This document provides a review of the techniques and therapies used in gait rehabilitation after stroke. It also examines the possible benefits of including assistive robotic devices and brain-computer interfaces in this field, according to a top-down approach, in which rehabilitation is driven by neural plasticity.The methods reviewed comprise classical gait rehabilitation techniques , functional electrical stimulation (FES), robotic devices, and brain-computer interfaces (BCI).From the analysis of these approaches, we can draw the following conclusions. Regarding classical rehabilitation techniques, there is insufficient evidence to state that a particular approach is more effective in promoting gait recovery than other. Combination of different rehabilitation strategies seems to be more effective than over-ground gait training alone. Robotic devices need further research to show their suitability for walking training and their effects on over-ground gait. The use of FES combined with different walking retraining strategies has shown to result in improvements in hemiplegic gait. Reports on non-invasive BCIs for stroke recovery are limited to the rehabilitation of upper limbs; however, some works suggest that there might be a common mechanism which influences upper and lower limb recovery simultaneously, independently of the limb chosen for the rehabilitation therapy. Functional near infrared spectroscopy (fNIRS) enables researchers to detect signals from specific regions of the cortex during performance of motor activities for the development of future BCIs. Future research would make possible to analyze the impact of rehabilitation on brain plasticity, in order to adapt treatment resources to meet the needs of each patient and to optimize the recovery process. Stroke is one of the principal causes of morbidity and mortality in adults in the developed world and the leading cause of disability in all industrialized countries. Stroke incidence is approximately one million per year in the European Union and survivors can suffer several neurological deficits or impairments, such as hemiparesis, communication disorders, cognitive deficits or disorders in visuo-spatial perception ,2.These impairments have an important impact in patient's life and considerable costs for health and social services . MoreoveHemiplegia is one of the most common impairments after stroke and contributes significantly to reduce gait performance. Although the majority of stroke patients achieve an independent gait, many do not reach a walking level that enable them to perform all their daily activities . Gait reTraditional approaches towards rehabilitation can be qualified as bottom-up approaches: they act on the distal physical level (bottom) aiming at influencing the neural system (top), being able to rehabilitate the patients due to the mechanisms of neural plasticity. How these mechanisms are established is still unkown, despite existing several hypotheses that lead to the description of several physical therapies. Recently some authors  argue abAn increasing number of researchers are pursuing a top-down approach, consisting on defining the rehabilitation therapies based on the state of the brain after stroke. This paper aims at providing an integrative view of the top-down approaches and their relationships with the traditional bottom-up in gait recovery after stroke. Besides, the article aim at examining how an integrative approach incorporating assistive robotic devices and brain-computer interfaces (BCI) can contribute to this new paradigm.According to the aim of this review, this document is organized as follows. First, we cover the neurophysiology of gait, focusing on the recent ideas on the relation among cortical brain stem and spinal centers for gait control. Then, we review classic gait rehabilitation techniques, including neurophysiological and motor learning approaches. Next, we present current methods that would be useful in a top-down approach. These are assistive robotic devices, functional electrical stimulation (FES), and non-invasive BCIs based on the electroencephalogram (EEG) and functional near infrared spectroscopy (fNIRS). Finally, we present our conclusions and future work towards a top-down approach for gait rehabilitation.Subsequently this paper is structured as follows: First there is an introduction to the physiology of gait. Then there is a review of current rehabilitation methodologies, with special emphasis to robotic devices as part of either a top-down or bottom-up approaches. Finally, we review the potential use of BCIs systems as key components for restructuring current rehabilitation approaches from bottom-up to top-down.Locomotion results from intricate dynamic interactions between a central program and feedback mechanisms. The central program relies fundamentally on a genetically determined spinal circuit capable of generating the basic locomotion pattern and on various descending pathways that can trigger, stop and steer locomotion. The feedback originates from muscles and skin afferents as well as some senses  that dynamically adapt the locomotion pattern to the requirements of the environment . For insIn post-stroke patients, the function of cerebral cortex becomes impaired, while that of the spinal cord is preserved. Hence, the ability to generate information of the spinal cord required for walking can be utilized through specific movements to reorganize the cortex for walking . The dysThe basic motor pattern for stepping is generated in the spinal cord, while fine control of walking involves various brain regions, including cerebral motor cortex, cerebellum, and brain stem . The spiRecent work has stressed the importance of peripheral sensory information  and descThe study of brain control over gait mechanisms has been hampered by the differences between humans and other mammals in the effects on gait of lesioning supraspinal motor centers. It is common knowledge that brain lesions profoundly affect gait in humans . TherefoThere is also increasing evidence that the motor cortex and possibly other descending input is critical for functional walking in humans: in adults the role of supraspinal centers on gait parameters has been studied mainly by magnetic or electric transcranial stimulation (TMS) ,22, by eSeveral research areas have provided indirect evidence of cortical involvement in human locomotion. Positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) have demonstrated that during rhythmic foot or leg movements the primary motor cortex is activated, consistent with expected somatotopy, and that during movement preparation and anticipation frontal and association areas are activated . FurtherAlexander et al. , by analIn closing, gait, as simple as it might seem, is the result of very complex interactions and not at all sustained by an independent automatic machine that can be simply turn off and on . The spiDespite the exact role of the motor cortex in control of gait is unclear, available evidence may be applied to gait rehabilitation of post-stroke patients.Restoring functions after stroke is a complex process involving spontaneous recovery and the effects of therapeutic interventions. In fact, some interaction between the stage of motor recovery and the therapeutic intervention must be noticed .The primary goals of people with stroke include being able to walk independently and to manage to perform daily activities . ConsistSeveral general principles underpin the process of stroke rehabilitation. Good rehabilitation outcome seems to be strongly associated with high degree of motivation and engagement of the patient and his/her family . SettingFurthermore, learning skills and theories of motor control are crucial for rehabilitation interventions. Motor adaptation and learning are two processes fundamental to flexibility of human motor control . AccordiAt present, gait rehabilitation is largely based on physical therapy interventions with robotic approach still only marginally employed. The different physical therapies all aim to improve functional ambulation mostly favouring over ground gait training. Beside the specific technique used all approaches require specifically designed preparatory exercises, physical therapist's observation and direct manipulation of the lower limbs position during gait over a regular surface, followed by assisted walking practice over ground.According to the theoretical principles of reference that have been the object of a Cochrane review in 2007 , neuroloThe neurophysiological knowledge of gait principles is the general framework of this group of theories. The physiotherapist supports the correct patient's movement patterns, acting as problem solver and decision maker so the patient beings a relatively passive recipient . Within ♦ Bobath  is the m♦ The Brunnström method  is also ♦ Proprioceptive neuromuscular facilitation (PNF) ,48 is wi♦ The Vojta method  has been♦ The Rood technique  focuses ♦ The Johnstone method  assumes Just opposite to the passive role of patients implied in neurophysiological techniques, motor learning approaches stress active patient involvement . Thus paThe motor learning approach has been applied by different authors to develop specific methodologies:♦ The Perfetti method  is widel♦ Carr and Shepherd in their motor relearning method  consider♦ Conductive education or Peto method  focuses ♦ The Affolter method  assumes ♦ Sensory integration or Ayres method  emphasisNeurorehabilitation principles and techniques have been developed to restore neuromotor function in general, aiming at the restoration of physiological movement patterns . NeverthSeveral systematic reviews have explored whether high-intensity therapy improves recovery -79. AlthAccording to these data, Salbach et al  suggesteConventional gait training does not restore a normal gait pattern in the majority of stroke patients . Robotici) precisely controllable assistance or resistance during movements, ii) good repeatability, iii) objective and quantifiable measures of subject performance, iv) increased training motivation through the use of interactive (bio)feedback. In addition, this approach reduces the amount of physical assistance required to walk reducing health care costs [These devices provide safe, intensive and task-oriented rehabilitation to people with mild to severe motor impairments after neurologic injury . In prinre costs ,91 and pre costs ,90,92.Because of robotic rehabilitation is intensive, repetitive and task-oriented, it is generally in accordance with the motor re-learning program ,63, moreThe efficacy of the human-robot interactions that promote learning depends on the actions either imposed or self-selected by the user. The applied strategies with available robotic trainers aim at promoting effort and self initiated movements. The control approaches are intended to i) allow a margin of error around a target path without providing assistance, ii) trigger the assistance in relation to the amount of exerted force or velocity, iii) enable a compliance at level of the joint and iv) detrend the robotic assistance by means of what has been proposed as a forgetting factor. In the former approach, the assumption is that the human resists applied forces by internally modelling the force and counteracting to it.Regarding current assistance strategies employed in robotic systems, the assist-as-needed control concept has emerged to encourage the active motion of the patient. In this concept, the goal of the robotic device is to either assist or correct the movements of the user. This approach is intended to manage simultaneous activation of efferent motor pathways and afferent sensory pathways during training. Current assist-as-needed strategies face one crucial challenge: the adequate definition of the desired limb trajectories regarding space and time the robot must generate to assist the user during the exercise. Supervised learning approaches that pre-determine reference trajectories have been proposed to this purpose. Assist-as-needed approach has been applied as control strategy for walking rehabilitation in order to adapt the robotic device to varying gait patterns and levels of support by means of implementing control of mechanical impedance. Zero-impedance control mode has been proposed to allow free movement of the segments. Such approach, referred to as "path control" has been proposed with the Lokomat orthosis,   resultinRegarding rehabilitation strategies, the most common robotic devices for gait restoration are based on task-specific repetitive movements which have been shown to improve muscular strength, movement coordination and locomotor retraining in neurological impaired patients ,96. RoboA fundamental aspect of these devices is hence the presence of an electromechanical system for the BWS that permits a greater number of steps within a training session than conventional therapy, in which body weight is manually supported by the therapists and/or a walker ,101. ThiHowever, some end-effector devices, such as the Gait Trainer, imposes the movements of the patient' feet, mainly in accordance to a bottom-up approach similar to the passive mobilizations of Bobath method  instead Several studies support that retraining gait with robotic devices leads to a more successful recovery of ambulation with respect to over ground walking speed and endurance, functional balance, lower-limb motor recovery and other important gait characteristics, such as symmetry, stride length and double stance time ,91,103.In these studies, BWS treadmill therapy has sometimes been associated, from a clinical point of view, to the robotic therapies, even if treadmill should not be considered as a robot for their substantial engineering differences. In fact, in a recent Cochrane, electromechanical devices were defined as any device with an electromechanical solution designed to assist stepping cycles by supporting body weight and automating the walking therapy process in patients after stroke, including any mechanical or computerized device designed to improve walking function and excluding only non-weight-bearing devices .Visintin et al [in et al  reportedLuft et al [ft et al  comparedMayr et al [yr et al  found moPeshkin et al [On the other hand, in et al  attempteAs most complex robots need to be permanently installed in a room, patients have to be moved from their beds to attend the rehabilitation. This is the main reason why therapy cannot be provided as soon as possible after stroke. In order to overcome this limitation, a robotic platform was developed by Monaco et al ,109 thatOn the other hand, robotic feedback training is an emerging but promising trend to constitute an active rehabilitation approach and novel methods to evaluate motor function. Forrester et al  tested tHuang et al  assessedHowever, other studies have provided conflicting results regarding the effectiveness of robotic devices for ambulatory and/or chronic patients with stroke ,115. A rMore recently, Morone et al  have proAt the light of all the above studies, the efficacy of each robotic device in neurorehabilitation seems to be related to a correct identification of the target population, in accordance with a generalization of the assist-as-needed strategy. Furthermore, it seems clear that a deeper knowledge about the proper selection of robotic devices, their training parameters and their effects on over ground walking performance for each patient can surely increase awareness of the potentialities of robotic devices for walking training in rehabilitation . It is hFunctional Electrical Stimulation (FES) is a useful methodology for the rehabilitation after stroke, along or as a part of a Neuro-robot .FES consists on delivering an electric current through electrodes to the muscles. The current elicits action potentials in the peripheral nerves of axonal branches and thus generates muscle contractions .FES has been used in rehabilitation of chronic hemiplegia since the 1960s.The firsts applications of FES in stroke recovery were focused on drop-foot correction, later researchers began to selectively stimulate the muscles for dorsiflexion of the foot as well as other key muscle groups in the affected leg .Stanic et al  found thBogataj et al  applied Later studies established the beneficial effects on the gait pattern of ambulatory patients, which, however, were likely to disappear after a few months .Kottink et al  performeOn the other hand there is strong evidence that FES combined with other gait retraining strategies results in improvements in hemiplegic gait, faster rehabilitation process and enhancement of the patients' endurance ,124,126.Lindquist et al  comparedMaple et al  attempteTong et al  reportedBoth robotic devices and FES can be controlled or triggered by biological signals recorded from the patient. For example, signals recorded from muscles  can provide information on the level of residual activation and on the neural control strategies. In these applications, the patient actively participates in intensive and repetitive task-oriented practice while task support (by robotic devices or FES) is triggered by residual myoelectric activity during volitional control. With respect to passive movements, it has been shown that motor learning is promoted by the use of residual EMG activity to trigger external devices assisting the movement . The ratSimilar mechanisms are supposed to be triggered by decoding the patient intention directly from the brain activity. This approach, which is referred to as brain-computer interfacing (BCI), requires more complex decoding methods than those based on muscular activities but provides a direct link with the neural circuitries activated during movement following the principles of a top-down approach.Brain-Computer Interface (BCI) systems record, decode, and translate some measurable neurophysiological signal into an effector action or behavior . TherefoBCIs establish a direct link between a brain and a computer without any use of peripheral nerves or muscles , therebyThere are several methods available to detect and measure brain signals: systems for recording electric fields  or magnetic fields , functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and functional near-infrared spectroscopy (fNIRS) ,140. AltIn the last decades, an increasing number of BCI research groups have focused on the development of augmentative communication and control technology for people with severe neuromuscular disorders, including those neurologically impaired due to stroke ,141,142.Daly et al.  explaine• Better understanding of the characteristics and possible uses of brain signals.• The widely recognition of activity-dependent plasticity throughout the CNS and its influence on functional outcomes of the patient.• The growth of a wide range of powerful low-cost hardware and software programs for recording and analyzing brain signals during real-time activities.• The enhancement of the incidence and consideration of the people with severe motor disabilities.One of the most popular neurophysiological phenomena used in BCI research is modulation of sensorimotor rhythms through motor imagery (MI) . ImaginaMental simulation of movement, engages the primary motor cortex in a similar way that motor execution does . Motor iIn particular, for motor recovery after stroke, MI has been extensively exploited to promote neuroplasticity in combination with traditional physiotherapy and robot-aided therapy . For exaIn an early report on BCI control by stroke patients, Birbaumer et al.  reportedAfter this report, other research groups presented reports on future prospects of BCIs and the role of BCIs in neurological rehabilitation.Buch et al.  reportedRecently, Broetz et al. ,165 repoAccording to the literature, MEG and fMRI are better at locating stroke lesions and the neural networks involved in MI, thus, making those techniques the best choice for assessing changes in the motor activity that could foster and improve motor function ,166-169.Nowadays, there are only a few reports of Electroencephalograpy (EEG)-based BCIs for rehabilitation in stroke patients. The major part of these reports for stroke recovery focus on the rehabilitation of upper limbs, specifically of hand movements. Moreover, most of these reports focus on BCI performance of stroke patients and only a few of them have shown a real effect of BCI usage on motor recovery. Ang et al.  presenteIn contrast, Platz et al.  found a Daly et al.  presentePrasad et al. ,175 presTan et al.  reportedThere is enough evidence to support the assumption that BCIs could improve motor recovery, but there are no long term and group studies that show a clear clinical relevance.There is also evidence that MI of lower limbs, e.g. dancing or foot sequences, helps to improve gait ,178 and Functional near infrared spectroscopy (fNIRS) is a non-invasive psycho-physiological technique that utilizes light in the near infrared range (700 to 1000 nm) to determine cerebral oxygenation, blood flow, and metabolic status of localized regions of the brain. The degree of increase in regional cerebral blood flow (rCBF) exceeds that of increases in regional cerebral oxygen metabolic rate (rCMRO2) resulting in a decrease in deoxygenated haemoglobin (deoxyHb) in venous blood. Thus, increase in total haemoglobin and oxygenated haemoglobin (oxyHb) with a decrease in deoxygenated haemoglobin is expected to be observed in activated brain areas during fNIRS measurement. fNIRS uses multiple pairs or channels of light sources and light detectors operating at two or more discrete wavelengths. The light source is usually a light emitting diode. Three techniques are available for NIRS signal acquisition, continuous-wave spectroscopy, time-resolved spectroscopy and frequency-domain techniques . ContinuThe favorable properties of the fNIRS approach are its simplicity, flexibility and high signal to noise ratio. fNIRS provides spatially specific signals at high temporal resolution and it is portable and less expensive than fMRI. Human participants can be examined under normal conditions such as sitting in a chair, without their motion being severely restricted. However, the depth of brain tissue which can be measured is only 1-3 cm, restricting its applications to the cerebral cortex. With exciting developments in portable fNIRS instruments incorporating wireless telemetry , it is nAs this paper focuses on rehabilitation of gait after stroke, the next sections will analyze the literature regarding gait performance using fNIRS and its application in stroke rehabilitation.Increasing evidence indicates that fNIRS is a valuable tool for monitoring motor brain functions in healthy subjects and patients. Less sensitivity of fNIRS to motion artifacts allows the experimenters to measure cortical hemodynamic activity in humans during dynamic tasks such as gait.Miyai and colleagues  recordedA study from Suzuki et al  exploredSimilarly, Mihara and colleagues  reportedA recent fNIRS study  showed tAltogether the studies on healthy participants reported an association between the PFC, SMA and SMC and control of gait speed. Moreover, the involvement of the left PFC might depend on an age-related decline in gait capacity in the elderly .Thus far, few studies utilized fNIRS to assess cortical activation patterns in stroke patients. Cortical activation during hemiplegic gait was assessed in six non-ambulatory patients with severe stroke, using an fNIRS imaging system . PatientFurthermore, Mihara and colleagues  comparedOverall, these studies demonstrate the suitability of fNIRS for detecting brain activity during normal and impaired locomotion and subsequently as being part of a top-down strategy for rehabilitation.Coyle et al.  and SitaSitaram et al  reportedIn the study by Coyle and Ward et al.  a BCI syRecently, several studies reported fNIRS based BCI implementations -201. SitWith the objective of developing a specific fNIRS-BCI for rehabilitation of patients with lower limbs impairment, Rea and colleagues  assessedOverall these findings indicate that despite the inherent latency of the hemodynamic response fNIRS provides researchers with an excellent opportunity to use motor activities to detect signals from specific regions of the cortex for the development of future BCIs.After stroke, gait recovery is a major objective in the rehabilitation program, therefore a wide range of strategies and assistive devices have been developed for this purpose. However, estimating rehabilitation effects on motor recovery is complex, due to the interaction of spontaneous recovery, whose mechanisms are still under investigation, and therapy.The approaches used in gait rehabilitation after stroke include neurophysiological and motor learning techniques, robotic devices, FES, and BCIs.Despite being successful, the main principles of current rehabilitative approaches remain unclear , and proThese approaches have been studied and compared by many researchers and, although there is not full consensus, the following general conclusions can be drawn:• Regarding neurophysiological and motor learning techniques, there is insufficient evidence to state that one approach is more effective in promoting gait recovery after stroke than any other approach. Furthermore, none of the methods is specifically focused on gait rehabilitation ,50.• There is moderate evidence of improvement in walking and motor recovery using robotic devices including systems for BWSTT when compared to conventional therapy ,105-107.• The combination of different rehabilitation strategies seems to be more effective than over-ground gait training alone.• Robotic devices need further research to show their suitability for walking training and their effects on over-ground gait .• The use of FES combined with different walking retraining strategies has been shown to result in improvements in hemiplegic gait ,207,208.• Reports on EEG-based BCIs for stroke recovery are limited to the rehabilitation of upper limbs, specifically of hand movements. Moreover, only a few of them have shown a real effect of BCI usage on motor recovery ,176,209.• There is enough evidence to support the assumption that BCIs could improve motor recovery after stroke, but there are no long term and group studies that show a clear clinical relevance.• Lower limbs and gait function have not been studied in combination with BCIs yet. However some works suggest that there is a common mechanism influencing upper and lower limb recovery simultaneously, independently of the limb chosen for the rehabilitation therapy ,181.• Despite the inherent latency of the hemodynamic response fNIRS enables researchers to detect signals from specific regions of the cortex during the performance of motor activities for the development of future BCIs -190,195.• Future research would make possible the analysis of the impact of rehabilitation on brain plasticity in order to adapt treatment resources to meet the needs of each patient and optimize the recovery process.The authors declare that they have no competing interests.JB structured the paper contents and made the revision of the whole article. SM wrote the draft of the manuscript and revised the integration of the different sections. IB provided the framework for the introduction. JM contributed to the robotic devices section, especially exoskeletons and JP revised it and looked for the consistency of this part in the whole paper. DF revised the paper for consistency in the neurophysiological aspects. MM and FT wrote the neurophysiology of gait and classic gait rehabilitation techniques sections. MI revised the manuscript for consistency in the gait rehabilitation approach. AR and AC wrote about fNIRS based BCIs and MR revised it. TS and CB wrote and revised the section of EEG based BCIs. All authors read and approved the final manuscript.


To investigate associations between cancer survivors’ sex, age, and diagnosis in relation to their (1) need for rehabilitation, (2) participation in rehabilitation activities, and (3) unmet needs for rehabilitation in a 14-month period following date of diagnosis.A population-based cohort study was performed on incident cancer patients diagnosed from 1 October 2007 to 30 September 2008. Fourteen months after diagnosis, participants completed a questionnaire developed to measure the aspects of rehabilitation. Logistic regression analyses were used to explore the association between sex, age, and diagnosis, and the outcome variables for rehabilitation.A total of 3,439 patients participated, yielding an overall response rate of 70%. One third of the cancer patients reported a need for physical rehabilitation and one third for psychological rehabilitation. Half of the patients participated in at least one activity. Unmet needs were most often reported in psychological, sexual, and financial areas. Women expressed more needs, participated more often in rehabilitation activities, and had, to a higher extent, their emotional needs fulfilled. Breast cancer patients participated more often in physical rehabilitation. Elderly who expressed rehabilitation needs more often had them unresolved.A substantial variation in rehabilitation needs, participation in activities, and unmet needs in relation to sex, age, and cancer type was observed. Cancer care ought to systematically address the wide range of needs in all groups through integration of systematic needs assessment and targeted supply of offers. Cancer survivors experience physical, psychological, work-related, and financial challenges and are potentially in need of individual and targeted rehabilitation , 2. The n = 147) found that 26% of the patients indicated a need for rehabilitation [n = 1,325) observed that 63% of the cancer patients reported a need for at least one rehabilitation service [There is little knowledge about the overall number of cancer patients in need of rehabilitation efforts at different time points in the cancer trajectory. A Dutch cross-sectional study of patients with breast and bowel cancer n = 17 found tn = 132 resp. n = 731) have assessed cancer patients’ actual utilization of rehabilitation activities, and typically, a third of the patients participated in an activity [Besides needs, aspects of rehabilitation can also be illustrated through participation in rehabilitation and unmet needs. Two studies including only one or a few types of cancers  need for rehabilitation, (2) participation in rehabilitation activities, and (3) unmet needs for rehabilitation in a 14-month period following date of diagnosis.We performed a population-based cohort study including incident cancer patients, except patients with non-melanoma skin cancer, diagnosed from 1 October 2007 to 30 September 2008 in the Regions of Southern and Central Denmark (2.4 million residents) by obtaining information from hospital-based and national administrative registers. Information about rehabilitation issues was obtained from a patient questionnaire administered 14 months after diagnosis.The Danish health care system is primarily a publicly funded system . More thCancer patients may participate in rehabilitation activities outside the public healthcare system and the municipalities. The Danish Cancer Society is a private patient organization offering patient support and counseling free of charge at counseling centers all over the country . FurtherThe study population was defined by the following means:All residents of Denmark are assigned a unique ten-digit personal identification number, which includes information on date of birth and sex. This Personal Identification Number (CPR) is the key variable in linkages between public, health, and disease registries in the country .From the regional hospitals’ Patient Administrative System , we obtaFollowing identification by the administrative sampling procedure, each patient’s GP was mailed a questionnaire to confirm that a cancer was diagnosed.This cohort of incident cancer patients was established for the use in several research projects, and 6 months following date of diagnosis, patients were mailed a questionnaire, which included a request for them to confirm that they had cancer for the first time and giving them the possibility of declining the use of the information given by their GP. Prior to the distribution of the 14-month patient questionnaires, vital status and postal address were confirmed by linkage to the Civil Registration System , which iThe patient questionnaire comprised 171 items and was designed to give information about various aspects of cancer rehabilitation , includiThe questionnaire was pilot-tested and revised in a three-step procedure. Researchers active in the field of cancer rehabilitation were asked to comment on content, layout, volume, and intelligibility of the draft. Subsequently, ten cancer patients were asked to fill in the questionnaire, and in a semi-structured interview with the first author, these patients provided comments on content, layout, volume, and intelligibility. Based on all these pilot activities, questions were revised and a new draft was completed. The last pilot study included 100 cancer patients, who were asked to fill in a mailed version, enabling us to examine discrimination and acceptability and make the final corrections. The overall participation rate in this pilot study was 75%.The rehabilitation variables covered self-assessed perceived need for rehabilitation, participation in rehabilitation activities, and unmet rehabilitation needs from time of cancer diagnosis until the day of filling in the questionnaire.“Need for rehabilitation during the 14-month period” and “unmet rehabilitation after 14 months” were asked for thematically, i.e., “physical,” “emotional,” “family oriented,” “sexual,” “work-related,” and “financial” areas.To what extent from diagnosis and until now have you needed professional help with physical problems? Until now, to what extent have you had your needs fulfilled in terms of help with physical problems? As an example, the following questions were asked with regard to physical needs:Similar questions were asked for all areas. Patients were categorized as having an “unmet need” if they had expressed a “need for rehabilitation” during the 14-month period, and the need, to some extent, was not fulfilled after 14 months.3.Have you from diagnosis and until now participated in any of the following activities due to problems caused by your cancer disease? (listing of possible providers/activities)“Participation in rehabilitation activities” was assessed by asking:Three categories of activities were defined based on profession of the provider/activity: (1) “Physical activities” , (2) “Psychological activities” , and (3) “work-related/financial activities” . Furthermore, the variable “Participation in one or more activities” was defined based on the above-mentioned categories.Need for rehabilitation was dichotomized into “no need”  and “need” , and similarly, unmet need was dichotomized into “unmet need” and “met need.” Answers in the “not relevant” category were excluded from the analyses. For analyses regarding “unmet rehabilitation needs after 14 months,” only patients expressing a “need for rehabilitation” were included.p < 0.05 was considered statistically significant. Adjusted odds ratios (ORadj) are presented with 95% confidence intervals (95% CI). Analyses were performed using Stata Release 11 .Univariate and multiple logistic regression analyses were used to explore the association between sex, age, and diagnosis, and the rehabilitation variables. In presence of an interaction between age group and sex, the analyses were stratified on sex. All tests were two-sided, and Of the 4,947 subjects eligible at 14 months, 3,439 returned the questionnaire (70%)  and least common for physical problems (17%) . The two most pronounced differences were that of the primarily included patients 19% had breast cancer compared with 16% in the full sample. This could have slightly overestimated our results regarding need for and participation in rehabilitation. Secondly, an under-sampling of the oldest patients (+80 years) was seen compared with the full sample (13% vs. 17%). This could have underestimated the absolute figures for unmet rehabilitation needs. However, as we calculated the relative associations and adjusted for differences in sex, age, and cancer type, this selection bias can be regarded as negligible.The response rate was high among the patients who received the questionnaire. Among responders, there was a slight overrepresentation of women, the 60–69-year-olds and patients with breast cancer Table . Hence, Participants were asked about needs and activities for a 14-month period, but the cancer disease and cancer-related activities are likely to be of major importance to most patients and therefore remembered for a long time. Hence, recall bias is supposedly low.An important question is whether our results are generalizable. In Denmark, there are relatively small regional differences with respect to organization of the health care system and prevalence of diseases. Therefore, we assume that our results are generalizable to all of Denmark. Furthermore, our findings that patient characteristics were associated with needs, participation, and unmet needs are likely to be generalizable to other countries with similar health care systems. It must be kept in mind that the results represent cancer patients who survived for more than 1 year and therefore may not apply to short-time survivors.n = 1,325) showed that need for physical rehabilitation followed by psychological rehabilitation was the most frequent [Similar to our results, a Norwegian study including the ten most frequent cancer types n = 1,32 showed tfrequent , and a mOur results showed that, compared with men, women, to a higher extent, expressed needs and participated in rehabilitation. This could reflect that women to a higher extent have a need for rehabilitation, more often articulate a need for help, or that current rehabilitation offers appeal more to women, i.e., activities match female demands and values. A cross-sectional study of 1,876 Danish cancer survivors participating in a 1-week residential rehabilitation course, offered to all cancer patients, support this explanation, as 85% of the participants were women . In anotOnly a few smaller cross-sectional studies have assessed utilization of rehabilitation activities among cancer patients , 8. As tn = 1,490) found that half of the patients who needed psychological counseling did not receive it [A Danish survey including cancer patients at various sites n = 1,40 found tceive it , 30, 31.In conclusion, one third of the total group of cancer patients reported a need for physical rehabilitation and in one third, a need for psychological rehabilitation. Half of the patients participated in one or more rehabilitation activities. Unmet needs were most often reported in the psychological, sexual, and financial areas. We observed a substantial variation in these matters pertinent to disease and patient characteristics. This study suggests that cancer care ought to systematically address the wide range of needs in all groups through integration of systematic needs assessment and targeted supply of offers. Emphasis should be put on development of assessment and monitoring tools for use in everyday clinical practice.


Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β Picornaviridae family of positive single stranded RNA viruses and is implicated in a variety of respiratory disorders ranging from the common cold to the induction of exacerbations of respiratory diseases. Of the respiratory viruses that cause asthma exacerbations, RV accounts for about two thirds of all viral-induced asthma exacerbations 2 agonists are the most common therapies for asthma management and, during acute exacerbations, including those caused by respiratory viruses, β2 agonists are a commonly used rescue medication Acute exacerbations of asthma are the major cause of morbidity, mortality and health costs related to the disease. Respiratory viral infections trigger approximately 85% of asthma exacerbation in adults and children and the mechanisms by which this occurs remain unclear 2 agonists, however there have been reports that airway obstruction does not improve with β2 agonists during virally induced asthma exacerbations 2 agonists despite having good asthma control prior to infection, and a good response to β2 agonists prior to achieving good asthma control 2 agonist therapy in response to a viral-induced exacerbation in which RV was the most frequently identified virus 2 agonists during these exacerbations of asthma may be unique to a viral infection. The exact causes of exacerbations of asthma are unknown, however it possible that functional impairment of the β2 adrenoceptor (β2 AR) may disrupt intrinsic bronchodilation through circulating epinephrine and thus result in airflow limitation characteristic of an exacerbation.Under normal circumstances, airway obstruction in asthma improves in response to inhaled βIn vivo, the epithelial cells form a physiological barrier in the airways and are the principal cell type infected by RV in the lower airways 2 agonist therapy may be due to desensitization of the β2 AR on airway smooth muscle cells (ASMCs) in vitro model to show that RV infection of epithelial cells produces a conditioned medium, containing unknown substances, that when applied to ASMCs, causes internalisation of the β2 AR, and results in reduced generation of cyclic adenosine monophosphate (cAMP) in response to a β2 agonist in vitro phenomenon may translate to the possible reason why asthmatic patients with RV-induced asthma exacerbations do not respond to β2 agonists clinically, however the mechanism by which it occurs, or the identity of the RV-induced epithelial derived substance remains unknown.Previous research has suggested that clinical impairment to β2 can cause ASMC relaxation by the induction of cAMP 2 can cause heterologous desensitization of the β2 AR by either activation of common G protein coupled receptor (GPCR) kinases, for example GPCR that share common Gs alpha subunits, or alternatively by direct activation of protein kinase A (PKA) 4 causes ASMC contraction by activating cystLT1 receptors which are coupled to Gq alpha subunit proteins and activate the protein kinase C (PKC) pathway which ultimately increases intracellular calcium. Surprisingly, LTD4 can also cause desensitization of the β2 AR Eicosanoids are lipid mediators which incorporate the two large families of prostaglandins and leukotrienes, and their levels are increased in asthma and during clinical RV infections 2 AR desensitization in ASMCs were investigated using an in vitro epithelial-ASM β2 AR model using human primary bronchial epithelial cells (HBEC) and human primary ASMCs. In the airways, fibroblasts are structural support cells that lie in close proximity to the epithelium and smooth muscle, and during RV infection could contribute to RV induced β2 AR desensitization by their release of mediators such as prostaglandins. For this reason, the ability of airway structural cells including ASMCs, epithelial cells and lung fibroblasts to produce prostaglandins in response to RV infection and the ability of prostaglandins to cause β2 AR desensitization in ASMCs were also investigated. We found that RV infection of epithelial cells resulted in an increase in viral RNA which could activate toll-like receptors (TLRs) on the ASMC. This resulted in the generation of COX-2 induced prostaglandins from ASMCs which in turn caused β2 AR desensitization. Since RV infection of various airway structural cells also produces prostaglandins, this could further contribute to β2 AR desensitization.In this study, the identity of the unknown substance/s responsible for or the mechanism that causes βThe study was approved by the Ethics Review Committee of the Sydney South West Area Health Service, Royal Prince Alfred Hospital and The University of Sydney human research ethics committee. All volunteers provided written informed consent.Primary HBEC, ASMC and parenchymal fibroblasts were isolated from macroscopically normal tissue of patients undergoing lung resection for thoracic carcinoma or transplantation for end stage lung disease. All patient details are shown in 2 flasks with bronchial epithelial growth medium (BEGM) . In doing so this exposes the underlying bands of smooth muscle, which were then gently separated from the underlying connective tissue in small bundles (smooth muscle cell explants). The explants were transferred into 25 cm2 flasks and covered with a minimal amount of Dulbecco’s Modified Eagle Medium (DMEM) containing 1% antibiotics: 20 U/L of penicillin, 20 µg/mL of streptomycin, 2.5 µg/mL of amphotericin B, and 10% Fetal Bovine Serum (FBS) . Fibroblasts were isolated from resected lung tissue by seeding 1–2 mm3 pieces of parenchymal tissue into DMEM supplemented with 10% FBS and 1% antibiotics in 75 mm2 flasks. Both cell types were incubated at 37°C in a humidified atmosphere of 5% CO2-95% air.HBEC and ASMC were obtained from human bronchial airways and fibroblasts from lung parenchyma by methods previously described The medium was replenished every 5 days for the first 10–20 days, and within this time, cell growth occurred. The cells were passaged (split 1∶3) with a solution of trypsin [0.05% wt/vol in HBSS] containing 1 mM EDTA. The cells were maintained in DMEM supplemented with 1% antibiotics in 10% FBS whilst HBEC were maintained in BEGM. All cells were tested for mycoplasma contamination and only mycoplasma free cells were selected and passaged once they reached confluence. ASMCs and fibroblasts at passages 3–7 and HBEC at passages 2–3 were used for experiments.µL of sample/well at a distance of 5 cm from a 30 W UV light source  for 15 minutes. Successful UV inactivation of RV was established by a virus titration and was used as a non-infectious virus control.Human RV serotype-16 were propagated in Ohio HeLa cells and purified using a 100,000 kDa molecular weight cut off (MWCO) filter as previously described 4 cells/mL in BEGM, while fibroblasts and ASMCs were seeded at 3.2×104 cells/mL in 10%FBS/DMEM in 6 well plates for 72 hours. The total number of cells after 72 hours was assessed and wells were then either left uninfected (control) or exposed to UV inactivated RV or live RV at a multiplicity of infection (MOI) of 1. The cells were placed on a shaker (100 rpm) for 15 minutes at room temperature before being incubated at 37°C at 5% CO2. After 1 hour the cells were washed with HBSS and replaced with fresh BEGM or 10%FBS DMEM before being incubated at 37°C in a humidified atmosphere of 5% CO2-95% air. After 24 hours, the supernatant was purged with nitrogen, collected and stored at −80°C for ELISA and the number of remaining viable cells was estimated for each well using trypan blue exclusion and manual cell counting to normalize prostaglandin release to cell number.HBEC were seeded at a concentration of 6.4×102, PGD2, PGF2α, PGI2 metabolite 6ketoPGF1α, CystLT and LTB4 ELISAs were purchased from Cayman Chemicals, Michigan, USA. The assays were carried out according to the manufacturer’s instructions. The detection range for each assay was PGE2 7–1000 pg/mL; PGD2 78–5000 pg/mL; PGF2α 3–500 pg/mL; 6ketoPGF1α 1–1000 pg/mL; CystLT 7–1000 pg/mL and LTB4 3–500 pg/mL. Prostaglandin ELISA results were normalised to cell number and expressed as concentration per 1×106cells.PGEELISA kits for IL-6 and IL-8 were purchased from R&D Systems  and BD Biosciences  respectively. ELISAs were carried out according to the manufacturers’ instructions. The detection limits of these assays were: 7–1000 pg/mL (IL-6) and 15–2000 pg/mL (IL-8).2, E2, F2α, the PGI2 analogues: MRE-269 (selective) and Beraprost (non-selective) – both of which are being used in clinical trials for the treatment of pulmonary hypertension, the selective COX-2 inhibitor celecoxib , indomethacin and 3-isobutyl-1-methylxanthine (IBMX) were dissolved in dimethyl sulfoxide (DMSO)  and stored at −80°C prior to use. Isoprenaline (Sigma-Aldrich) was dissolved in water before use. The protease trypsin was dissolved in Hank’s balanced salt solution (HBSS) (both from Invitrogen) prior to use and to inhibit its digestive activity during experimentation 0.1% bovine serum albumin (BSA) (Invitrogen) was used. The TLR 3 agonist polyinosinic : polycytidylic acid (Poly I:C) (Sigma-Aldrich) and TLR 7/8 agonist imiquimod  were dissolved in DMSO and H2O respectively and stored at −20°C. The pro-opiomelanocortin (POMC) protein was purchased ready to use from ABCAM  and the 1.2 kDa “substance X” peptide was synthesized by Auspep  and dissolved in H2O. Both protein and peptide were stored at −80°C before use. The prostaglandin receptor antagonists AH6809 ; CAY10441 (IP); AL8810 (FP); BWA868C (DP2); L-161,982 (EP4) were purchased from Cayman Chemicals and dissolved in DMSO and stored at −80°C prior to use. In experiments where a single or mix of prostaglandin receptor antagonists or vehicle were used, the final concentrations and formulation for experimentation were chosen according to its use in previous studies to successfully inhibit their specified receptor and they consisted of AH6809 (10−5 M) −6 M) −5 M) −5 M) −6 M) Prostaglandins (PG) DIn some experiments, cell viability was assessed by means of measuring mitochondrial activity using the 3--2,5-diphenyltetrazolium (MTT) (Sigma) assay as previously described 4 cells/mL in 75 cm2 culture flasks with 10 mL of BEGM until >90% confluent. Briefly, RV or UVi-RV (MOI = 2) or BEGM was added to the confluent cell monolayers for 1 hour with orbital shaking at 37°C. The cells were then washed with HBSS and fresh BEGM was added and the cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 24 hours. In experiments with the COX inhibitor indomethacin, infection occurred in the presence of the drug at 1×10−5 M in BEGM and replacement BEGM also contained the drug at 1×10−5 M. Control, UVi-RV and RV generated conditioned medium was collected 1 day after infection. All conditioned medium  were UV treated by placing 250 µL of conditioned medium into each well of a 12-well tissue culture plate at a distance of 5 cm from a UV lamp for 5 minutes to ensure that all RV was inactivated before being applied undiluted on primary human ASMCs to assess β2 AR function.Primary HBEC were seeded at a concentration of 5×102 AR desensitization, treatments were applied in BEGM or control conditioned medium. In experiments utilizing prostaglandin receptor antagonists to determine the prostaglandin responsible for β2 AR desensitization, ASMCs were pretreated with the antagonist 1 hour prior and during the 3 day incubation period with conditioned medium. β2 AR function on ASMCs was assessed by the functional cAMP assay and where appropriate ASMC viability was assessed using an MTT assay.Primary human ASMCs were incubated with control, UVi-RV and RV conditioned medium for 3 days. In experiments assessing whether prostaglandins, TLR agonists or total extracted RNA from conditioned medium can cause βEvidence of epithelial RV infection was assessed by the measurement of RV induced IL-6 by ELISA. The induction of proteins present in RV conditioned medium was assessed using the Quantipro BCA assay kit (Sigma-Aldrich) and 1-D protein gel electrophoresis with silver staining.2 AR function cells were stimulated with the β agonist isoprenaline (10−7 M) for 5 minutes in the presence of the phosphodiesterase inhibitor IBMX (10−5 M) in HBSS. The cells were then lysed in a solution of H2O with 0.03% (v/v) Tween-20 and 5 mM HEPES buffer by vigorous pipetting. The amount of isoprenaline induced cAMP in the lysate samples was quantified using an Alphascreen cAMP Assay Kit  according to the manufacturer’s instructions.To assess βProtein concentration in conditioned medium was determined using the bicinchoninic acid (BCA) assay kit according to the manufacturer’s instruction.Conditioned medium was prepared as follows for gel electrophoresis: 5 µL of loading buffer (2% sodium dodecyl sulphate (SDS), 7.5% glycerol, 31.25 mM Tris-HCl (pH 6.8), 0.0025% bromophenol blue, 200 mM DTT  was added to 25 µL of conditioned medium and denatured at 95°C for 5 minutes. Five µL of Precision plus Protein Dual Xtra Molecular Standard ladder  was added in a separate lane of each gel to indicate the size of the visualised bands, while 20 µL of prepared protein samples was added to each separate lane.20) and separated by SDS-polyacrylamide electrophoresis on a 10% polyacrylamide gel (25% (v/v) SDS Tris pH 8.8; 25% (v/v) 37.5∶1 acrylamide; 0.1% (v/v) tetramethylethylenediamine; 0.1% (v/v) ammonium persulfate; in Milli-Q H20) at 150 V for 90 minutes before they were prepared for silver staining. Gels were visualized and analysed using an IS4000MM Kodak imaging system and software .Twenty µL of protein samples were loaded onto a 4% polyacrylamide stacking gel (25% (v/v) SDS Tris pH 6.8; 10% (v/v) 37.5∶1 acrylamide (Bio-Rad); 0.1% (v/v) tetramethylethylenediamine (Sigma-Aldrich); 0.1% (v/v) ammonium persulfate (Sigma-Aldrich); in Milli-Q H2O) for 30 minutes on an orbital shaker. The fixative solution was then removed and three washes of 50% ethanol in Milli-Q H2O were carried out each for 20 minute intervals. The gels were then washed three times with Milli-Q H2O and incubated with 20% w/v thiosulfate solution for 1 minute then washed three times again with Milli-Q H2O. The gels were incubated in 0.2% w/v silver nitrate in the dark for 20 minutes before being washed twice in Milli-Q H2O and then incubated in developer solution (6% (w/v) Na2CO3, 0.05% (v/v) 37% formaldehyde, 2% (v/v) of thiosulfate solution as described above, in Milli-Q H2O) until the bands were clear. The development was stopped by three washes in Milli-Q H2O and the bands were captured using the Kodak imaging system.After protein electrophoresis, the gels were transferred into a 50 mL fixative solution (50% (v/v) methanol, 12% (v/v) glacial acetic acid, 0.05% (v/v) 37% formaldehyde (Sigma-Aldrich), Milli-Q H2 AR desensitization, large proteins were digested using the protease trypsin and the conditioned medium was reassessed for its ability to induce β2 AR desensitization on ASMCs.In order to assess whether RV-induced epithelial derived proteins in the conditioned medium may be responsible for causing βInitial experiments involved the optimization of multiple variables including the amount of protease required for digestion, time for sufficient digestion of conditioned medium proteins and the appropriate concentration of inhibitor substance to avoid cytotoxic effects on ASMCs. Protein gel electrophoresis followed by silver staining of digested products validated the digestion process, while MTT assays were used to validate the safety of optimized concentrations of protease and time of digestion on ASMC viability over 3 days. As the protein concentration in conditioned medium varied from batch to batch, control and RV conditioned medium from multiple batches were pooled in these experiments.2 AR desensitization.Since endogenous proteases are necessary for homeostatic cell function, the use of protease inhibitors to stop the exogenous protease digestion may affect ASMC function and viability. Therefore since trypsin digestion can be safely stopped using saturation with BSA instead of the use of protease inhibitors, trypsin instead of other proteases was selected for enzymatic digestion of the conditioned medium to assess β2 AR desensitization using a functional cAMP assay.Briefly, 3–4 batches of control and RV conditioned medium were pooled to a total of 4–6 mL and trypsin was added (500 µg/mL) and the mixture was allowed to incubate in a water bath at 37°C for 24 hours. After the digestion, 0.1% BSA was added to the mixture to saturate and stop the enzymatic digestion. Since the presence of trypsin and BSA may affect the outcome of results, trypsin (>20 kDa) was also inactivated by removal using the Amicon ultra-15 centrifugal filtration unit  with 3 kDa molecular weight cut off (MWCO). The undiluted trypsin digested conditioned medium was applied to ASMCs to assess β2 columns filled with 1 mL of PGE2 affinity sorbent  were purchased from Cayman Chemicals. PGE2 purification and extraction was carried out according to the manufacturer’s instruction. Briefly, the column was first washed with 0.1 M phosphate buffer solution, and then 4 mL of pooled control or RV conditioned medium was loaded onto the column. The flow-through product free of PGE2 (confirmed by PGE2 ELISA) was collected for experimentation. The column was then washed twice with 2 mL of distilled water, and a 2 mL solution containing 95% absolute ethanol and 5% distilled water was used to elute the column bound PGE2. The eluted solution was completely evaporated by vacuum centrifugation for 2 hours at 37°C and purified PGE2 was reconstituted in 1 mL of BEGM for experimentation. Depletion of PGE2 was confirmed by ELISA.PGE2 AR desensitization.Amicon ultra-15 centrifugal filtration units of 100, 50, 30, 10, 3 kDa MWCO filters were purchased from Millipore. Initially, 15 mls of control or RV conditioned medium was pooled and loaded into the initial 100 kDa MWCO filter unit and centrifuged at 4000×g for 30 minutes. One mL of the flow-through product was collected for experimentation and the remainder was loaded into the 50 kDa filter unit and centrifuged as before. The sequential filtration, fractionation and sampling were carried out from the 100 kDa to the 3 kDa MWCO filter unit. Flow-through fractions were used to treat ASMCs to assess the relationship between the size fraction and its ability to cause βet alIn order to identify whether there was a difference in the peptide profiles between the control and RV conditioned medium that were in the <3 kDa fraction, mass spectrometry (MS) was carried out as previously described by Ly Peak lists were generated using Analyst QS 2.0 and were searched using Mascot v2.3  against the LudwigNR 2012 Q1 database. The following criteria were used: precursor and product ion tolerances 0.2 Da; variable modifications of methionine oxidation; trypsin and one allowed missed cleavage specified; and decoy search enabled. Similar searches were performed but with no enzyme specified.Total RNA including micro RNA was purified using the miRNeasy Mini purification kit  as per the manufacturer’s instructions. Total RNA concentration and quality was determined using a spectrophotometer (Nanodrop ND-1000).P value of less than or equal to 0.05 was considered statistically significant.All data were checked for normal distribution and when the results were not parametrically distributed, the dataset was log transformed prior to statistical analysis using GraphPad Prism Version 5 . Results were analysed by paired Student’s t-tests and 1 or 2-way analysis of variance (ANOVA) where appropriate and Bonferroni post test comparison with corresponding controls. In all cases, a 2 AR desensitization , UVi-RV or replication competent RV. Since RV infection of HBECs induces IL-6, measurement of IL-6 induction was used as a positive control marker to indicate viral infection  p<0.05, . RV-indu p>0.05, .2 AR desensitization was investigated by size fractionation of the conditioned medium. ASMCs treated with RV-induced conditioned medium that was either not fractioned or was size fractioned through 100, 50, 30, 10 or 3 kDa molecular weight cut off (MWCO) filters, all decreased isoprenaline induced cAMP compared to the control conditioned medium from the corresponding fraction  and IL-8 (8 kDa)  and furt2 AR desensitization was not trypsin digestible and had a molecular weight of <3 kDa.ASMCs treated with RV-induced conditioned medium that was trypsin digested and fractionated through the 3 kDa filter to remove trypsin still caused decreased isoprenaline induced cAMP compared to their respective controls p<0.05, . RV-indu2, PGF2α, PGD2 and the PGI2 metabolite 6ketoPGF1α from all cell types when compared to basal control levels , caused a concentration dependent decrease in isoprenaline induced cAMP compared to the respective vehicle controls  caused a concentration dependent decrease in isoprenaline-induced cAMP compared to the respective vehicle controls , however this effect was absent when ASMCs was treated with indomethacin, celecoxib or a combination of prostaglandin receptor antagonists in the presence of RV-induced conditioned medium .A comparative proteomic analysis of uninfected, UVi-RV and RV-induced conditioned medium resulted in the identification of a 1.2 kDa peptide with an 11 amino acid sequence “GDEQPLTENPR” that was present only in the RV-induced conditioned medium. The peptide is a fragment of the pro-opiomelanocortin (POMC) protein, which is a polypeptide that is cleaved to give rise to a super family of neuro-peptides. The 1.2 kDa peptide was synthesized and along with POMC, did not influence ASMC viability, PGE2 AR desensitization on ASMCs.Toll-like receptors (TLR) are pathogen pattern recognition receptors that are present on ASMCs 2 and β2 AR desensitization on ASMCs were investigated.When conditioned medium generated from HBEC (n = 3) was pooled, the total RNA measured in RV-induced conditioned medium (300.25 ng/µL) was almost 6 times higher than RNA in control conditioned medium (53.91 ng/µL). RV infection produces both ssRNA and dsRNA during replication and whether activation of TLRs 3 and 7/8 using synthetic analogues can result in induction of PGE2 from ASMCs compared to BEGM alone (p<0.05) and did not alter isoprenaline induced cAMP  or imiquimod (30 µg/mL) induced PGEced cAMP  A&B. Tre p<0.05,  A&B.2 release and decreased isoprenaline induced cAMP from ASMCs compared to the control treatment without RNA  released from RV infected epithelial cells cause β2AR desensitization on ASMCs 2 AR desensitization. It was deduced that the responsible mediator was less than 3 kDa which meant it could potentially have been a lipid such as a prostaglandin. We showed that RV infected airway cells produce prostaglandins and, since PGE2 is known to desensitize the β2 AR 2 AR was examined in vitro. It was found that PGF2α, PGE2 and MRE-269 caused β2 AR desensitization and with the use of pharmacological tools it was deduced that β2 AR desensitization occurred via autocrine prostaglandins from ASMCs. Mass spectrometry analysis yielded a single peptide present in the less than 3 kDa conditioned medium fraction; however it alone had no influence on β2 AR function. Since RNA was detected at greater levels in RV-induced conditioned medium than the control conditioned medium, we investigated RNA as the potential mediator causing β2 AR desensitization via the activation of TLR receptors. Using synthetic analogs of double stranded (ds) and single stranded (ss) RNA it was found that their combination caused PGE2 induction from ASMCs and β2 AR desensitization and similarly, total RNA extracted from RV-induced conditioned medium also caused PGE2 induction from ASMCs and β2 AR desensitization. Since human endogenous RNA extracted from HBEC did not cause this effect but RNA extracted from RV did, it was deduced that viral RNA produced during RV replication was required to cause β2 AR desensitization.Using an 2 AR occurs physiologically to prevent over activation of the receptor. This can be achieved by the mechanism of homologous desensitization i.e. activation of the receptor by its own defined agonist, for example, activation of the β2 AR by a β2 agonist causing β2 AR desensitization. Alternatively this can also occur by heterologous desensitization i.e. activation of another GPCR resulting in β2 AR desensitization because of common downstream cross-regulation of the signal pathways of the G protein subunits 2 can cause heterologous desensitization of the β2 AR by means of downstream cross talk of GPCR kinases (GRK) but also by activation of the PKA pathway through the EP2 and EP4 receptors Like all G protein coupled receptors (GPCRs), desensitization of the βin vitro model as used in this study to explore and understand why patients with naturally occurring RV-induced asthma exacerbations do not respond well to β2 agonists. We previously deduced that the substance was released only from RV infected HBEC 2 AR desensitization. In this study our data indicates that the responsible mediator for β2 AR desensitization was less than 3 kDa and not affected by trypsin digestion and therefore could potentially be a lipid or a trypsin-resistant peptide.Trian et al. first developed this in vivo RV infection results in the up regulation of COX-2 enzymes as detected by immunostaining of bronchial mucosal biopsies et al reported that RV infection of alveolar macrophages induces the release of PGE2et al found that RV infection of epithelial cells resulted in the induction of PGE2, and RV-induced PGE2 could potentially influence airway remodelling via its effect on altering neighbouring cellular behaviour such as cell migration 2, to show that RV infection of primary HBECs, ASMCs and lung fibroblasts can induce the release of various prostaglandin isotypes other than just PGE2. The amount of each prostaglandin released in response to RV infection varied amongst the various airway cell types tested, and although this issue is beyond the scope of this study, it could potentially highlight differences in the contribution of each individual prostaglandin as infection progresses from the epithelium to sub-mucosal cells. This raises the possibility of a pool of potential candidates singly or in combination that may be responsible for desensitization of the β2 AR.It has already been reported by Seymour et al that 2 can cause desensitization of the β2 AR, which is in keeping with the study by Penn et al. who showed that PGE2 can cause desensitization of the β2 AR, 2α can also cause desensitization of the β2 AR, suggesting it may share common GRKs with the β2 AR. Similarly, the PGI2 analogue MRE-269 also has the ability to cause β2 AR desensitization suggesting this desensitization may involve PKA as well as GRK cross talk. Therefore it is possible that RV infection may result in an increase of prostaglandins in the vicinity and may result in β2 AR desensitization on ASMCs. The ability of PGF2α to induce β2 AR desensitization was minimal compared to those by MRE-269 and PGE2, and since PGI2 is extremely unstable and only its inactive metabolite could be measured, it was considered unlikely to be sufficiently stable in the model to cause β2 AR desensitization on ASMCs. Since we showed PGE2 caused β2 AR desensitization most prominently compared to other prostaglandins, it was investigated whether HBEC derived PGE2 could be responsible by its depletion from conditioned medium. However, removal of PGE2 from conditioned medium did not affect β2 AR desensitization on ASMCs. It was then hypothesized that the autocrine action of ASMC derived prostaglandins may instead be responsible for β2 AR desensitization on the ASMCs, as PGE2 depleted RV-induced conditioned medium further induced approximately 10 fold higher levels of PGE2 in comparison to control conditioned medium. This suggested that the unidentified mediator, although not HBEC derived PGE2, induces PGE2 and potentially other prostaglandins from ASMCs.The investigation showed that RV infection of airway cells produced prostaglandins and PGE2 AR desensitization, ASMCs was treated with indomethacin and this prevented β2 AR desensitization from occurring when ASMCs were exposed to RV-induced HBEC conditioned medium. These findings were similar to the study by Guo et al, who showed that exogenously applied interleukin (IL)-1β induced COX-2 induction of PGE2 caused β2 AR desensitization and this was also preventable with indomethacin treatment in primary human ASMCs 2 AR desensitization as treatment with celecoxib also prevented this effect. The hypothesis that autocrine prostaglandins were causing β2 AR desensitization was also confirmed when ASMCs treated with a combination of prostaglandin antagonists prevented β2 AR desensitization in response to RV-induced HBEC conditioned medium exposure. Therefore this suggests that RV infection of HBEC may be producing unidentified mediators which cause increased activity of COX-2 and production of prostaglandins from ASMCs which act autocrinely to cause β2 AR desensitization.To investigate if ASMC derived prostaglandins were responsible for β2 AR desensitization by pre-treating ASMCs with different prostaglandin receptor antagonists only to find that none of the antagonists alone prevented β2 AR desensitization. The receptor antagonists available are designed to be selective for individual prostaglandin receptors however, in some circumstances; the antagonists are not selective enough and lack the ability to differentiate between the very similar receptors of PGE2 and PGD2. In addition, prostaglandins are also very pleotropic and can cross activate each other’s receptors 2 AR desensitization.An attempt was made to determine which prostaglandin isotype was responsible for β2 AR desensitization on ASMCs, suggesting they are not the responsible mediators or that a combination of mediators are required.Using mass spectrometry it was found that a 1.2 kDa peptide was present in RV-induced conditioned medium, but not in the control conditioned medium. The 1.2 kDa peptide is a fragment from the POMC protein which is a precursor for various hormones and ultradian rhythm neuro-peptides 2 AR desensitization through an immuno-stimulatory process such as via the process of autocrine COX-2 induction, and the unidentified mediator was small, it was hypothesized that the responsible mediator might be viral RNA or viral RNA fragments.Since RV-induced conditioned medium cause β2 release from ASMCs but did not alter isoprenaline induced cAMP. This result is in agreement with that of Cooper et al, who also showed that poly I:C promotes inflammatory mediator release 2 AR desensitization as well as an additive induction of PGE2 from ASMCs equivalent to those levels produced by RV-induced conditioned medium. This was further explored by showing that RNA extracted from an equal volume of RV-induced HBEC conditioned medium was higher than the control conditioned medium, and that RNA collected from RV-induced conditioned medium caused ASMC β2 AR desensitization. Total RNA extracted from RV-induced conditioned medium could include both endogenous human RNA as well as RV RNA and it is difficult to distinguish these small nucleotide fragments. It was confirmed that equal concentrations of RNA extracted from pure RV-16 caused PGE2 induction and β2 AR desensitization on ASMCs but this was not the case for endogenous human RNA extracted from HBEC lysate, which suggests that foreign viral RNA is required.Results of the study showed that the use of the synthetic TLR 7/8 agonist imiquimod and the TLR 3 agonist poly I:C alone slightly increased PGE−3 and 10−4 errors/nucleotide/cycle of replication 2 AR desensitization. Whilst UVi-RV contains RV RNA, the virions cannot replicate and therefore there is no increase in viral RNA. Whether β2 AR desensitization is restricted to asthma, or not is not known. It has been reported that greater RV replication occurs in epithelial cell derived from people with asthma 2 AR desensitization can occur during colds and remains clinically unnoticed. It is uncertain whether full length RV RNA or RNA fragments activate ASMCs to cause β2 AR desensitization, however both are possibilities, as it has been shown that nucleotide fragments as small as 5 nucleotides long can activate TLR 7/8 RV is a positive sense ssRNA virus and during replication, dsRNA are formed in order to generate more copies of the ssRNA genome to be packed into new virions. However, like many RNA viruses, RV replication is susceptible to RNA polymerase error with a rate of 10in vitro model attempted to simulate the events of an in vivo RV infection of the lung, which occurs principally in the airway epithelium and may result in ASMC β2 AR desensitization. However there are limitations to the current model that need to be acknowledged. Primarily, the in vitro model involved only 2 cell types, whilst physiologically there could be more cell types involved during infection. For example inflammatory cells and or perhaps other structural cells such as fibroblasts could play a role. For this reason it is possible that other cells may be as influential in causing β2 AR desensitization on ASMCs as epithelial cells. Furthermore, when other cells are considered, the system can become much more complex, and protein mediators such as IL-1β which can also cause β2 AR desensitization 2 AR desensitization, it does not necessarily mean that it is only one small mediator that was responsible. It is possible that there is a combination of multiple sized mediators trapped in each fraction which could activate GPCRs and cause β2 AR desensitization.This 2 AR desensitization was due to RV RNA activating TLRs, in our investigation RNA was extracted using a trizol methodology and for this reason small RNA mediators such as RV-induced HBEC derived micro (mi) RNA which may be able to activate TLRs 2 AR modulation could also be responsible and requires further investigation. It is theoretically possible to degrade the RNA in RV-induced conditioned medium using exogenous ribonucleases and assess for β2 AR desensitization. The limitation to this is that RNA that has been degraded to as small as 5 nucleotides long may still be or become immuno-stimulatory 2 AR desensitization, as it has been shown previously with LTD42 AR desensitization and should be investigated in future studies.Although it was deduced in the current model that βin vitro model is the use of primary human ASMCs and BEC which were obtained from multiple disease tissue origins. The use of primary human cells is a better representation of human cellular behaviour than some transformed cell lines because they reflect more accurately the true nature of biological heterogeneity of cellular responses, however since the current model utilized two cell types which were not diseased matched, and in some cases, HBEC derived conditioned medium was pooled from multiple diseases, it might be possible that asthma specific effects were missed.The second limitation of the 2 AR desensitization as viral RNA. During RV replication, viral RNA increases and activates TLRs and as a result induces COX-2 mediated prostaglandin production which causes β2 AR dysfunction. Interestingly, the findings in this study also raise the possibility that β2 AR desensitization may not be entirely unique to RV infection but could also be caused by other respiratory viruses that actively infect and replicate. By extrapolating the current findings to the clinical situation, the potential use of COX-2 inhibitors to restore β2 agonist efficacy in asthmatic patients during viral infection is possible and could be investigated clinically.Results from the present study identify the potential mediator responsible for RV-induced βFigure S1Trypsin digestion of conditioned medium removes IL-6 and IL-8. Conditioned medium pooled from HBEC (n = 3) that were uninfected (Control) or treated with: UV inactivated RV (UVi-RV) or replication competent RV (RV) at an MOI = 2 for 24 hours was digested in the presence of 500 µg/mL of trypsin for 24 hours at 37°C and the reaction was stopped with 0.1% BSA. IL-6 and IL-8 were measured using ELISA with n = 1 experimental repeat.(TIF)Click here for additional data file.Figure S22 adrenoceptor desensitization.Trypsin digestion of conditioned medium still causes ASMC β Conditioned medium pooled from HBEC (n = 3) that were uninfected (Control) or exposed to: UV inactivated RV (UVi-RV) or replication competent RV (RV) at an MOI = 2 for 24 hours was digested in the presence of 500 µg/mL of trypsin for 24 hours at 37°C and the reaction was stopped with 0.1% BSA. ASMCs (n = 6) were treated with trypsin digested conditioned medium for 3 days. Isoprenaline induced cAMP was measured using a cAMP functional assay. Data represent mean ± SEM. Statistical differences were detected using a 1-way ANOVA with Bonferroni post test comparisons to control conditioned medium *p<0.05.(TIF)Click here for additional data file.Figure S32 adrenoceptor desensitization.Individual prostaglandin antagonists do not prevent β ASMCs (n = 6) were pretreated for 1 hr with vehicle (0.1% DMSO), CAY10441 (10−6 M) (A), AH6809 (10−5 M) (B), AL8810 (10−5 M) (C), BWA868C (10−5 M) (D) or L-161,982 (10−6 M) (E) and maintained for a further 3 days in the presence of conditioned medium from HBEC (n = 2) that were uninfected (Control) or infected with replication competent RV (RV) at an MOI = 2 for 24 hours. Isoprenaline induced cAMP was measured using a cAMP functional assay. Data represent mean ± SEM. Statistical differences were detected using 1-way ANOVA with Bonferroni post test comparisons to the control conditioned medium pretreatment in each group *p<0.05.(TIF)Click here for additional data file.


Kr) is modulated by α- and β-adrenergic stimulation. However, the role and mechanism regulating IKr through β2-adrenoreceptor (β-AR) stimulation in heart failure (HF) are unclear.Increasing evidence indicates that the rapid component of delayed rectifier potassium current  caused a 17% decrease in IKr. In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in IKr. These effects were not modified by the incubation of myocytes with CGP-20712A, a β1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a β2-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of IKr in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD90) repolarization in HF ventricular myocytes.In the present study, we investigated the effects of fenoterol, a highly selective βKr induced by β2-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes.The results indicate that inhibition of I Heart failure (HF) is associated with significant mortality, with nearly 50% of deaths occurring suddenly, primarily from ventricular tachycardia to ventricular fibrillation KCNH2) KrKr is modulated by catecholamines. There is increasing evidence that hERG/IKr channels are modulated by various G protein-coupled receptors including α- and β-ARs, which act through the intracellular signaling modulators cAMP, protein kinase A (PKA), and protein kinase C (PKC) Kr tail currents in guinea pig ventricular myocytes Kr tail currents are enhanced by 100 nM isoprenaline in canine ventricular myocytes 1-AR is downregulated and β2-AR is relatively preserved in HF. In addition, experimental studies have demonstrated that canine ventricular response to β2-agonists is increased in tachypacing failure L-type calcium current to β2-AR stimulation is increased in rats with HF induced by ligation of the coronary artery 2-AR activation in IKr in HF have not been previously assessed. Accordingly, the present study was designed to examine the role and possible mechanisms of β2-AR activation in IKr in left ventricular (LV) myocytes from HF guinea pigs using the whole cell patch clamp technique.The human ether-a-go-go-related gene  and fractional shortening (FS) of the heart of guinea pigs were significantly decreased; LV end-diastolic diameter, LV end-systolic diameter, the QT interval, and corrected QT interval were significantly increased while there were no changes in heart rate . Cell cakr tail currents of guinea pig LV myocyte were completely abolished by the specific Ikr blocker, dofetilide (1 µm) .Ie (1 µm) , indicatcontrols . Figure 2-AR agonist fenoterol (10 µM) decreased Ikr tail currents by 17±5%  into the bath. After washout of fenoterol, Ikr tail current reached 83.5±4.5% and 67.8±6.7% of the control and HF value, respectively. The inhibitory effect of fenoterol was almost not reversible , whereas β2-AR mRNA levels remained unchanged. β1-AR protein showed a significant decline, as illustrated in 2-AR protein remained unchanged . These results suggest that IKr inhibition induced by β2-AR stimulation involves cAMP activation.It is known that the hERG Kkr decrease was largely abolished by pretreatment of HF myocytes with KT5720 , supporting an involvement of PKA activation.We further examined the effect of PKA inhibition on the inhibitory effects of fenoterol. As illustrated in Kr would lead to substantial changes in the repolarization process. We therefore examined the effects of fenoterol on AP in LV myocytes. 90) was significantly prolonged in HF myocytes compared with control . Fenoterol caused significant prolongation of APD90 in HF myocytes , whereas no significant prolongation of APD90 was observed in control myocytes . Fenoterol did not influence the AP amplitude and resting potentials in control and HF ventricular myocytes.Because action potential duration (APD) is dependent on the balance between depolarizing inward and repolarizing outward currents, an alteration in the amplitude of major repolarizing current such as I2-AR stimulation produces an inhibitory effect on IKr in ventricular myocytes of guinea pigs with descending aortic banding-induced heart failure, and this effect is induced by the activation of the cAMP/PKA pathway.The present study demonstrates, for the first time, that increased response to βkr) and is critical for the kinetics of cardiac AP repolarization Kr channels are modulated by various G protein-coupled receptors including α- and β-ARs, acting through the intracellular signaling modulators cAMP, PKA, and PKC 1-and β2-AR coexist in the heart. In large mammal hearts, β2-AR may account for approximately 40% of β-AR in total. The reported ratio of β1-/β2-AR was 85/15 in guinea pig hearts 1-AR was downregulated; however, β2-AR was unaltered. The increase of β2-AR density has been demonstrated in the LV of dogs with pacing-induced HF 2-AR stimulation exhibits a heightened response Kr to β2-AR stimulation is enhanced in HF guinea pigs ventricular myocytes. The effect of selective β2-AR agonist fenoterol (10 µM) on IKr was far less effective in control myocytes; whereas, IKr was significantly inhibited by 10 µM fenoterol in HF myocytes. The reduction of IKr tail current occurred within 2–4 min and reached saturation about 10 min after addition of fenoterol into the bath. After washout of fenoterol, the inhibitory effect on IKr tail current was almost irreversible. In order to identify the β2-AR specific effect, we used salbutamol (10 µM), another highly selective β2-AR agonist, and achieved similar results (data not shown). Moreover, the inhibitory effect of fenoterol was almost completely abolished by β2-AR antagonist, but not by the β1-AR antagonist. Therefore, our findings indicate that the IKr inhibition induced by fenoterol is due to β2-AR activation.The hERG channel (Kv11.1) underlies the rapid component of delayed rectifier potassium current , which leads to increased cellular cAMP, thus activating PKA the Xenopus laevis oocyte expression system that PKA can reduce hERG currents via direct phosphorylation of all four putative PKA consensus sites Kr was fully prevented by intracellular application of Rp-cAMPS, an inhibitory cAMP analog, suggesting that this effect may be mediated through a cAMP-dependent mechanism in HF ventricular myocytes. Furthermore, fenoterol-induced inhibition of IKr was partly attenuated by PKA inhibitor, implicating the involvement of PKA activation. The dual regulation of IKr by cAMP and PKA phosphorylation has been previously demonstrated 2-AR redistribution in HF can change cAMP compartmentation Stimulation of different G protein-coupled receptors, including α2-AR can couple with both Gs and Gi proteins. The complexity of the β2-AR signaling pathway, including functional compartmentalization of signaling mediated by Gi, phosphatidylinositol 3-kinase, and mitogen-activated protein kinases (MAPKs), has been documented et al. has reported that one or more A-kinase anchoring proteins (AKAPs) targets PKA to HERG channels and may contribute to the acute regulation of IKr by cAMP 2-ARs are found to be associated directly with Cav1.2 in a macromolecular signaling complex 2-adrenergic stimulation-induced inhibition of IKr for HF myocytes remains to be further elucidated. For example, it remains unclear whether Gi pathway or a β2-AR-macromolecular signaling complex mediates the inhibition of IKr via β2-AR in HF ventricular myocytes.Cardiac βto (transient outward K+ current ), IKs and IKr in heart failure 90 in failing ventricular myocytes, whereas no significant prolongation of APD90 was observed in control myocytes. The present study provides evidence that fenoterol-induced inhibition of IKr may result in delay in cardiac repolarization via stimulation of β2-AR in failing ventricular myocytes. Because the IKr is crucial for the repolarization of cardiac AP, inhibition of IKr induced by stimulation of β2-AR in failing ventricular myocytes may in part contribute to the delay in cardiac repolarization.Ventricular myocytes from the guinea pig heart possess a time- and voltage-independent Cl− current induced by β-adrenoceptor activation − current. Therefore, the effect of fenoterol on cardiac repolarization should be studied without ignoring other currents in the future.One of the most characteristic electrophysiological remodeling in failing heart is APD prolongation, which is believed to mainly result from the downregulation of repolarizing outward potassium currents, including IKr induced by β2-AR stimulation is increased in HF. The effect is likely to be mediated through a cAMP/PKA pathway mechanism in HF ventricular myocytes. This regulatory pathway of the β2-adrenergic system on Ikr current provides a possible relationship between stress and life-threatening arrhythmias in HF.In conclusion, the inhibitory effect of IAll experiments were performed in accordance with animal care protocols approved by the Nanjing Medical University Institutional Animal Care and Use Committee.Heart failure was induced in adult male guinea pigs (250 to 300 g) by subtotal descending thoracic aortic banding. After sodium pentobarbital anesthesia , the descending thoracic aorta was exposed sterilely through an incision in the left third intercostal space. A uniform degree of constriction around the descending thoracic aorta was produced by tightening a 2-0 surgical silk ligature around an 18-gauge needle. The needle was then withdrawn from the ligature, and the chest incision was closed. Sham animals underwent the same operation, with the exception that the aorta was not banded. Sham-operated animals were used as control. Aortic-banded animals and sham animals were housed and fed under identical conditions and were used 12 weeks after surgery. To monitor the progress of HF during the observation period, two-dimensional echocardiography  was carried out periodically. ECGs and two-dimensional echocardiography were obtained under anesthesia . The QTc was calculated by Bazett’s formula where QTc = QT/√RR. Following 12 weeks of aortic banding, guinea pigs showed signs of HF, including ventricular dilation, decreased ejection fraction (EF) and fractional shortening (FS), ascites, and pleural effusions.kr was determined using the following test pulse protocol: after a holding potential of −40 mV, test pulses were applied at various voltages from −40 to +40 mV  prior to returning to −40 mV for tail current recording Kr current was recorded at 37±0.5°C. Substance effects were investigated on tail currents after the test pulse was applied at +40 mV. Measurements were repeated every two min.Single left ventricular (LV) myocytes were enzymatically dissociated from the heart of guinea-pigs (550–600 g) as described previously 2 1.0, CaCl2 1.8, glucose 10, and HEPES 10; pH was adjusted to 7.4 with NaOH. Pipette solution (in mmol/L) contained potassium aspartate 120, KCl 20, MgCl2 2.0, HEPES 10, EGTA 10, Na2ATP 10; pH was adjusted to 7.2 with KOH. APs were evoked using whole-cell current-clamp mode by suprathreshold current pulse of 5 ms duration at the frequency of 1 Hz. APD was measured at 90% repolarization (APD90). APs were generated with the same amplifier and recorded at 37±0.5°C.For AP recording, extracellular solution (in mmol/L) contained NaCl 137, KCl 5.4, MgClet al. ks. Na2-ATP, EGTA, creatine phosphate, L-glutamic acid, HEPES, taurine, bovine serum albumin (BSA), nifedipine, isoproterenol, xamoterol, fenoterol, CGP20712A, ICI118551, and Rp-cAMPS were purchased from Sigma , collagenase II from Invitrogen , and KT5720 from Merck KGaA . Dofetilide, a specific blocker of Ikr or hERG, was kindly provided by Pfizer Inc . All other reagents were obtained from Amresco .Preparation of the solution was performed as described previously by Wang For stock solutions, chromanol and KT5720 were dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 1 mM and stored at −20°C until use. Final concentration of DMSO was less than 0.5% in the bath, which exerted no effect on the currents measured.GAPDH housekeeping gene was used as a reference for loading control. The primers for genes examined are shown in −△Ct method.RNA was extracted from 100 mg of LV tissue using the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA concentration was determined spectrophotometrically. Reverse transcription of 2 µg RNA was performed in a 20-µl reaction mixture using an RT kit (Takara Biotech). The 1-AR and β2-AR . After washing, blots were incubated for one h with appropriate peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology). Staining was determined with chemiluminescence and quantified using image acquisition and analysis software. All data were expressed relative to GAPDH staining for the same samples on the same gels.Membrane fractions were prepared as previously described t-test (paired or unpaired). Differences were considered statistically significant if p<0.05.The data were acquired using Pulse +Pulsefit V 8.53 and were expressed as mean ± SEM. Western blot band intensities were expressed after background subtraction as optical density units (ODUs) normalized against GAPDH signal intensity for the same sample. Data comparisons were made using Student’s