Interactive patient education via an audience response system in cardiac rehabilitation

Objectives: Patient education and compliance play an important role in the success of rehabilitation in cardiovascular diseases. The aim of this study is to analyze whether interactive learning methods, in this study, the audience response system with a “clicker,” can improve the learning success of patients during and after their rehabilitation process. Methods: In a randomized, prospective cohort study, a total of 260 patients were randomized to either an interactive training group using Athens audience response system or to a control group without the use of audience response system during the educational sessions. Patients were taught and tested on four different topics concerning their primary disease: heart failure, arterial hypertension, prevention of cardiovascular diseases, and coronary heart disease. After each session, the patients had to answer questions on the previously taught topics via questionnaires. These questions were asked again at the day of discharge, as well as 3 and 12 months after discharge. Additional information on the patients’ health, plus their mental status, was gathered with the help of further questionnaires (HADS and SF-12). Results: A total of 260 patients (201 men and 59 women) were recruited. The patients were on average 61.1 ± 11 years old. A significant short-term effect on the patients’ knowledge about their disease was found immediately after the educational sessions in the intervention group. However, there was no long-term effect in either the intervention or control group. Although there was no statistical significance found in any of the observations, a positive short-term effect on learning capacity as well as positive trends in mental and physical health after discharge could be found in patients after the use of audience response system during their rehabilitation. Conclusion: This study provides interesting and new data on the use of an interactive learning method for patients to gain knowledge about their primary disease and eventually improve their physical and mental health status in a long-term perspective. By implementing different and new ways of teaching and interaction during the hospitalization, not only patients, but also medical staff and caregivers could benefit.


Study of LX-2 activation through TGF-β1
To study the effect of GNS561 on TGF-β1 stimulation in LX-2 cells, cells were incubated for 24 h in medium containing 1% fetal bovine serum (FSB). GNS561 was then added at increasing concentrations (0.75-6 µM) 2 h before addition of 5 ng/μL TGF-β1. Cell lysates were prepared 24 h after the addition of GNS561 and analyzed by quantitative real-time PCR and western blotting.

Study of primary human hepatic stellate cells activation through TGF-β1
To study the effect of GNS561 on TGF-β1 stimulation in human hepatic stellate cells (HSC), cells were first plated during 24 h in medium containing 10% FBS to allow their correct attachment on plate surface. In a second time, cells were cultured in medium without FBS for 24 h. Then, cells were treated with increasing concentrations of GNS561 (0.75-1.5 µM) 2 h before addition of 5 ng/mL TGF-β1. Cells were harvest 24 h after the addition of GNS561 and analyzed by quantitative real-time PCR.

Caspase activity assay
The activity of caspases 3/7 was measured using the Caspase-Glo 3/7 Assay following the manufacturer's protocol (Promega). Briefly, LX-2 cells were plated in a 96-well plate (6,000 cells per well) in 90 µL of medium. Twenty-four hours after plating, cells were treated with 10 µL of GNS561 (0.75-9 µM) or GNS561 vehicle and incubated for 24 h. At the end of the treatment, 100 µL of Caspase-Glo 3/7 reagent was added to each well and cells were incubated for 1 h at room temperature. Then, luminescence was measured by an Infinite F200 Pro plate reader. Fold change of activation of caspases 3/7 was determined by comparing the luminescence in the treated groups with the luminescence observed in the negative control wells (vehicle treated cells), with the luminescence of blank wells subtracted. At each time point, in parallel with the activation of caspases 3/7, cell viability was also investigated using CellTiter-Glo Luminescent Cell Viability Assay. Each GNS561 concentration was tested in triplicate in three independent experiments.

Reactive oxygen species assay
Reactive oxygen species (ROS) were measured using ROS-Glo H2O2 Assay following the manufacturer's protocol (Promega). LX-2 cells were plated in a 96-well plate (6,000 cells per well) in 70 µL of medium.
Twenty-four hours after plating, the cells were treated with 10 µL of GNS561 (0.75-9 µM) or vehicle and incubated for 24 h. Six hours before the end of treatment, 20 µL of H2O2 substrate was added to each well. At the end of the treatment, 100 µL of ROS-Glo Detection Solution was added to each well and cells were incubated for 20 min at room temperature. Then, luminescence was measured by an Infinite F200 Pro plate reader. Fold change of ROS quantity was determined by comparing the luminescence in the treated groups to the luminescence observed in the control wells (vehicle treated cells). At each time point, in parallel, cell viability was also investigated using the CellTiter-Glo Luminescent Cell Viability Assay. Each GNS561 concentration was tested in triplicate in three independent experiments.

Lysosomotropism-mediated death study
The effect of lysosomotropism on anti-fibrotic activity was assessed as previously described 1 . LX-2 cells were plated in a 96-well plate (6,000 cells per well) in 80 µL of medium. Twenty-four hours after plating, cells were pre-treated with 10 µL of Baf (100 nM) or NH 4 Cl (10 mM) for 2 h and then treated with GNS561 (1.5-9 µM) or vehicle and incubated for 24 h. At the end of the treatment, cell viability was assessed using the CellTiter-Glo Assay. Cell viability was expressed as a percentage of the values obtained from the control cells (vehicle treated cells). Each condition was tested in triplicate, and three independent experiments were performed.

Autophagy assay
The autophagy pathway was studied as performed previously 1 . Twenty-four hours after LX-2 cell plating, the cells were treated with GNS561 (1.5-6 µM) for 24 h. Treatment with vehicle was used as a baseline for autophagic flux control. In specified conditions, Baf (100 nM) was added for the last 2 h of treatment.

Western blotting
In brief, cells were lysed with Mammalian Cell Lysis Buffer. cOmplete™ Protease Inhibitor Cocktail was added extemporaneously to the lysis buffer. Ten to twenty micrograms of protein from each sample was separated on a 15% or 4-15% SDS-PAGE gel, transferred to a PVDF membrane, and blotted with antibodies against LC3-II with Baf and without Baf. The LAP cleavage ratio was determined as the ratio between the LAP level and the pro-TGF-β1 level for each condition in comparison with the ratio obtained for the condition without GNS561 treatment. The α-SMA and COL1A1 fold changes were calculated as the ratio between α-SMA or COL1A1 level normalized against GAPDH level for each condition and α-SMA or COL1A1 level normalized by GAPDH level for the untreated condition (neither GNS561 nor TGF-β1 stimulation). The p-Smad/Smad ratio was determined as the ratio between the p-Smad and Smad level for each condition in comparison with the ratio obtained for the untreated condition (neither GNS561 nor TGF-β1 stimulation). The p-Erk1/2/Erk1/2 ratio was determined as the ratio between the p-Erk1/2 and Erk1/2 level for each condition in comparison with the ratio obtained for the untreated condition (neither GNS561 nor TGF-β1 stimulation). All the experiments were repeated at least three times.
Representative autoradiograms are shown.

Cathepsin activity assay in cellular lysates
Twenty-four hours after LX-2 cell plating, the cells were treated with GNS561 (1.5-9 µM) for 24 h.

Immunocytochemistry assay
4.5×10 4 LX-2 cells were plated on glass coverslips, placed in 24-well dishes containing 1% FBS and incubated for 24 hours at 37°C. Cells were then pre-treated during 2 h by GNS561 in increasing doses followed by 5 ng/mL TGF-β treatment. 24 h after GNS561 treatment, cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at 4°C. Blocking buffer (1X PBS, 5% FBS) was then added for 60 minutes at room temperature. 5 minutes before the end of blocking step, cells were permeabilized in 0.3% Triton X-100. Then, the cells were incubated with primary antibodies against Smad4 (1/500) and with Hoechst 33342 Solution (1 µg/mL) for nuclei staining during 1 h at room temperature. Alexa Fluor 488 secondary antibody for Smad4 labelling was then applied in PBS containing 5% FBS, for 30 minutes at room temperature. Cells were mounted between slide and slip cover with Mowiol diluted 1:2 in PBS. Image acquisition was performed 24 h after labelling on a LSM 800 Airyscan confocal microscope (Carl Zeiss, Oberkochen, Germany) and collected by Zen 3.0 (Blue Edition) software (Carl Zeiss). Three independent experiments were performed.

Rat model and treatment groups
Fourteen 6-week-old Fischer 344 male rats (Charles River, Wilmington, MA, USA) were housed in the Plateforme de Haute Technologie Animale animal facility (Jean Roget, University of Grenoble-Alpes, France). Rats were kept in individually ventilated cage (IVC) systems at constant temperature and humidity with 2-3 animals in each cage having free access to food (standard diet) and water during the entire study period. All rats were treated weekly with intra-peritoneal injections of 50 mg/kg of diethylnitrosamine (DEN) (Sigma-Aldrich), which were diluted in olive oil to obtain a cirrhotic liver with hepatocellular carcinoma after 14 weeks 2 . Rats were randomized into 2 groups (n=7/group) and treated over six weeks by daily oral gavages of GNS561 (15 mg/kg of GNS561, GNS561 group) or of vehicle (control group). The nutritional state was monitored by daily weighing of the rats. The food intake per cage was monitored during the last 6 weeks of the experiment. Food was withheld for 3-4 h before the animals were sacrificed. After six weeks of treatment treatment with GNS561 or vehicle, animals were anesthetized with isoflurane and euthanized with vena cava blood sampling.

Collagen and α-SMA detection in rat samples
Collagen was detected in paraffin-embedded sections with Direct Red 80 stain solution and the staining was subsequently quantified by MetaMorph software (Molecular Devices) in 10 randomly selected fields/section (10× magnification). α-SMA staining was detected on paraffin-embedded sections by α-SMA (D4K9N) rabbit monoclonal antibody followed by goat anti-rabbit AF647 secondary antibody.
Images were captured using an ApoTome microscope (Carl Zeissequipped with a camera AxioCam MRm and collected by AxioVision software (Carl Zeiss). Positive areas were quantified using ImageJ software (NIH, Bethesda, MD, USA) on 15 randomly selected fields/section (10× magnification). All analyses were performed in a double blinded manner.

Gene expression analysis by quantitative real-time PCR
Total RNA was isolated from LX-2 or primary human HSC cells using the RNeasy Mini kit (Qiagen, (PPH01094E). Data were analyzed with the LightCycler 480 SW 1.5 software (Roche) and fold changes were calculated as the relative expression of fibrogenic genes after normalizing to B2M used as housekeeping gene compared with the untreated condition (neither GNS561 nor TGF-β1 stimulation).
A melting curve analysis was done after amplification to verify the accuracy of the amplicon. Every analysis was done in duplicate and three independent experiments were performed.
Total RNA from frozen rat liver tissue samples was extracted using the RNeasy Mini Kit (Qiagen).