Material and Methods
Materials : All chemical reagents and solutions were purchased from Fisher Scientific (Göteborg, Sweden), Saveen & Werner (Limhamn, Sweden) or Sigma-Aldrich (Stockholm, Sweden) unless otherwise stated. Primers for qPCR were purchased from Eurofins (Ebersberg, Germany).
Animals: This investigation conforms with the Guide for Care and Use of Laboratory Animals published by the European Union (Directive 2010/63/EU) and the ARRIVE 2.0 guidelines. All animal care and experimental protocols were approved by the institutional animal ethics committee at Lund University (Dnr.: 5.8.18-08003/2017; 5.8.18-04938/2021) and were conducted in accordance with European animal protection laws. Commercially available male wild-type mice (12-14 weeks old; C57BL/6N) were purchased from Taconic (Lyngby, Denmark). All mice were housed under a standard 12h:12h light−dark cycle and had access to standard chow and water ad libitum . In the clinic, research into sex differences showed that HF prevalence is about 1.5-2x higher in men above 55 years of age compared to women [22]. Moreover, women have a higher probability of survival [23]. Females are therefore more protected from HF than males. For this reason, male mice that generally show a stronger phenotype were used in this study.
To ensure blinding, experiments were performed after the animals and samples had received codes that did not reveal the identity of the treatment. HF animals were assigned to vehicle or treatment groups using block randomization. To obey the rules for animal welfare, experimental groups were designed to minimize stress and guarantee maximal information using the lowest group size possible when calculated with a type I error rate of α = 0.05 (5%) and power of 1-β > 0.8 (80%) based on earlier studies [5, 24].
Myocardial infarction (MI): HF in mice was induced by experimental MI generated by permanent surgical ligation of the left anterior descending (LAD) coronary artery [12]. Briefly, mice were anesthetized with isoflurane (1.5-2% in air), intubated with a 22-gauge angiocatheter, and ventilated with room air at a rate of 120 bpm, 250 µl tidal volume, and 3 cm positive end expiratory pressure. The thorax and pericardium were opened, and the LAD was permanently ligated with 7-0 silk suture (Ågnthos, Sweden). Sham control mice underwent the same procedure without LAD ligation. Mice received pain medication (2 µl/g mouse buprenorphine 0.05 mg/ml) for up to three days post-surgery. This model shows stable cardiac injury 6 weeks after MI [12]. CFTR corrector treatment was initiated 10 weeks after MI (Supplemental Fig. 1 ). For 2 weeks, mice received daily intraperitoneal (i.p.) injections of Lumacaftor (Lum; 3 mg/kg in DMSO diluted 1:10 with sterile polyethylene glycol (PEG) in deionized (DI) water (50:50)) or were instilled with 50 µl Lum (18 mg/ml in DMSO diluted 1:10 in sterile PBS) 5 times during the treatment period (orotracheal; o.t.). Group sizes were as follows: N=8 for sham, N=10 for HF, N=10 for HF + Lum, N=6 for HF + Lum i.p., N=8 for HF + Lum o.t. Not all animals were used for histology experiments.
Cardiac function assessment: Cardiac function was assessed using magnetic resonance (MR) imaging on a 9.4 T MR horizontal MR scanner equipped with Bruker BioSpec AVIII electronics, a quadrature volume resonator coil (112/087) for transmission and a 20 mm linear surface loop coil for reception (Bruker, Ettlingen, Germany), operating with ParaVision 6.0.1. Mice were anaesthetized with isoflurane in room air with 10% oxygen and kept at a respiration of 70-100 bpm and at 36-37°C body temperature (sequence details in supplement). Image-based determination of ejection fraction (EF), stroke volume, cardiac output, end diastolic volume, end systolic volume, and left ventricle mass was performed with Segment (Medviso, Lund, Sweden).
Fluorescence activated cell sorting: After trans-cardiac perfusion, lung-heart blocks were extracted, and a broncho-alveolar lavage was performed by instilling sterile PBS. The left lung was cut into pieces and enzymatically digested in a DNAse-Collagenase XI mix under continuous agitation. After centrifugation, red blood cells were lysed, and the cell pellets were reconstituted in Fc block prior to antibody staining (Supplemental Table 1 ). Data acquisition was carried out on a BD LSR II cytometer using FacsDiva software Vision 8.0 (BD Biosciences). Data analysis was performed with FlowJo software (version 10, TreeStar Inc., USA). Cells were plotted on forward versus side scatter and single cells were gated on FSC-A versus FSC-H linearity. Pulmonary macrophages were identified as Live, CD45+, B220-, CD11b+, F4/80+ cells (gating strategy: Supplemental Figure 2 ). Non-alveolar macrophages were identified as Live, CD45+, B220-, CD11b+, F4/80+, SiglecF- cells while alveolar macrophages were identified as Live, CD45+, B220-, CD11b+, F4/80+, SiglecF+ cells [25-27].
For CFTR staining, pulmonary cells were incubated with CFTR antibody and live/dead staining dye without reconstitution in Fcblock. After washing and centrifugation, cells were resuspended and incubated with a secondary goat anti-mouse AF488 antibody (Supplemental Table 1 ).
Hydroxyproline assay: Hydroxyproline content was measured using the “Hydroxyproline Assay Kit” as per manufacturer’s instructions.
Cell culture: Murine macrophages (RAW246.7, ATCC TIB-71) were cultivated in high glucose DMEM supplemented with 10% heat inactivated fetal bovine serum and 1% Penicillin/Streptomycin. Cells were activated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, AdipoGen) for 48 h followed by a 24 h rest period before they were incubated with 10 µM Lum (Cayman Chemicals) for 24 h. In a second approach, Lum treatment was started at the same time as PMA-induced activation. Cells were harvested after 96 h and subjected to flow cytometry to determine CFTR surface expression.
Western Blotting, qPCR, and histological experiments: Standard procedures were utilized for experiments involving reverse transcription polymerase chain reaction (PCR), quantitative PCR, Western blotting, and histological experiments. See supplement for methodological details.
Data and Statistical Analysis: The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology [28]. All data are expressed as mean ± SEM, where N is the number of independent measures. Data were analysed using GraphPad Prism 8 software (San Diego, California). Data distribution was determined using Shapiro-Wilk test. For comparisons of 2 independent groups, a Student’s t-test or Mann Whitney test was used. For comparison of multiple independent groups, one-way analysis of variance (ANOVA) or a Kruskal Wallis test was used, followed by a Tukey or Dunnettpost-hoc test. Differences were considered significant at p < 0.05.