1. Introduction
Heart failure (HF) currently affects 64 million people worldwide with increasing prevalence 1. Thus, health care expenditures are substantial; and considering our ageing population, they will continue to rise. HF morbidity and mortality are still high despite remarkable advancements in prevention and therapy 2. Moreover, quality of life remainspoor for HF patients 3as HF causesinjury and dysfunction of target organs, including the lung 4-7. Although thisaffectsprimary disease management and outcome, the mechanisms underlying target organ injury in HF remain incompletely understood and hence, safe and efficient treatment strategies are limited.Regarding HF-associated lung complications, progress has been made in understanding the pathophysiology of pulmonary oedema, but other pulmonary complications of HF continue to challenge patients and clinicians alike.
Similar to several chronic lung diseases 8, elevated biomarker levels of inflammation are features of chronic HF. An augmentation in pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α) 9, has been demonstrated to play a role duringHF progression, suggesting an involvement of inflammation during HF-mediated target organ damage 10.We previously showed that therapeutically scavenging TNF-α using Etanercept attenuates target organ dysfunction in a mouse model of HF 7.Therapeutic interventions aimed at limiting TNF-α-mediated inflammation in chronic HF or lung diseases have yielded controversial results 11.Considering this, we invested in understanding the molecular mechanism by which TNF-α signalling promotes target organ function during experimental HF 12. Particularly, we showed that elevated TNF-α levels lead to considerable downregulation of the cystic fibrosis transmembrane regulator (CFTR) in the murine vasculature, heart, brain, and lung tissue 5, 13. The importance of proper CFTR function is appreciated in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Here, CFTR protein dysfunction is common in the airways of affected patients 14. In contrast to the genetic origin in CF, CFTR dysfunction in COPD is acquired since neutrophil elastase can induce alterations of CFTR expression, which correlate with disease severity 15. Besides epithelial 16and smooth muscle cells 5, 6, CFTR expression has been documented in several immune cells 17, 18. Peripheral blood monocytes isolated from patients heterozygous for the F508del CFTR mutation showed enhanced interleukin (IL)-8 secretion after activation compared to non-CF controls 19. The latter was corroborated in macrophages isolated from Cftr knockout mice 20, suggesting a hyperinflammatory phenotype. Interestingly, pharmacological CFTR inhibition in macrophages increased secretion of pro-inflammatory cytokines 18, suggesting that acquired CFTR dysfunction (e.g., induced by HF, smoking or neutrophil elastase 15, 21, 22) may contribute to hyperinflammatory immune responses. Since dysregulation of inflammation represents a hallmark of multiorgan manifestationsof many diseases, including HF, we tested the hypothesis that murine HF associates with pulmonary CFTR dysfunction and concurrent tissue inflammation,which is correctable byCFTR targeting therapy.
2. Material and Methods
2.1. Materials
All chemical reagents and solutions were purchased from Fisher Scientific (Gothenburg, Sweden), Saveen & Werner (Limhamn, Sweden) or Sigma-Aldrich (Stockholm, Sweden) unless otherwise stated. Primers for qPCR were purchased from Eurofins (Ebersberg, Germany).
2.2. 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 23. Moreover, women have a higher probability of survival 24. 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 randomisation. To obey the rules for animal welfare, experimental groups were designed to minimise 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, 25.
2.3. Myocardial infarction (MI)
HF in mice was induced by experimental MIgeneratedby permanent surgical ligation of the left anterior descending (LAD) coronary artery 12. Briefly, mice were anaesthetised 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 miceunderwent the same procedure withoutLAD ligation. Mice received pain medication (2 µl/g mouse buprenorphine0.05 mg/ml) for up to three days post-surgery. This model showsstable cardiac injury 6 weeksafter MI 12.CFTR correctortreatmentwas initiated 10 weeks after MI(Supplemental Fig. 1).For 2 weeks,mice receiveddaily 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 treatmentperiod(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.
2.4. Cardiacfunctionassessment
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 anaesthetised 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). Additional details for cardiac function assessment are provided in the Supplementary material online.
2.5. Fluorescence activated cell sorting
After transcardiac 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 Fcblock 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 (FSC) 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 26-28.
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 2).
2.6. Hydroxyproline assay
Hydroxyproline content was measured using the “Hydroxyproline Assay Kit” as per manufacturer’s instructions.
2.7. Cell culture
Murine macrophages (RAW246.7, ATCC TIB-71) were cultivated in high glucose DMEM supplemented with 10% heat inactivated foetal 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 incubation 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.
2.8. Western Blot analysis
2.9. RNA extraction and quantitative real-time PCR
For total RNA isolation, the right middle lobe was homogenised in 1 ml Trizol(Invitrogen) and isolated according to the manufacturer’s manual. 1 µg of mRNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit in an T100TM Thermal Cycler (Bio-Rad). The resulting cDNA was diluted 12.5x andsubsequently used for PCR reactions. The PCR protocol consisted of 40 cycles of 30 s denaturation (95°C); 45 s primer annealing (60°C) and 45 s primer extension (72°C) using a CFX384TM Real-Time System with a C1000 TouchTM Thermal Cycler (Bio-Rad). A list of the primers utilisedis provided in
Supplemental Table 3.
2.10. Histology
Lungs were fixed in 4% PFA (Histolab) overnight and transferred into paraffin using a EprediaTMSTP 120 Spin Tissue Processor (Fisher Scientific). Afterwards, samples were embedded into paraffin blocks using an EC 350-1 (Especialidades Médicas Myr, S.L.). 4 µm thin sections were cut with a microtome (HM 355S, Thermo Scientific) and fitted onto superfrost glass slides. Paraffin sections were deparaffinised and rehydrated beforethey were subjectedto Haematoxylin &Eosinand Masson-Trichromestaining. Additional details about immunohistochemical staining are provided in the online supplementary material.For quantification, vessel wall thickness in at least 3 images from 4 animals was manually assessed using the “straight line” tool in ImageJ (
https://imagej.net/ImageJ) in scale adjusted images. For the qualitative quantification of collagen in Masson-Trichrome stained lung slides, the staining intensity and staining amount in comparable areas of the lungs (mainly around airways and vessels) was graded on scale from 1-5. At least 5 regions of interest per animal were graded and 8-10 animals per group were evaluated.
For immunofluorescence, paraffin sections were deparaffinised, rehydrated, and subjected to antigen retrieval in 0.1 M sodium citrate buffer (pH 6) for 20 min before blocking with blocking reagent (Roche) and primary antibody incubation in a humidity chamber over night at 4°C. Slides were subsequently washed with PBS, incubated with secondary antibody at RT and mounted with Fluoromount-G with DAPI. Staining was evaluated with a
Zeiss Axio Imager and Zen Pro 10 software. For the quantification, MOMA+ and DAPI+ cells in at least 3 images from 4 animals were manually counted using ImageJ (https://imagej.net/ImageJ). Positively stained cells for MOMA+ were reported as % of DAPI+ cells per vessel.
Lung samples were snap frozen in liquid nitrogen and stored at -80 °C until analysis. Samples were homogenised in 1x PBS using an Ultra-Turrax TP18-10 (Janke & Kunkel KG) and proteins lysed in RIPA buffer supplemented with phosphatase and protease inhibitors for 30 min on ice. Afterwards, samples were frozen at -80 °C and thawed on ice. Thereafter, protein extracts were centrifuged for 15 min at 20,000 g at 4 °C and stored at -20 °C. Protein content was measured using the Pierce™ BCA Protein Assay Kit according to manufacturer’s instructions. After SDS-PAGE, proteins were transferred onto PVDF membranes (VWR) using either wet transfer (5 mM Tris, 40 mM glycine, 20% methanol) or semi-dry transfer in TransBlot®TurboTM(Bio-Rad), blocked with 5% non-fat dry milk powder in PBS-T (1x PBS, 0.05% Tween 20) for 1 h at room temperature and incubated with the respective primary antibody overnight at 4°C. Blots were incubated with secondary, HRP-labelled antibodies for 1-2 h at room temperature and enhanced chemiluminescence was used to visualise proteins using a ChemiDocTMMP (Bio-Rad). Protein expression was quantified in relation to β-Tubulin expression and normalised to sham animals.