordingly, 22 transmural myocardial biopsies were obtained in 11 patients with ischemic ventricular dysfunction during bypass surgery, guided by transesophageal echocardiography. Patients underwent dobutamine echocardiography (DE) and rest scintigraphic studies before revascularization and DE at 3 to 4 months. &agr;- and &bgr;-receptor density (ARD and BRD) and extent of fibrosis were quantified from the myocardial biopsies. Of the 22 segments, 16 had abnormal rest function and 6 were normal. Severely hypokinetic or akinetic segments showed a 2.4-fold increase in ARD with a concomitant 50% decrease in BRD compared with normal segments. An increase in ARD, a decrease in BRD to a lesser extent, and thus an increase in ARD/BRD ratio were seen in dysfunctional segments with contractile reserve compared with normal segments and were most pronounced in those without contractile reserve (P <0.001). Similar findings were observed if recovery of function or scintigraphic uptake was analyzed as a marker for viability. No significant relation between either ARD or BRD and percent myocardial fibrosis was noted (r =0.37 and −0.39, respectively). Conclusions—Thus, graded and reciprocal changes in &agr;- and &bgr;-adrenergic receptor densities occur in viable, hibernating myocardium and may account in part for the observed depression in resting myocardial function and preserved contractile reserve in this entity.

Effects of Chan Su, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: Cardiotoxicity due to more than Na, K-ATPase blocking

Extracts of Chan Su, a traditional Chinese medication used as a topical anesthetic and cardiac medication, were incubated with cardiomyocytes that had been loaded with a calcium specific fluorescent probe. Calcium transients were measured by real-time fluorescence spectrophotometry following treatment. The transients were rapidly abolished following addition of a moderate concentration of the extract (400 ng/ml), resulting in high levels of intracellular calcium, while the lower amount (40 ng/ml) blocked the sodium-potassium adenosine triphosphatase. Treatments with ouabain and nifedipine were also made, either prior to, or after the addition of the Chan Su, in an attempt to better delineate the site(s) of action. The moderate concentration of Chan Su (400 ng/ml) extract caused the myocytes to cease beating within seconds of addition, even in experiments when saturating concentrations of nifedipine or ouabain had been previously added to the cells. As expected bufalin, the active component of Chan Su has similar effects. Our findings indicate that this compound is extremely cardiotoxic, even in small dose and acts rapidly to alter intracellular calcium stores in cardiomyocytes and possibly acts at sites other than the Na(+)+K(+) ATPase, either directly, or indirectly via changes in calcium concentrations.

The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid

SummarySmall amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.

Enteral glutamine but not alanine maintains small bowel barrier function after ischemia/reperfusion injury in rats.

We previously demonstrated that glucose and glutamine, solutes metabolized by the gut, replenish ATP and enhance gut function compared with alanine, a solute not metabolized by the gut, following mesenteric ischemia/reperfusion (I/R). The purpose of the present study was to determine if the nonmetabolizable solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R. At laparotomy, rats had jejunal sacs filled with 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mm, osmotic control) followed by superior mesenteric artery clamping for 60 min and 30 min of reperfusion or sham laparotomy. Jejunum was harvested for evaluation by deconvolution microscopy, fluorescent measurement of F:G actin ratio, or mounted in an Ussing chamber for determination of intestinal permeability. Deconvolution microscopy revealed that the actin cytoskeleton was preserved by enteral glutamine, comparable to shams, but disrupted by enteral alanine. Glucose and controls resulted in comparable disruption, which was less than that with alanine. The F:G actin ratio was highest for glutamine and lowest for alanine; glucose was comparable to controls. Intestinal permeability was highest for alanine and lowest for glutamine, which was comparable to shams. Permeability following glucose and controls was higher than that following glutamine but lower than that following alanine. The nonmetabolizable solute alanine resulted in disruption of the actin cytoskeleton and enhanced intestinal permeability under conditions of mesenteric I/R. The metabolizable solute glutamine was protective under these conditions, whereas glucose exerted minimal effect on the integrity of the cytoskeleton and intestinal permeability. The individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications when administered to critically injured patients.

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2.

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I–IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2‐dependent yet is gp91phox‐independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue‐derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum‐starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.