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.