Showing papers in "Pflügers Archiv: European Journal of Physiology in 2018"

Mitochondrial calcium uptake in organ physiology: from molecular mechanism to animal models

Abstract: Mitochondrial Ca2+ is involved in heterogeneous functions, ranging from the control of metabolism and ATP production to the regulation of cell death. In addition, mitochondrial Ca2+ uptake contributes to cytosolic [Ca2+] shaping thus impinging on specific Ca2+-dependent events. Mitochondrial Ca2+ concentration is controlled by influx and efflux pathways: the former controlled by the activity of the mitochondrial Ca2+ uniporter (MCU), the latter by the Na+/Ca2+ exchanger (NCLX) and the H+/Ca2+ (mHCX) exchanger. The molecular identities of MCU and of NCLX have been recently unraveled, thus allowing genetic studies on their physiopathological relevance. After a general framework on the significance of mitochondrial Ca2+ uptake, this review discusses the structure of the MCU complex and the regulation of its activity, the importance of mitochondrial Ca2+ signaling in different physiological settings, and the consequences of MCU modulation on organ physiology.

Alternative splicing isoforms in health and disease

Abstract: Alternative splicing (AS) of protein-coding messenger RNAs is an essential regulatory mechanism in eukaryotic gene expression that controls the proper function of proteins. It is also implicated in the physiological regulation of mitochondria and various ion channels. Considering that mis-splicing can result in various human diseases by modifying or abrogating important physiological protein functions, a fine-tuned balance of AS is essential for human health. Accumulated data highlight the importance of alternatively spliced isoforms in various diseases, including neurodegenerative disorders, cancer, immune and infectious diseases, cardiovascular diseases, and metabolic conditions. However, basic understanding of disease mechanisms and development of clinical applications still require the integration and interpretation of physiological roles of AS. This review discusses the roles of AS in health and various diseases, while highlighting potential AS-targeting therapeutic applications.

Intracellular thermometry with fluorescent sensors for thermal biology

Abstract: Temperature influences the activities of living organisms at various levels. Cells not only detect environmental temperature changes through their unique temperature-sensitive molecular machineries but also muster an appropriate response to the temperature change to maintain their inherent functions. Despite the fundamental involvement of temperature in physiological phenomena, the mechanism by which cells produce and use heat is largely unknown. Recently, fluorescent thermosensors that function as thermometers in live cells have attracted much attention in biology. These new tools, made of various temperature-sensitive molecules, have allowed for intracellular thermometry at the single-cell level. Intriguing spatiotemporal temperature variations, including organelle-specific thermogenesis, have been revealed with these fluorescent thermosensors, which suggest an intrinsic connection between temperature and cell functions. Moreover, fluorescent thermosensors have shown that intracellular temperature changes at the microscopic level are largely different from those assumed for a water environment at the macroscopic level. Thus, the employment of fluorescent thermosensors will uncover novel mechanisms of intracellular temperature-assisted physiological functions.

The role of the circadian clock system in physiology.

Abstract: Life on earth is shaped by the 24-h rotation of our planet around its axes. To adapt behavior and physiology to the concurring profound but highly predictable changes, endogenous circadian clocks have evolved that drive 24-h rhythms in invertebrate and vertebrate species. At the molecular level, circadian clocks comprised a set of clock genes organized in a system of interlocked transcriptional–translational feedback loops. A ubiquitous network of cellular central and peripheral tissue clocks coordinates physiological functions along the day through activation of tissue-specific transcriptional programs. Circadian rhythms impact on diverse physiological processes including the cardiovascular system, energy metabolism, immunity, hormone secretion, and reproduction. This review summarizes our current understanding of the mechanisms of circadian timekeeping in different species, its adaptation by external timing signals and the pathophysiological consequences of circadian disruption.

A look at the smelly side of physiology: transport of short chain fatty acids.

Abstract: Fermentative organs such as the caecum, the colon, and the rumen have evolved to produce and absorb energy rich short chain fatty acids (SCFA) from otherwise indigestible substrates. Classical models postulate diffusional uptake of the undissociated acid (HSCFA). However, in net terms, a major part of SCFA absorption occurs with uptake of Na+ and resembles classical, coupled electroneutral NaCl transport. Considerable evidence suggests that the anion transporting proteins expressed by epithelia of fermentative organs are poorly selective and that their main function may be to transport acetate-, propionate-, butyrate- and HCO3- as the physiologically relevant anions. Apical uptake of SCFA thus involves non-saturable diffusion of the undissociated acid (HSCFA), SCFA-/HCO3- exchange via DRA (SLC26A3) and/or SCFA--H+ symport (MCT1, SLC16A1). All mechanisms lead to cytosolic acidification with stimulation of Na+/H+ exchange via NHE (SLC9A2/3). Basolaterally, Na+ leaves via the Na+/K+-ATPase with recirculation of K+. Na+ efflux drives the transport of SCFA- anions through volume-regulated anion channels, such as maxi-anion channels (possibly SLCO2A1), LRRC8, anoctamins, or uncoupled exchangers. When luminal buffering is inadequate, basolateral efflux will increasingly involve SCFA-/ HCO3- exchange (AE1/2, SCL4A1/2), or efflux of SCFA- with H+ (MCT1/4, SLC16A1/3). Furthermore, protons can be basolaterally removed by NHE1 (SCL9A1) or NBCe1 (SLC4A4). The purpose of these transport proteins is to maximize the amount of SCFA transported from the tightly buffered ingesta while minimizing acid transport through the epithelium. As known from the rumen for many decades, a disturbance of these processes is likely to cause severe colonic disease.

The role of the mitochondrial calcium uniporter (MCU) complex in cancer.

Abstract: The important role of mitochondria in cancer biology is gaining momentum. With their regulation of cell survival, metabolism, basic cell building blocks, and immunity, among other functions, mitochondria affect not only cancer progression but also the response and resistance to current treatments. Calcium ions are constantly shuttled in and out of mitochondria; thus, playing an important role in the regulation of various cellular processes. The mitochondrial calcium uniporter (MCU) channel and its associated regulators transport calcium across the inner mitochondrial membrane to the mitochondrial matrix. Due to this central role and the capacity to affect cell behavior and fate, the MCU complex is being investigated in different cancers and cancer-related conditions. Here, we review current knowledge on the role of the MCU complex in multiple cancer types and models; we also provide a perspective for future research and clinical considerations.

Calcium- and voltage-gated BK channels in vascular smooth muscle.

Abstract: Ion channels in vascular smooth muscle regulate myogenic tone and vessel contractility. In particular, activation of calcium- and voltage-gated potassium channels of large conductance (BK channels) results in outward current that shifts the membrane potential toward more negative values, triggering a negative feed-back loop on depolarization-induced calcium influx and SM contraction. In this short review, we first present the molecular basis of vascular smooth muscle BK channels and the role of subunit composition and trafficking in the regulation of myogenic tone and vascular contractility. BK channel modulation by endogenous signaling molecules, and paracrine and endocrine mediators follows. Lastly, we describe the functional changes in smooth muscle BK channels that contribute to, or are triggered by, common physiological conditions and pathologies, including obesity, diabetes, and systemic hypertension.

Erythropoietin stimulates fibroblast growth factor 23 (FGF23) in mice and men.

Abstract: Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism.

Dawning of a new era in TRP channel structural biology by cryo-electron microscopy.

Abstract: Cryo-electron microscopy (cryo-EM) permits the determination of atomic protein structures by averaging large numbers of individual projection images recorded at cryogenic temperatures—a method termed single-particle analysis. The cryo-preservation traps proteins within a thin glass-like ice layer, making literally a freeze image of proteins in solution. Projections of randomly adopted orientations are merged to reconstruct a 3D density map. While atomic resolution for highly symmetric viruses was achieved already in 2009, the development of new sensitive and fast electron detectors has enabled cryo-EM for smaller and asymmetrical proteins including fragile membrane proteins. As one of the most important structural biology methods at present, cryo-EM was awarded in October 2017 with the Nobel Prize in Chemistry. The molecular understanding of Transient-Receptor-Potential (TRP) channels has been boosted tremendously by cryo-EM single-particle analysis. Several near-atomic and atomic structures gave important mechanistic insights, e.g., into ion permeation and selectivity, gating, as well as into the activation of this enigmatic and medically important membrane protein family by various chemical and physical stimuli. Lastly, these structures have set the starting point for the rational design of TRP channel-targeted therapeutics to counteract life-threatening channelopathies. Here, we attempt a brief introduction to the method, review the latest advances in cryo-EM structure determination of TRP channels, and discuss molecular insights into the channel function based on the wealth of TRP channel cryo-EM structures.

TRPs et al.: a molecular toolkit for thermosensory adaptations.

Abstract: The ability to sense temperature is crucial for the survival of an organism. Temperature influences all biological operations, from rates of metabolic reactions to protein folding, and broad behavioral functions, from feeding to breeding, and other seasonal activities. The evolution of specialized thermosensory adaptations has enabled animals to inhabit extreme temperature niches and to perform specific temperature-dependent behaviors. The function of sensory neurons depends on the participation of various types of ion channels. Each of the channels involved in neuronal excitability, whether through the generation of receptor potential, action potential, or the maintenance of the resting potential have temperature-dependent properties that can tune the neuron's response to temperature stimuli. Since the function of all proteins is affected by temperature, animals need adaptations not only for detecting different temperatures, but also for maintaining sensory ability at different temperatures. A full understanding of the molecular mechanism of thermosensation requires an investigation of all channel types at each step of thermosensory transduction. A fruitful avenue of investigation into how different molecules can contribute to the fine-tuning of temperature sensitivity is to study the specialized adaptations of various species. Given the diversity of molecular participants at each stage of sensory transduction, animals have a toolkit of channels at their disposal to adapt their thermosensitivity to their particular habitats or behavioral circumstances.

The extraordinary AFD thermosensor of C. elegans.

Abstract: The nematode C. elegans exhibits complex thermal experience-dependent navigation behaviors in response to environmental temperature changes of as little as 0.01°C over a > 10°C temperature range. The remarkable thermosensory abilities of this animal are mediated primarily via the single pair of AFD sensory neurons in its head. In this review, we describe the contributions of AFD to thermosensory behaviors and temperature-dependent regulation of organismal physiology. We also discuss the mechanisms that enable this neuron type to adapt to recent temperature experience and to exhibit extraordinary thermosensitivity over a wide dynamic range.

Cellular populations and thermosensing mechanisms of the hypothalamic thermoregulatory center.

Abstract: Temperature affects all aspects of life down to the diffusion rates of biologically active molecules and reaction rates of enzymes. The reciprocal argument holds true as well and every biological process down to enzymatic reactions influences temperature. In order to assure biological stability, mammalian organisms possess the remarkable ability to maintain internal body temperature within a narrow range, which in humans and mice is close to 37 °C, despite wide environmental temperature variations and different rates of internal heat production. Nevertheless, body temperature is not a static property but adaptively regulated upon physiological demands and in the context of pathological conditions. The brain region that has been primarily associated with internal temperature regulation is the preoptic area and the anterior portion of the hypothalamus. Similar to a thermostat, this brain area detects deep brain temperature, integrates temperature information from peripheral body sensors, and-based on these inputs--controls body temperature homeostasis. Discovered more than a century ago, we still know comparatively little about the molecular and cellular make-up of the hypothalamic thermoregulatory center. After a brief historic outline that led to the discovery of the thermoregulatory center, we here review recent studies that have considerably advanced our understanding of hypothalamic thermoregulation. We touch upon proposed mechanisms of intrinsic deep brain temperature detection and focus on newly identified hypothalamic cell populations that mediate thermoregulatory responses and that provide novel entry points not only to shed light on the mechanistic underpinnings of the thermoregulatory center but also to probe its therapeutic value.

Catalase and nonalcoholic fatty liver disease.

Abstract: Obesity and insulin resistance are considered the main causes of nonalcoholic fatty liver disease (NAFLD), and oxidative stress accelerates the progression of NAFLD. Free fatty acids, which are elevated in the liver by obesity or insulin resistance, lead to incomplete oxidation in the mitochondria, peroxisomes, and microsomes, leading to the production of reactive oxygen species (ROS). Among the ROS generated, H2O2 is mainly produced in peroxisomes and decomposed by catalase. However, when the H2O2 concentration increases because of decreased expression or activity of catalase, it migrates to cytosol and other organelles, causing cell injury and participating in the Fenton reaction, resulting in serious oxidative stress. To date, numerous studies have been shown to inhibit the pathogenesis of NAFLD, but treatment for this disease mainly depends on weight loss and exercise. Various molecules such as vitamin E, metformin, liraglutide, and resveratrol have been proposed as therapeutic agents, but further verification of the dose setting, clinical application, and side effects is needed. Reducing oxidative stress may be a fundamental method for improving not only the progression of NAFLD but also obesity and insulin resistance. However, the relationship between NAFLD progression and antioxidants, particularly catalase, which is most commonly expressed in the liver, remains unclear. Therefore, this review summarizes the role of catalase, focusing on its potential therapeutic effects in NAFLD progression.

Circulating chromogranin A and its fragments as diagnostic and prognostic disease markers.

Abstract: Chromogranin A (CgA), a secretory protein released in the blood by neuroendocrine cells and neurons, is the precursor of various bioactive fragments involved in the regulation of the cardiovascular system, metabolism, innate immunity, angiogenesis, and tissue repair. After the original demonstration that circulating CgA can serve as a biomarker for a wide range of neuroendocrine tumors, several studies have shown that increased levels of CgA can be present also in the blood of patients with cardiovascular, gastrointestinal, and inflammatory diseases with, in certain cases, important diagnostic and prognostic implications. Considering the high structural and functional heterogeneity of the CgA system, comprising precursor and fragments, it is not surprising that the different immunoassays used in these studies led, in some cases, to discrepant results. Here, we review these notions and we discuss the importance of measuring total-CgA, full-length CgA, specific fragments, and their relative levels for a more thorough assessment of the pathophysiological function and diagnostic/prognostic value of the CgA system.

Calcium channel gating

Abstract: Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a "gate releasing" movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close.

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

Abstract: Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11–491 and STIM11–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

Heart regeneration and the cardiomyocyte cell cycle.

Abstract: Cardiovascular disease and in particular, heart failure are still main causes of death; therefore, novel therapeutic approaches are urgently needed. Loss of contractile substrate in the heart and limited regenerative capacity of cardiomyocytes are mainly responsible for the poor cardiovascular outcome. This is related to the postmitotic state of differentiated cardiomyocytes, which is partly due to their polyploid nature caused by cell cycle variants. As such, the cardiomyocyte cell cycle is a key player, and its manipulation could be a promising strategy for enhancing the plasticity of the heart by inducing cardiomyocyte proliferation. This review focuses on the cardiac cell cycle and its variants during postnatal growth, the different regenerative responses of the heart in dependance of the developmental stage and on manipulations of the cell cycle. Because a therapeutic goal is to induce authentic cell division in cardiomyocytes, recent experimental approaches following this strategy are also discussed.

Relevance of TRPA1 and TRPM8 channels as vascular sensors of cold in the cutaneous microvasculature

Abstract: Cold exposure is directly related to skin conditions, such as frostbite. This is due to the cold exposure inducing a vasoconstriction to reduce cutaneous blood flow and protect against heat loss. However, a long-term constriction will cause ischaemia and potentially irreversible damage. We have developed techniques to elucidate the mechanisms of the vascular cold response. We focused on two ligand-gated transient receptor potential (TRP) channels, namely, the established “cold sensors” TRP ankyrin 1 (TRPA1) and TRP melastin (TRPM8). We used the anaesthetised mouse and measured cutaneous blood flow by laser speckle imaging. Two cold treatments were used. A generalised cold treatment was achieved through whole paw water immersion (10 °C for 5 min) and a localised cold treatment that will be potentially easier to translate to human studies was carried out on the mouse paw with a copper cold probe (0.85-cm diameter). The results show that TRPA1 and TRPM8 can each act as a vascular cold sensor to mediate the vasoconstrictor component of whole paw cooling as expected from our previous research. However, the local cooling-induced responses were only blocked when the TRPA1 and TRPM8 antagonists were given simultaneously. This suggests that this localised cold probe response requires both functional TRPA1 and TRPM8.

Neurosecretion: what can we learn from chromaffin cells

Abstract: Many of the molecular players in the stimulus-secretion chain are similarly active in neurosecretion and catecholamine release. Therefore, studying chromaffin cells uncovered many details of the processes of docking, priming, and exocytosis of vesicles. However, morphological specializations at synapses, called active zones (AZs), confer extra speed of response and another layer of control to the fast release of vesicles by action potentials. Work at the Calyx of Held, a glutamatergic nerve terminal, has shown that in addition to such rapidly released vesicles, there is a pool of “Slow Vesicles,” which are held to be perfectly release-competent, but lack a final step of tight interaction with the AZ. It is argued here that such “Slow Vesicles” have many properties in common with chromaffin granules. The added complexity in the AZ-dependent regulation of “Fast Vesicles” can lead to misinterpretation of data on neurosecretion. Therefore, the study of Slow Vesicles and of chromaffin granules may provide a clearer picture of the early steps in the highly regulated process of neurosecretion.

Roles of Na + , Ca 2+ , and K + channels in the generation of repetitive firing and rhythmic bursting in adrenal chromaffin cells

Abstract: Adrenal chromaffin cells (CCs) are the main source of circulating catecholamines (CAs) that regulate the body response to stress. Release of CAs is controlled neurogenically by the activity of preganglionic sympathetic neurons through trains of action potentials (APs). APs in CCs are generated by robust depolarization following the activation of nicotinic and muscarinic receptors that are highly expressed in CCs. Bovine, rat, mouse, and human CCs also express a composite array of Na+, K+, and Ca2+ channels that regulate the resting potential, shape the APs, and set the frequency of AP trains. AP trains of increasing frequency induce enhanced release of CAs. If the primary role of CCs is simply to relay preganglionic nerve commands to CA secretion, why should they express such a diverse set of ion channels? An answer to this comes from recent observations that, like in neurons, CCs undergo complex firing patterns of APs suggesting the existence of an intrinsic CC excitability (non-neurogenically controlled). Recent work has shown that CCs undergo occasional or persistent burst firing elicited by altered physiological conditions or deletion of pore-regulating auxiliary subunits. In this review, we aim to give a rationale to the role of the many ion channel types regulating CC excitability. We will first describe their functional properties and then analyze how they contribute to pacemaking, AP shape, and burst waveforms. We will also furnish clear indications on missing ion conductances that may be involved in pacemaking and highlight the contribution of the crucial channels involved in burst firing.

Sensing the heat with TRPM3.

Abstract: Heat sensation, the ability to detect warm and noxious temperatures, is an ancient and indispensable sensory process. Noxious temperatures can have detrimental effects on the physiology and integrity of cells, and therefore, the detection of environmental hot temperatures is absolutely crucial for survival. Temperature-sensitive ion channels, which conduct ions in a highly temperature-dependent manner, have been put forward as molecular thermometers expressed at the endings of sensory neurons. In particular, several temperature-sensitive members of the transient receptor potential (TRP) superfamily of ion channels have been identified, and a multitude of in vivo studies have shown that the capsaicin-sensitive TRPV1 channel plays a key role as a noxious heat sensor. However, Trpv1-deficient mice display a residual heat sensitivity suggesting the existence of additional heat sensor(s). In this chapter, we provide evidence for the role of the non-selective calcium-permeable TRPM3 ion channel as an additional heat sensor that acts independently of TRPV1, and give an update of the modulation of this channel by various molecular mechanisms. Finally, we compare antagonists of TRPM3 to specific blockers of TRPV1 as potential analgesic drugs to treat pathological pain.

Late sodium current associated cardiac electrophysiological and mechanical dysfunction

Abstract: Late sodium current (INaL) is a small sustained inward current observed during the cardiac action potential plateau phase following decay of the early peak INa. The endogenous INaL is relatively small in normal hearts but exerts functionally significant effects on cardiomyocyte repolarization with potentially pro-arrhythmic effects in hearts with reduced repolarization reserve. Enhanced INa,L occurs in long QT syndrome 3 (LQTS 3) patients, and under a number of pathological and pharmacological cardiovascular conditions, including bradycardia, myocardial ischemia, reperfusion injury, and heart failure. It may there play important roles in arrhythmogenesis and mechanical dysfunction. Experimental and clinical research suggests that INaL inhibition may prevent and treat cardiac arrhythmias and improve ventricular pump function. Selective INa,L inhibitors, exemplified by ranolazine, GS-967 and GS-458967 have little or no effect on peak sodium current and/or IKr, and carry no or minimal pro-arrhythmic risk compared to those associated with administration of classical class I or III antiarrhythmic drugs, particularly in patients with ischemic heart disease. This increased understanding of INaL may be encouraging to clinicians in use of INaL inhibitors to treat cardiac arrhythmias and mechanical dysfunction directly associated with enhanced INaL such as LQTS type 3, and myocardial ischemia. This review discusses the roles of endogenous and enhanced INaL in arrhythmogenesis and mechanical dysfunction, and the basic and clinical research of INaL inhibitors.

Sarcopenia in a mice model of chronic liver disease: role of the ubiquitin–proteasome system and oxidative stress

Abstract: Sarcopenia is the loss of muscle mass and strength produced by aging or secondary to chronic diseases such as chronic liver disease (CLD). Although not all types of sarcopenia involve the same features, the most common are decreased fiber diameter and myosin heavy chain (MHC) levels, increased activity of ubiquitin–proteasome system (UPS) and reactive oxygen species (ROS). In this study, we aim to characterize the development of sarcopenia secondary to CLD induced by the hepatotoxin 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). For this purpose, four-months-old male C57BL6 mice were fed with normal diet or DDC supplemented diet for 6 weeks. Functional tests to evaluate muscle strength, mobility, and motor skills were performed in alive mice. The muscle strength in isolated gastrocnemius was also assayed via electrophysiological measurements. Morphometric measures of fibers’ diameter, total and ubiquitinated protein levels of myosin heavy chain (MHC), E3 ubiquitin ligases, ROS, and oxidation-dependent modified proteins in gastrocnemius tissue were also determined. Our results demonstrated that mice fed the DDC diet developed muscle wasting as evidenced by a loss of muscle mass and decreased muscle strength. The muscles of mice fed with DDC diet have a decreased diameter of fibers and MHC levels, also as increased MuRF-1 and atrogin-1 protein levels, ROS levels, and oxidation-modified protein levels. Additionally, control and DDC mice have the same food and water intake as well as mobility. Our results demonstrate mice with CLD develop sarcopenia involving decreased levels of myofibrillar proteins, increased UPS, and oxidative stress, but not for impaired caloric intake or immobility.

Chromogranins: from discovery to current times

Abstract: The discovery in 1953 of the chromaffin granules as co-storage of catecholamines and ATP was soon followed by identification of a range of uniquely acidic proteins making up the isotonic vesicular storage complex within elements of the diffuse sympathoadrenal system. In the mid-1960s, the enzymatically inactive, major core protein, chromogranin A was shown to be exocytotically discharged from the stimulated adrenal gland in parallel with the co-stored catecholamines and ATP. A prohormone concept was introduced when one of the main storage proteins collectively named granins was identified as the insulin release inhibitory polypeptide pancreastatin. A wide range of granin-derived biologically active peptides have subsequently been identified. Both chromogranin A and chromogranin B give rise to antimicrobial peptides of relevance for combat of pathogens. While two of the chromogranin A-derived peptides, vasostatin-I and pancreastatin, are involved in modulation of calcium and glucose homeostasis, respectively, vasostatin-I and catestatin are important modulators of endothelial permeability, angiogenesis, myocardial contractility, and innate immunity. A physiological role is now evident for the full-length chromogranin A and vasostatin-I as circulating stabilizers of endothelial integrity and in protection against myocardial injury. The high circulating levels of chromogranin A and its fragments in patients suffering from various inflammatory diseases have emerged as challenges for future research and clinical applications.

PACAP signaling in stress: insights from the chromaffin cell.

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified in hypothalamus, based on its ability to elevate cyclic AMP in the anterior pituitary. PACAP has been identified as the adrenomedullary neurotransmitter in stress through a combination of ex vivo, in vivo, and in cellula experiments over the past two decades. PACAP causes catecholamine secretion, and activation of catecholamine biosynthetic enzymes, during episodes of stress in mammals. Features of PACAP signaling allowing stress transduction at the splanchnicoadrenomedullary synapse have yielded insights into the contrasting roles of acetylcholine's and PACAP's actions as first messengers at the chromaffin cell, via differential release at low and high rates of splanchnic nerve firing, and differential signaling pathway engagement leading to catecholamine secretion and chromaffin cell gene transcription. Secretion stimulated by PACAP, via calcium influx independent of action potential generation, is under active investigation in several laboratories both at the chromaffin cell and within autonomic ganglia of both the parasympathetic and sympathetic nervous systems. PACAP is a neurotransmitter important in stress transduction in the central nervous system as well, and is found at stress-transduction nuclei in brain including the paraventricular nucleus of hypothalamus, the amygdala and extended amygdalar nuclei, and the prefrontal cortex. The current status of PACAP as a master regulator of stress signaling in the nervous system derives fundamentally from the establishment of its role as the splanchnicoadrenomedullary transmitter in stress. Experimental elucidation of PACAP action at this synapse remains at the forefront of understanding PACAP's role in stress signaling throughout the nervous system.

SLC2A9 (GLUT9) mediates urate reabsorption in the mouse kidney

Abstract: Uric acid (UA) is a metabolite of purine degradation and is involved in gout flairs and kidney stones formation. GLUT9 (SLC2A9) was previously shown to be a urate transporter in vitro. In vivo, humans carrying GLUT9 loss-of-function mutations have familial renal hypouricemia type 2, a condition characterized by hypouricemia, UA renal wasting associated with kidney stones, and an increased propensity to acute renal failure during strenuous exercise. Mice carrying a deletion of GLUT9 in the whole body are hyperuricemic and display a severe nephropathy due to intratubular uric acid precipitation. However, the precise role of GLUT9 in the kidney remains poorly characterized. We developed a mouse model in which GLUT9 was deleted specifically along the whole nephron in a tetracycline-inducible manner (subsequently called kidney-inducible KO or kiKO). The urate/creatinine ratio was increased as early as 4 days after induction of the KO and no GLUT9 protein was visible on kidney extracts. kiKO mice are morphologically identical to their wild-type littermates and had no spontaneous kidney stones. Twenty-four-hour urine collection revealed a major increase of urate urinary excretion rate and of the fractional excretion of urate, with no difference in urate concentration in the plasma. Polyuria was observed, but kiKO mice were still able to concentrate urine after water restriction. KiKO mice displayed lower blood pressure accompanied by an increased heart rate. Overall, these results indicate that GLUT9 is a crucial player in renal handling of urate in vivo and a putative target for uricosuric drugs.

Recent advances in pharmacological, hormonal, and nutritional intervention for sarcopenia.

Abstract: Sarcopenia, the age-related loss of skeletal muscle mass, is characterized by a deterioration of muscle quantity and quality leading to a gradual slowing of movement, a decline in strength and power, increased risk of fall-related injury, and often frailty. This review focuses on the recent advances of pharmacological, hormonal, and nutritional approaches for attenuating sarcopenia. The article is composed of the data reported in many basic and some clinical studies for mammalian muscles. Resistance training combined with amino acid-containing supplements is the gold standard to prevent sarcopenia. Supplementation with proteins (amino acids) only did not influence sarcopenic symptoms. A myostatin-inhibiting strategy is the most important candidate to prevent sarcopenia in humans. Milder caloric restriction (CR, 15-25%) would also be effective for age-related muscle atrophy in humans. Supplementation with ursolic acid and ghrelin is an intriguing candidate to combat sarcopenia, although further systematic and fundamental research is needed on this treatment.

Gating mechanism of Kv11.1 (hERG) K + channels without covalent connection between voltage sensor and pore domains

Abstract: Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Transcripts of Kv7.1 and MinK channels and slow delayed rectifier K+ current (IKs) are expressed in zebrafish (Danio rerio) heart

Abstract: Zebrafish are increasingly used as a model for human cardiac electrophysiology, arrhythmias, and drug screening. However, K+ ion channels of the zebrafish heart, which determine the rate of repolarization and duration of cardiac action potential (AP) are still incompletely known and characterized. Here, we provide the first evidence for the presence of the slow component of the delayed rectifier K+channels in the zebrafish heart and characterize electrophysiological properties of the slow component of the delayed rectifier K+current, IKs. Zebrafish atrium and ventricle showed strong transcript expression of the kcnq1 gene, which encodes the Kv7.1 α-subunit of the slow delayed rectifier K+ channel. In contrast, the kcne1 gene, encoding the MinK β-subunit of the delayed rectifier, was expressed at 21 and 17 times lower level in ventricle and atrium, respectively, in comparison to the kcnq1. IKs was observed in 62% of ventricular myocytes with mean (± SEM) density of 1.23 ± 0.37 pA/pF at + 30 mV. Activation rate of IKs was 38% faster (τ50 = 1248 ± 215 ms) than kcnq1:kcne1 channels (1725 ± 792 ms) expressed in 3:1 ratio in Chinese hamster ovary cells. Microelectrode experiments demonstrated the functional relevance of IKs in the zebrafish heart, since 100 μM chromanol 293B produced a significant prolongation of AP in zebrafish ventricle. We conclude that AP repolarization in zebrafish ventricle is contributed by IKs, which is mainly generated by homotetrameric Kv7.1 channels not coupled to MinK ancillary β-subunits. This is a clear difference to the human heart, where MinK is an essential component of the slow delayed rectifier K+channel.

CFTR supports cell death through ROS-dependent activation of TMEM16F (anoctamin 6)

Abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is the essential chloride and bicarbonate channel in the apical membrane of epithelial cells. CFTR was also proposed earlier to conduct glutathione (GSH) out of airway epithelial cells to be enriched in the apical airway surface liquid to neutralize reactive oxygen species (ROS). Although earlier studies suggested that release of GSH by wild type (wt) CFTR may lead to an increase in cytosolic ROS, we did not detect different ROS levels in cells expressing wt-CFTR and mutant F508del-CFTR, independent of CFTR-activation or exposure to the ROS donor tert-butyl hydroperoxide. The Ca2+-activated phospholipid scramblase and ion channel TMEM16F (anoctamin 6, ANO6) is also expressed in airway cells. ANO6 produced outwardly rectifying Cl- currents (ORCC) and scrambled plasma membrane phospholipids when activated by increase in cytosolic ROS and consecutive peroxidation of plasma membrane lipids. ANO6 activity is enhanced by CFTR, probably through translocation of signaling proteins to the plasma membrane. The present data suggest that enhanced cell death in CFTR-expressing cells is due to upregulation of ANO6-activity. In ANO6 knockout mice, the number of apoptotic cells in the intestinal epithelium was strongly reduced, supporting the role of ANO6 for cell death. Thus, ANO6 and CFTR act cooperatively on ROS-mediated cell death, which is not further augmented by cAMP-dependent stimulation. We propose that ANO6 supports cell death correlated with expression of CFTR, possibly by inducing ferroptosis.