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Highlights from the Literature

Edited by Christopher D. Verrico

Nominated by Olaf Andersen
Editor, Journal of General Physiology
Cornell University
sparre{at}med.cornell.edu

Question: How does phosphorylation of titan affect cardiac function?

Background: Sarcomeres are the repeating contractile subunits from which the myofibrils of striated muscle are built. Titin is a large sarcomeric protein involved in muscle elasticity and myofibril scaffolding, which provides passive tension to muscle based on physiological demands. In mammalian cardiac tissue, there are two isoforms of titan that differ in the number of elastic elements (modules) in the sequence. Titin isoforms with fewer elastic elements (N2B) have increased passive tension, whereas isoforms with more elastic elements (N2BA) have decreased passive tension.

ß-Adrenergic stimulation activates PKA to increase myocardial contractile force and accelerate relaxation. Although this is known to induce phosphorylation of a number of proteins, accumulating evidence suggests that ß-adrenergic modulation of myocardial properties involves, in part, myofilaments. Granzier and colleagues recently reported that ß-adrenergic stimulation of PKA causes phosphorylation of N2B, which reduces the passive (diastolic) force in cardiomyocytes, but the physiological role of titan phosphorylation in diastolic function is not well defined, and the role of titan phosphorylation in the active (systolic) cardiac function is unknown.

Observations: In the current report, the effects of titan phosphorylation on diastolic and systolic function were explored. PKA-dependent phosphorylation was found to decrease both the restoring force and the passive force. The attenuated passive force involved phosphorylation of both N2B and N2BA but was more pronounced in tissues with higher N2B expression. The physiological relevance of the PKA-dependent phosphorylation was explored through ß-adrenergic stimulation, which decreases the diastolic force and increases titin phosphorylation.

Significance: Fukuda et al. provide new mechanistic insight into how ß-adrenergic stimulation modulates diastolic and systolic function. The significance of this report is underscored by the observations that expression ratios of N2B and N2BA are altered in coronary artery disease and dilated cardiomyopathy, a disease in which the heart muscles become thin and flaccid and the heart becomes enlarged. Thus understanding how changes in titan isoform expression alter the functions of the heart has important implications for treating cardiac diseases.

Nominated by Michael Caplan
Yale University School of Medicine
michael.caplan{at}yale.edu

Question: What mediates rapid responses to estrogen?

Background: The steroid hormone estrogen acts through rapid signaling pathways outside the nucleus and with two known nuclear receptors (ER and ERß) that control transcription of target genes. The role of the nuclear receptors in mediating the rapid responses to estrogen is controversial. However, there is some evidence that the G protein-coupled receptor, GPR30, mediates rapid estrogen signaling, but a functional mechanism has not been determined.

Observations: Revankar et al. report that GPR30 mediates rapid estrogen signaling in a number of cell types. Perhaps unexpectedly, they found that GPR30 was localized to the membrane of the endoplasmic reticulum, not the plasma membrane. Nonetheless, they determined that upon estrogen binding to GPR30, calcium is mobilized and nuclear phosphatidylinositol 3,4,5-trisphosphate is synthesized to mediate the fast responses.

Significance: The localization of GPR30 to the endoplasmic reticulum is in contrast to hypotheses that position it in the plasma membrane. However, the membrane-permeability of estrogen supports the possibility that it facilitates rapid nongenomic signaling responses via interactions with intracellular GPR30. As GPR30 appears to represent the sole estrogen-responsive receptor in some cancer cells, such as breast cancer SKBr3 cells, these results may have implications for pathological estrogen physiology.

Nominated by Michael Caplan
Yale University School of Medicine
michael.caplan{at}yale.edu

Question: Is a high-throughput approach to studying dynamic protein-protein interactions (PPI) in mammalian cells feasible?

Background: PPI are essential for many cellular processes and are implicated in the occurrence and intensity of signal transduction network outputs. However, identifying signaling proteins that interact with a specific protein to produce complex signaling networks and physiological effects has proven difficult. To date, analysis of dynamic PPI networks in vertebrates has not been explored.

Observations: Barrios-Rodiles et al. report a novel, high-throughput technique to tag proteins that allows comprehensive mapping of PPI networks. This was applied to the morphogen transforming growth factor-ß (TGF-ß) pathway as a model of cell signaling. Approximately 12,000 experiments were required to analyze all of the possible interactions in different signaling contexts for 18 core members of the TGF-ß pathway with 518 different proteins. The results revealed ~950 interactions that formed a dynamic interconnected signaling network, which included posttranslational modification-dependent interactions, constitutive protein interactions, and transmembrane receptor interactions.

Significance: The ability to systematically analyze the PPI involved in mammalian cell signaling represents a significant advancement for the scientific community. In addition to revealing previously unknown connections in signaling pathways, the ability to detect protein interactions with transmembrane receptors is particularly noteworthy given their importance as drug targets and the difficulty of studying them with traditional high-throughput approaches.

Nominated by Amira Klip
The Hospital for Sick Children
amira{at}sickkids.ca

Question: Can the relationship between ghrelin secretory dynamics and secretion of other hormones be better understood by frequent sampling?

Background: Synthesized predominantly in the stomach, ghrelin is a peptide hormone that acts within hypothalamus to stimulate appetite and in the anterior pituitary to activate the growth hormone secretagogue receptor (GHS-R), thus potently inducing the secretion of growth hormone (GH). Ghrelin also affects the secretion of a number of other nutritional hormones. In humans, plasma ghrelin concentrations decrease after consumption of a meal and subsequently increase during fasting. Patients with anorexia nervosa (AN, a state of negative energy balance) have higher than normal fasting plasma levels of ghrelin and GH, although the physiological relationship between ghrelin, GH, and other hormones is unclear.

Observations: To understand, more comprehensively, the secretory dynamics of ghrelin, and because ghrelin levels fluctuate as a function of meal consumption, Misra et al. obtained frequent blood samples from adolescent girls with AN overnight. They demonstrated that increased levels of ghrelin in AN is a consequence of increased secretory burst mass and thus pulsatile and total secretion, which improved with weight gain. Additionally, several correlations were drawn between ghrelin and the many changes in neuroendocrine function observed during malnourishment. Insulin resistance was found to be most predictive of ghrelin levels, and ghrelin, in turn, was an independent predictor of GH secretion, GH secretory burst frequency, and cortisol burst frequency. Thus, there was an inverse correlation between insulin, leptin, and ghrelin that was positively correlated with GH and cortisol.

Significance: That increased concentrations of ghrelin in blood of AN patients correlates with other observed physiological changes in circulating hormones is clearly demonstrated, but the contribution of ghrelin, and/or other nutritional status parameters, to the pathophysiology of AN needs to be defined. Additionally, it is not known whether the appetite response of AN patients to ghrelin is overridden or whether the patients are ghrelin resistant. Nonetheless, the innovative methodology employed here more precisely delineated the physiological relationship between ghrelin and other nutritionally relevant hormones and will likely become a standard for future investigations.

Nominated by Lawrence Palmer
Associate Editor, Journal of General Physiology
Cornell University
lgpalm{at}med.cornell.edu

Question: Is the Nrl^–/^– mouse a viable model to investigate mechanisms of cone functions?

Background: Photons reflected or emitted from objects enter the eye through the pupil to form an image of an object on the retina. There, photoreceptors convert the photons into electrochemical signals, which are processed by neural circuits in the retina and transmitted to the brain. In humans, nighttime vision depends on sensitive rod photoreceptors, whereas daytime color vision depends on cone photoreceptors. Mammalian cones coexpress two visual pigments (opsins), a UV or S- and an M/L-pigment. This is paradoxical, as coexpression could potentially attenuate the capacity to discriminate lights based on their spectral content.

To comprehend the functional consequences of this coexpression, a mammalian model of cones would ideally allow genetic manipulations, characterization of the genetic products, and functional analysis. Although this prototype has been elusive thus far, the Nrl^–/^– mouse (null for the neural retina leucine zipper transcription factor), which produces no rods, has some promising attributes that make it a potential candidate.

Observations: The authors address the electrophysiological properties of single photoreceptors from the Nrl^–/^– mouse. For the first time, they demonstrate that both S- and M/L-cone opsins are functionally coexpressed. Additionally, using conventional and novel recording configurations, the authors characterized the dim-flash responses (responses to flashes of light in a dim background) from cells obtained from mice lacking Grk1, the only G protein receptor kinase expressed in mouse cones, and Nrl function. These double-knockout cells exhibited differences in recoveries to stimuli that activate the M/L- and S-pigment; the M/L-pigment was considerably more retarded.

Significance: Strikingly, Nikonov et al. provide incontrovertible evidence that Grk1 is essential for inactivation of both opsins, although S-opsins also have a Grk1-independent inactivation pathway, and that both coexpressed photopigments mediate phototransduction in individual cones. The finding that Nrl^–/^– photoreceptors are indeed a species of cone importantly establishes the validity of this model for the study of cone function and for exploration of the many questions provoked by the current observations.

Nominated by Pontus Perrson
Editor, American Journal of Physiology-Regulatory,
Integrative, and Comparative Physiology
Humboldt University
pontus.perrson{at}charite.de

Question: Does skin temperature affect sleep onset latency (SOL)?

Background: The ventrolateral preoptic nucleus of the hypothalamus is involved in circadian rhythms, and the ventromedial preoptic area (POAH) is involved in thermoregulation; together they play a key role in arousal-state regulation. Both sleep and body temperature have day-night rhythms, and onset of sleep usually follows the maximal rate of core body temperature decline, although the decline in body temperature occurs independently of sleep. However, because afferents that relay skin temperature affect firing rates of the POAH and because skin temperature increases as conditions become conducive to sleeping, it may be that the thermoregulatory state of the body affects sleep. The hypothesis that skin temperature modulates sleep onset was tested.

Observations: Raymann et al. determined that SOL was affected by very subtle manipulations of skin temperature, but not of core temperature. This suggests that it is not the decline of core temperature itself that is related to sleep propensity, as has been suggested so often, but rather the mechanism underlying this decline, i.e., heat loss by increased skin blood flow and consequently skin warming. In other words, the associated warming of the skin may be the signal that decreased SOL.

Significance: This study is the first of its kind to simultaneously apply direct experimental manipulations of core and skin temperatures independently, with the aim of modifying SOL. Strikingly, the decrease in SOL observed following subtle changes of skin temperature are comparable to the observed decrease in SOL following pharmacological interventions. Given the abuse potential of hypnotics, this approach to decrease SOL warrants further investigation and may provide insight into nonpharmacological or other pharmacological approaches that do not have abuse potential.

Nominated by Michael Romero
Case Western Reserve University
mfr2{at}po.cwru.edu

Question: What parameter(s) initiates the compensatory secretion of H⁺ from kidney following a decrease in blood pH?

Background: Pulmonary and renal functions are critical for maintaining blood concentrations of H⁺ within narrow physiological limits. Thus, under normal conditions, the CO₂ and H⁺ produced during aerobic metabolism are counterbalanced by a concomitant increase in renal H⁺ secretion. However, the equilibrium between CO₂, HCO₃^–, and H⁺ (CO₂ + H₂O HCO^–₃ + H⁺) has made empirical analysis of this compensatory mechanism problematic and has impeded identification of the underlying parameter(s) that mediates this mechanism.

Observations: Using methodology previously developed by Boron and colleagues for generating out-of-equilibrium CO₂/HCO^–₃ solutions, Zhou et al. independently varied basolateral CO₂ concentration, HCO₃^– concentration, and pH in renal proximal tubules (PT) to determine volume reabsorption (J_V), HCO^–₃ reabsorption (J_HCO3; this is comparable with H⁺ secretion), and reabsorption of solutes other than NaHCO₃ (J_other). Interestingly, they found that J_HCO3 and J_other did not respond to acute manipulations of pH. However, acutely increasing basolateral HCO^–₃ concentration was offset by a reduction in J_HCO3 and an increase in J_other, whereas acutely increasing basolateral CO₂ concentration was compensated for by an increase in J_HCO3 and a decrease in J_other. In other words, changes in J_HCO3 mediate the maintenance of physiological blood pH, whereas a compensatory reciprocal change of J_other stabilizes J_V.

Significance: It was previously hypothesized that cell acid-base chemosensitivity and balance was a pH-dependent phenomenon. In contrast, the evidence provided by Zhou et al. suggests that, in PT at least, there is no single parameter that determines acute chemosensitivity. Accordingly, sensors are hypothesized to monitor alterations in HCO₃^– and CO₂ and, as a result, adjust reabsorption rates of HCO^–₃ and other solutes, which ultimately maintain pH homeostasis. As the signaling mechanisms of HCO^–₃ and CO₂ in the maintenance of this acid-base equilibrium are discovered, so too may be drugs used to treat specific acid-base abnormalities.

Nominated by Gary Sieck
Editor, Journal of Applied Physiology
gary.sieck{at}mayo.edu

Question: Can mechanoelectric feedback (MEF)-induced arrhythmias be produced without direct stretch of cardiac cells?

Background: Under normal physiological conditions, the sinus node in the right atrium of the heart generates electrical impulses, which propagate throughout the heart and cause synchronous contractions. Following a brief resting interval, the sinus node generates the next stimulus. During a reentrant arrhythmia, electrical impulses move about the heart as circulating waves in a continuous, self-sustained manner, resulting in asyncrhonous and nonuniform contractions. MEF is the process whereby nonuniform mechanical strain applied to cardiac myocytes or tissues results in altered action potential morphology and, occasionally, the generation of arrhythmias. The physiological consequences of MEF are attributed largely to longitudinal stretch-induced activation of stretch-activated channels (SAC) in cardiac cells but could arise from other forms of mechanical stimuli such as intramyocardial pressure and shear stress.

Observations: To investigate the ability of highly localized deformations to initiate electrical waves, Tung and colleagues developed and then simultaneously employed several novel methodologies. Pulsing with a solution against the surface of confluent cardiac myocyte monolayers, electrophysiological responses were recorded by optically mapping fluorescence signals. Mechanical stimulation triggered excitation that induced a propagated excitatory wavefront, which could cause a reentrant arrhythmia. The mechanical induction of wavefront activity was largely suppressed by SAC blockers.

Significance: Relative to other experimental preparations, Kong et al. demonstrated the feasibility of inducing MEF and reentrant activity in monolayers of cardiac cells that form a small engineered tissue sample. Another significant distinction between this and other studies is the use of optical mapping to study MEF in the absence of pharmaceuticals that are commonly used to inhibit contraction-related artifacts, which can affect tissue physiology. Hence, subcellular deformations caused by mechanical stimuli other than stretch can trigger electrical activity.

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