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Edited by Christopher D. Verrico
Axle-less F1-ATPase rotates in the correct direction.
Furuike S, Hossain MD, Maki Y, Adachi K, Suzuki T, Kohori A, Itoh H, Yoshida M, Kinosita K Jr. Science 319: 955–958, 2008.
Nominated by Michael
Caplan Associate Editor, Physiology
Yale University School of Medicine
michael.caplan{at}yale.edu
Question: Do F1-ATPases require an axle to generate torque?
Background: F-Type ATPase is a transmembrane protein that uses a proton gradient to drive ATP synthesis. F-ATPase consists of two domains: the Fo domain, which is integral in the membrane, and the F1 domain, which is peripheral. The F1 domain consists of 
3β3
subunits where the central subunit rotates in, and is supported by, the static 3β3 cylinder. Furuike et al. set out to determine whether the shaft is even necessary for rotation.
Observations: By producing several truncated mutants until the remaining rotor head simply sat on the concave entrance of the stator orifice, Furuike et al. determined that all the truncated mutants rotated in the correct direction. It is noteworthy, however, that the average speed of the rotor was low, and the short mutants exhibited more irregular motion.
Significance: All the truncation mutants likely generate torque from ATP hydrolysis since they rotated in the correct direction, albeit in a less efficient manner. Nevertheless, these results suggest that a fixed pivot and a rigid axle are not required for rotation of F1-ATPases. As noted by the authors, elucidating the physiological process(es) that underlie the ability of the orifice to prevent backward diffusion and ensure generation of effective torque may provide a new paradigm for the design of molecular machines.
Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.
Huang B, Wang W, Bates M, Zhuang X. Science 319: 810–813, 2008.
Nominated by Michael Caplan
Associate Editor, Physiology
Yale University School of Medicine
michael.caplan{at}yale.edu
Question: Can a three-dimensional nanoscale imaging technique be developed to resolve intracellular structures?
Background: Conventional light microscopy is limited by the diffraction barrier, ~200–300 nm in the lateral dimensions and 500–800 nm in the axial dimension, which does not allow one to resolve many intracellular organelles and molecular structures. Although the diffraction limit has been surpassed by several techniques, including in 2006 by Zhuang and colleagues who developed stochastic optical reconstruction microscopy (STORM) with a two-dimensional resolution of 20–30 nm, most cellular structures cannot be resolved without imaging in all three dimensions.
Observations: Huang et al. used an optical astigmatism method to achieve super-resolution in all three dimensions with STORM. When images are collected to generate a two-dimensional STORM image of an individual molecule, those images also contain information regarding the third dimension. For example, by introducing a cylindrical lens into the imaging path, two different focal planes for the x and y directions are created, which allows the z coordinate to be determined. Using this approach, the authors achieved ~20-nm lateral and ~50-nm axial resolutions in 3D STORM.
Significance: This significant advance in imaging biological specimens will allow researchers to examine the three-dimensional morphology of some of the smallest cellular structures. Although any imaging center with a fluorescence microscope setup can readily acquire the instrumentation needed for 3D STORM, the data analysis needed to generate an image is more complex. The authors hope that a user-friendly 3D STORM analysis software packet will be accessible to other researchers soon. Ultimately, by speeding up the 3D STORM imaging process, it may be possible to observe some physiological processes in action.
Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization.
Shimada M, Yanai Y, Okazaki T, Noma N, Kawashima I, Mori T, Richards JS. Development 135: 2001–2011, 2008.
Nominated by Hsiao Chang Chan
The Chinese University of Hong Kong
hsiaocchang{at}cuhk.edu.hk
Question: What role do toll-like receptors (TLRs) have in ovulated cumulus-oocyte complexes (COCs)?
Background: TLRs recognize molecules derived from microbes and are thus members of the pathogen recognition receptor surveillance pathway. The TLR system is expressed in cumulus cells of ovulated COCs; however, their physiological role in ovulated COCs is unknown, as is their endogenous ligands. Hyaluronan (HA), which is one of the chief components of the extracellular matrix and is necessary for extrusion of COCs and ovulation, is known to activate TLR2 and TLR4. Thus Shimada et al. hypothesized that, during the fertilization process, sperm-secreted hyaluronidase could generate small HA fragments, which activate TLR2 and/or TLR4 on cumulus cells.
Observations: Interestingly, the authors found that hyaluronidase and sperm activate TLR2/TLR4 on cumulus cells. In turn, these activated cumulus cells, but not sperm itself, release chemokines that enhance sperm capacitation (the next to last step in the maturation of spermatozoa) and fertilization. They also determined that, when TLR2/TLR4 was neutralized or hyaluronan was blocked, in vitro fertilization of COC-enclosed oocytes was reduced.
Significance: These studies have identified an endogenous ligand for the TLRs and their physiological role in ovulated COCs, which collectively suggests that a functional regulatory loop exists between sperm and ovulated COCs. In addition, these results suggest that it may be possible to improve in vitro fertilization protocols in not only mice but also for human infertility care by adding CC chemokines to fertilization medium.
Comparative analysis of neonatal and adult rat carotid body responses to chronic intermittent hypoxia.
Pawar A, Peng YJ, Jacono FJ, Prabhakar NR. J Appl Physiol 104: 1287–1294, 2008.
Nominated by Jerry Dempsey
Editor, Journal of Applied Physiology
University of Wisconsin
jdempsey{at}wisc.edu
Question: Does the carotid body response to chronic intermittent hypoxia (CIH) differ between neonates and adults?
Background: The carotid body is a sensory organ located near the bifurcation of the carotid artery and is responsible for detecting changes in the partial pressure of oxygen in arterial blood. In adult rodents, during CIH, the carotid body is theorized to evoke the reflexes that mediate cardiorespiratory changes and results in a long-lasting increase in baseline sensory activity known as sensory long-term facilitation (LTF). In contrast to adults, the carotid bodies of neonatal rodents are immature and respond poorly to hypoxia. To better understand the physiology of neonatal carotid bodies, Pawar et al. explored whether CIH evokes sensory LTF and whether the effects of CIH are reversible in neonatal carotid bodies.
Observations: CIH augmented the hypoxic sensory response in both adults and neonates; however, the neonates displayed more susceptibility to the effects of CIH, resulting in significantly greater hypoxic sensitization than the adults. Additionally, CIH-induced hypoxic sensitization was reversed in adults after only 10 days of exposure to normal levels of oxygen. In contrast, the sensitization persisted in the neonates into adulthood (2 months). Moreover, CIH induced hyperplasia of the neonates carotid body type I glomus cells, an effect that was absent in adults. Finally, the CIH-evoked sensory LTF that occurs in adults upon exposure to acute intermittent hypoxic conditions did not occur in the neonates.
Significance: In response to CIH, neonates displayed striking differences in the function and morphology of carotid bodies, including greater sensitization to hypoxia, which could be attributed to multiple factors. However, given that the current experiments were carried out ex vivo, it is unlikely that cardiovascular variables account for this observed phenomenon. What is indicated by the present set of experiments is that the greater sensitization could be attributed to hyperplasia of the carotid body cells. Because the CIH-induced effects on neonates persisted into adulthood, it may mean they are irreversible. These findings suggest that 1) it is possible to augment carotid body function in neonates with apneic episodes and 2) the persistence of the effects of neonatal CIH on carotid body O2 sensing may have consequences in adult life.
Munc13-2–/– baseline secretion defect reveals source of oligomeric mucins in mouse airways.
Zhu Y, Ehre C, Abdullah LH, Sheehan JK, Roy M, Evans CM, Dickey BF, Davis CW. J Physiol 586: 1977–1992, 2008.
Nominated by Michael Joyner
Associate Editor, Journal of Physiology
Mayo College of Medicine
joyner.michael{at}mayo.edu
Question: Is there another source of mucins besides goblet cells?
Background: Mucins are heavily glycosylated proteins found in mucus. Mucins are secreted by airway goblet cells, which are regulated, in part, by Ca2+ interacting with C2-domain-containing proteins, such as Munc13. Unlike larger mammals, the mouse possesses very few goblet cells under control conditions and therefore has no obvious source of mucins; despite this, the mouse remains a popular model for allergic mucous metaplasia. Hence, in this study, mice deficient for the exocytic priming protein Munc13-2 were utilized to test whether it mediates agonist-related mucin secretion in mice.
Observations: Perhaps surprisingly, Zhu et al. discovered that Munc13-2-deficient mice had mucin-secreting cells under control conditions. Moreover, stimulated release of some, but not all, mucins was achieved, which suggests Munc13-4 is capable of supporting agonist-regulated secretion. The mucin Muc5b was found in Clara cells of wild-type control mice, and was elevated in the knockout mice.
Significance: This study offers the first direct evidence that Clara cells synthesize mucins under control conditions and release them tonically, thus contributing significantly to airway mucus levels in addition to the known production of mucus by submucosal glands. The findings also imply that there may be differing physiological/pathophysiological roles, respectively, for Muc5b produced normally by Clara cells as opposed to Muc5a that is upregulated dramatically in the setting of inflammation.
A GRK5 polymorphism that inhibits beta-adrenergic receptor signaling is protective in heart failure. Liggett SB, Cresci S, Kelly RJ, Syed FM, Matkovich SJ, Hahn HS, Diwan A, Martini JS, Sparks L, Parekh RR, Spertus JA, Koch WJ, Kardia SL, Dorn GW 2nd. Nat Med 14: 510–517, 2008.[CrossRef][Web of Science][Medline]
Nominated by Litsa Kranias
University of Cincinnati College of Medicine
litsa.kranias{at}uc.edu
Question: What accounts for the conflicting results of the use of β-blockers to treat heart failure?
Background: Blockade of β-adrenergic receptors (βAR) slows the heart rate and lowers blood pressure to decrease the heart’s workload, which is why this is the standard therapy for heart failure and myocardial ischemia. The G protein-coupled receptor kinase (GRK) GRK2 phosphorylates βARs, which prevents downstream signaling. GRK5 is also highly expressed in cardiac tissue, and when overexpressed it depresses βAR responsiveness. The roles of GRK2 and GRK5 do not appear to be redundant; GRK2 is thought to acutely regulate βAR signaling, whereas GRK5 may be more important for chronic regulation.
Observations: Liggett et al. searched the DNA sequence of the GRK2 and GRK5 genes in people of European-American, African-American, or Chinese descent to look for differences. The majority of people had exactly the same DNA sequence in GRK2 and GRK5. However, a variation called GRK5-Leu41 was found in more than 40% of African-Americans. Patients with the GRK5-Leu41 variant who did not take β blockers lived almost twice as long as those with the more common version of the GRK5 gene. Although β blockers prolonged life to the same degree as the GRK5 variant, it did not prolong survival of those with the variant.
Significance: These findings suggest that the often conflicting reports as to whether β blockers help African-American patients is because of the GRK5 variant, which mimics the effects of β blockers. This supports the idea that genetic testing could be used to identify people who can benefit from particular therapies and that someday patients will receive individualized therapy based on their genetic makeup.
Ca2/calmodulin-dependent protein kinase II delta and protein kinase D over-expression reinforce the histone deacetylase 5 redistribution in heart failure.
Bossuyt J, Helmstadter K, Wu X, Clements-Jewery H, Haworth RS, Avkiran M, Martin JL, Pogwizd SM, Bers DM. Circ Res 102: 695–702, 2008.
Nominated by Litsa Kranias
University of Cincinnati College of Medicine
litsa.kranias{at}uc.edu
Question: Is Ca2+/calmodulin (CaM) protein kinase II (CaMKII) or protein kinase D (PKD) signaling to histone deacetylases (HDACs) nuclear export elevated in heart failure (HF)?
Background: Cardiac hypertrophy occurs in response to prolonged physiological stressors, which can become pathological and lead to HF. Class II HDACs have been implicated in this process since they are phosphorylated in response to stressful stimuli, allowing activation of a hypertrophic program of gene expression. Recently, PKD has been identified as a type II HDAC kinase, although its role in the heart is relatively unexplored. In addition, CaM and CaMKII have been implicated in cardiac hypertrophy and HF. In fact, both CaMKII and PKD are involved in HDAC5 phosphorylation and nuclear export.
Observations: Bossuyt et al. sought to determine the expression and activity levels of PKD and whether CaMKII signaling was elevated in a model of HF. They found that PKD and CaMKII 
C expression and activation state are increased in rabbit and human HF. By inhibiting either CaMKII or PKD in HF myocytes, they were able to partially restore the distribution of HDAC5. In contrast, overexpressing PKD or CaMKII caused the distribution to shift toward what is observed in HF.
Significance: These findings suggest that upregulation of CaMKII and/or PKD may contribute to the altered gene expression associated with HF. Although a more extensive understanding of these kinases in heart function is necessary, these results suggest that pharmacologically interfering with CaMKII or PKD phosphorylation could prove beneficial to limiting the dysfunction associated with HF.
Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl– transport in biliary epithelial cells through a PKCzeta-dependent pathway.
Woo K, Dutta AK, Patel V, Kresge C, Feranchak AP. J Physiol 586: 2779–2798, 2008.
Nominated by Hugh Matthews
Associate Editor, Journal of Physiology Cambridge
University hrm1{at}cam.ac.uk
Question: What is the stimulus involved in biliary ATP release?
Background: ATP is present in bile, where it acts to stimulate the secretion of fluid and Cl– from the epithelial cells that line the bile ducts (cholangiocytes) via activation of purinergic (P2) receptors. In other cell types, mechanical forces that stretch, deform, apply pressure, or sheer stress to the plasma membrane cause ATP release. However, the physiological stimulus that underlies cholangiocyte ATP release is unknown.
Observations: Using a multidisciplinary approach, the authors investigate how fluid-flow (shear) is coupled to transepithelial ion transport in biliary epithelia. They found that human biliary cells and cholangiocyte monolayers from rat increased ATP release when exposed to fluid flow, which was proportional to the shear stress and desensitized by repeated exposures. The fluid flow increased intracellular Ca2+ and permeability of Cl– via stimulation of ATP release and P2 receptors. Finally, flow-stimulated ATP release was found to be regulated by PKC<zeta.
Significance: This is the first study to identify a mechanism that causes biliary ATP release: the mechanical force of flow at the apical membrane. This is also the first study to find that Cl– channels are activated by flow in biliary epithelial cells. These findings provide important information concerning the regulation of biliary secretion, which could lead to novel strategies to treat cholestatic liver disorders. Additionally, this novel mechanism of coupling fluid flow to transepithelial ion transport might serve as an important regulatory mechanism in other epithelial tissues.
Loading effect of fibroblast-myocyte coupling on resting potential, impulse propagation, and repolarization: insights from a microstructure model.
Jacquemet V, Henriquez CS. Am J Physiol Heart Circ Physiol 294: H2040–H2052, 2008.
Nominated by Alberto Nasjletti
Editor, American Journal of Physiology—Heart and Circulatory Physiology
New York Medical College alberto_nasjletti{at}nymc.edu
Question: What electrical interactions occur between myocytes and fibroblasts in the myocardium?
Background: Cardiac tissue is comprised of myocytes and nonmyocytes. The nonmyocytes are made up of fibroblasts, which are a connective tissue that makes up the myocardial extracellular matrix. Recently, fibroblasts were shown to affect impulse propagation in vitro, but the coupling conductance was unknown (it has been measured only in cell pairs, not yet in tissue), which makes it more difficult to predict what will happen in vivo if such coupling was present.
Observations: Utilizing a computer to simulate the load-induced electrophysiological coupling of myocytes and fibroblasts Jacquemet and Henriquez varied parameters, which illustrated the complex interactions of the model. They determined that when weakly coupled, conduction velocity is increased, but when coupling between fibroblasts and myocytes is strong, conduction velocity is decreased. They also found that action potential duration is prolonged, but the resting potential and density of the fibroblasts determines the amount of prolongation.
Significance: This work extends previous findings regarding fibroblast-myocyte coupling conductance, which is important because the actual coupling conductance between myocytes and fibroblasts is unknown. Moreover, because proliferation of fibroblasts occurs in pathological conditions, such as mitral valve disease and congestive heart failure, understanding the effects of fibroblasts on propagation of cardiac impulse could have significance for elucidating how this facilitates cardiac disease pathology.
Reduced phospholamban phosphorylation is associated with impaired relaxation in left ventricular myocytes from neuronal NO synthase-deficient mice. Zhang YH, Zhang MH, Sears CE, Emanuel K, Redwood C, El-Armouche A, Kranias EG, Casadei B. Am Circ Res 102: 242–249, 2008.[CrossRef]
Nominated by Ulrich Pohl
Associate Editor, Physiology
Physiologisches Institut
upohl{at}lmu.de
Question: What is the mechanism by which nitric oxide (NO) released by a neuronal NO synthase (nNOS) hastens myocardial relaxation?
Background: In the left ventricular (LV) myocardium, NO is synthesized by both the endothelial NO synthase (eNOS) and nNOS. Endothelium-derived eNOS-derived NO facilitates relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity. Recent evidence suggests that NO derived from a myocardial nNOS isoform is also involved in the regulation of LV relaxation. However, the mechanism by which nNOS-derived NO mediates this effect is unclear.
Observations: The findings by Zhang et al. suggest that nNOS-derived NO hastens LV relaxation in a cGMP-independent manner by promoting PKA-mediated phospholamban (PLB) phosphorylation. In nNOS–/– myocytes or after pharmacological inhibition of nNOS, they found the level of PLB phosphorylation was reduced. Moreover, the activity of protein phosphatase type 1 (PP1) and PP type 2A (PP2A) was higher in the nNOS–/– hearts than in their wild-type litter-mates (nNOS+/+). Pharmacological inhibition of PP1 or PP2A increased phosphorylation of PLB and decreased the time to 50% relaxation in nNOS–/– but not nNOS+/+ myocytes.
Significance: These findings identify a novel mechanism by which myocardial constitutive NO production regulates LV relaxation and the rate of decay of the intracellular calcium transient by modulating PKA-dependent phosphorylation of PLN, and thus SERCA2A activity, via subcellular targeting of protein phosphatase activity. This mechanism may play an important part in the protective role of myocardial nNOS in infarcted hearts.
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