Animals

All described animal experiments were in accordance with the national guidelines and approved by the local animal care and use committee (LANUV Recklinghausen, Germany, License no AZ: 84-02.04.2014.A220, AZ: 81-02.04.2020.A190) iCMp38αKO mice were generated by crossing C57Bl/6 mice homozygously expressing floxed p38 MAPKα (p38flox/flox, Exon 2 and 3) [59] with heterozygous mice expressing the tamoxifen-inducible Cre-recombinase merCremer under control of α-MHC promotor [53, 60]. For KO induction mice received 500 µg 4OH-Tamoxifen (Tx) (5 mg/ml in peanut oil, Sigma #H6278) i.p. for 10 days at 6 weeks age and were allowed to recover from injections for 4 weeks. Animals used for experiments were male mice at the age of 12 weeks. As control, p38flox/flox littermates, equally injected with 4OH-Tx, were used.

Mice received 1.5 mg/kg/d angiotensin II (Sigma, #A9525) via Alzet osmotic mini pumps (model 1003D) for maximum 2 days.

Radiotelemetric blood pressure measurements in conscious mice were performed by implanting pressure-sensing catheters (Data Sciences International, Saint Paul, MN, USA) into the left common carotid artery. After 7 days of recovery and reestablishment of diurnal blood pressure variation, blood pressure measurements were collected continuously with sampling every 20 min for 10-s intervals.

Echocardiographic measurements were performed with Vevo 2100 High Frequency Ultrasound System (Visualsonics, Toronto, Canada) using transducer MS400 (18–38 MHz) as described before [19]. Ejection fraction, end diastolic- and end systolic volume were calculated via Simpson’s protocol from parasternal short and long axis B-Mode images. Left ventricular mass was calculated from left ventricular inner diameter (LVID), left ventricular posterior (LVPW) and anterior wall (LVAW) (diastole) from short axis M-Mode.

For organ harvest mice were sacrificed by cervical dislocation. Hearts were then dissected, the apex of the heart was frozen in liquid nitrogen for RNA and protein analysis, the rest of the heart was OCT embedded and snap frozen in − 40 °C cold isopentane.

The following groups were analyzed: timeline analysis: four different groups of KO and control animals were either sacrificed at baseline (without AngII treatment), or after 12, 24 and 48 h of AngII treatment. Atglistatin treatment: all animals, KO and control, either Atglistatin or vehicle treated, were sacrificed after 48 h of AngII treatment. Propranolol treatment: all animals, KO and control, either propranolol or vehicle treated, were sacrificed after 48 h of AngII treatment. Anti-Ly6G treatment: all animals, KO and control, either anti-Ly6G or isotype treated, were sacrificed after 48 h of AngII treatment.

Western blot analyses

Proteins were isolated from heart apex in 150 mM NaCl, 10 mM Tris, 0.1% NP 40, pH 7.4, 4 °C using a tissue homogenizer (TissueRuptor, Qiagen). Protein content was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher #23225). SDS-PAGels were loaded with 25–50 μg total protein. Blotting was performed using Pierce™ G2 Fast Blotter (Thermo Fisher #62287) with preprogrammed method for mixed molecular weight (25 V, 1.3 mA, 7 min).

Primary antibodies were used in 5% BSA in TBST and are listed in Supplemental Table S1.

Normalization was performed using REVERT Total Protein Stain (Li-COR) according to the manufacturer’s recommendation as not otherwise specified.

Histological analyses

For immunofluorescence staining 8 µm frozen tissue sections were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 and permeabilized with 0.2% saponin in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 × 2H2O, 1.76 mM KH2PO4, pH 7.4). Blocking buffer (10% normal goat serum (NGS) in 0.2% saponin in PBS) was incubated for 60 min, primary antibody (in 2% NGS) overnight, 4 °C, secondary antibody for 3 h at room temperature in the dark. Mounting was performed with Pro long gold antifade reagent with DAPI (#P-36935, Invitrogen).

Primary antibodies: Anti-ADFP antibody (#ab52356), Abcam, (1:100), Monoclonal Mouse anti-Ly6g antibody (#551461), BD Pharming (1:50).

Wheat germ agglutinin: Lectin from Tritium vulgaris (wheat) (#W11261), Invitrogen, (10 µg/ml).

Secondary antibodies: Rhodamine Red-X-AffiniPure Goat Anti-Rat IgG (H + L) (#112-295-167), Jackson Immunoresearch Laboratories, Inc., (1:600), Cy3-AffiniPure Goat Anti-Rabbit IgG (H + L) (#111-165-144), Jackson Immunoresearch Laboratories, Inc. (1:600). For lipid staining frozen tissue sections were stained after fixation with Bakers formol–calcium fixative (3.6% Formol, 1% CaCl2) with filtrated sudan red staining solution (0.3% Sudan Red 7B (#201618, Sigma) in 60% isopropanol).

Tunel Assay was performed using Click-iT™ Plus TUNEL Assay for in situ Apoptosis Detection, Alexa Fluor™ 488 dye, ThermoFisher Scientific (C10617) according to the manufacturer’s recommendation.

Microscopy was performed with fluorescence microscope Keyence BZ 9000 (Keyence) or Zeiss Imager.M2. Overviews were taken using the “multi image-function”, where 4× magnifications were automatically put together.

Due to the obvious phenotype of iCMp38αKO mice analysis of histological sections could not be performed in a blinded manner.

Measurement of lipid profiles

The frozen heart tissue samples (80–120 mg) were homogenized in 2 mL destilled water by precellys ceramic beads using the Minilys homogenizer (Bertin Technologies). For tandem mass spectrometric analysis (Xevo-TQS, Waters) the neutral lipids were extracted from heart tissues by methanol/MTBE (methyl-tert-butylether) procedure (1:5, v,v) [29]. A flow injection analysis method was used and the mobile phases consisted of 50% A: methanol including 50 mM ammonium acetate and 50% B: acetonitrile/2-propanol/chloroform (45:45:10, v,v,v). An internal standard TG 21:1 d5-(17:0/17:1/17:0) was used (Avanti). The analysis of TG’s was performed in the positive ion mode. A neutral loss detection of 8 common acyl fragments in TG’s was performed [40]. The TG species were detected as ammonium adducts [M + NH4]+. Following mass transitions were used for fatty acids: C16:0 m/z 273; C16:1 m/z 271; C18:0 m/z 301; C18:1 m/z 299; C18:2 m/z 297; C18:3 m/z 295; C20:4 m/z 321; C22:6 m/z 345.

Gene expression profiling

RNA was isolated from the heart apex using Fibrous Tissue RNeasy Kit, Qiagen (#74704) according to manufacturer’s recommendation and used for real time-PCR and array and RNA-sequencing analyses.

Microarray analyses Total RNA preparations were checked for RNA integrity by Agilent 2100 Bioanalyzer quality control. All samples in this study showed high quality RNA integrity numbers (RIN; > 9). RNA was further analysed by photometric Nanodrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies).

Synthesis of cDNA and subsequent fluorescent labelling of cRNA was performed according to the manufacturers’ protocol (One-Color Microarray-Based Gene Expression Analysis/Low Input Quick Amp Labeling; Agilent Technologies). Briefly, 100 ng of total RNA were converted to cDNA, followed by in vitro transcription and incorporation of Cy3-CTP into nascent cRNA. After fragmentation labelled cRNA was hybridized to Agilent SurePrint G3 Mouse GE 8 × 60K Microarrays for 17 h at 65 °C and scanned as described in the manufacturers’ protocol.

Signal intensities on 20 bit tiff images were calculated by Feature Extraction software (FE, Vers. 11.0.1.1; Agilent Technologies). Data analyses were conducted with GeneSpring GX software (Vers. 12.5; Agilent Technologies). Probe signal intensities were quantile normalized across all samples to reduce inter-array variability [2]. Input data pre-processing was concluded by baseline transformation to the median of all samples.

To improve signal-to-noise ratio, a given transcript had to be expressed above background (i.e., called “detected” by FE) in all four replicates in any one of two, or both conditions to be further analysed in pairwise comparisons. Differential gene expression was statistically determined by moderated T tests (Benjamin–Hochberg FDR corrected, p(corr) < 0.05).

Microarray data were analysed using the IPA Ingenuity Pathway Analysis software Package (Qiagen Inc. 2016). The data set was subject to core analysis applying “Experimentally observed” as analysis filter. Canonical pathways, upstream regulators and causal networks tools were used to identify affected regulatory networks.

RNA-Seq analyses For transcriptome analyses total RNA samples were quantified using Qubit RNA HS Assay (Thermo Fisher Scientific). Quality was measured by capillary electrophoresis using the Fragment Analyzer and the ‘Total RNA Standard Sensitivity Assay’ (Agilent Technologies, Inc. Santa Clara, USA). All samples in this study showed high quality RNA Quality Numbers (RQN; mean = 9.0). Library preparation was performed with the ‘VAHTS™ Stranded mRNA-Seq Library Prep Kit’ for Illumina® according to the manufacturer’s recommendations. 850 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification. Bead purified libraries were normalized and finally sequenced on the NextSeq 550 system (Illumina Inc. San Diego, USA) with a read setup of 1 × 75 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well as for adapter trimming and demultiplexing.

Data analyses on fastq files were performed with CLC Genomics Workbench (version 21.0.4, QIAGEN, Venlo, NL). The reads of all probes were adapter trimmed (Illumina TruSeq) and quality trimmed (using the default parameters: bases below Q13 were trimmed from the end of the reads, ambiguous nucleotides maximal 2). Mapping was done against the Mus musculus (mm10; GRCm38.86; March 24, 2017) genome sequence. After grouping of samples (three biological replicates each) according to their respective experimental condition, a differential expression analysis of the two groups was made and statistically determined using the Wald test. The statistics are based on the fit of a Generalized Linear Model with a negative binomial distribution. The resulting P values were corrected for multiple testing by FDR and Bonferroni-correction. A p value of ≤ 0.05 was considered significant.

Quantitative real time PCR 1 µg mRNA was transcribed into cDNA using QuantiTect Reverse Transcription Kit, Qiagen (#205313). Real-time quantitative PCR (qPCR) was performed using Maxima SYRB Green/ROX qPCR Master Mix, Thermo Scientific (#K0223) and StepOne Plus Real-Time PCR Detection System (Applied Biosystems). All genes were amplified with 1 cycle at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The specificity of amplicons was verified by melting curve analysis starting with 95 °C for 15 s, then 60 °C for 60 s. Thereafter the temperature was increased 0.3 °C every 15 s, until the final temperature of 90 °C was reached. Two technical replicates of every biological replicate (n = 4/group) were used for qPCR analysis.

Because other commonly used internal reference genes as Hprt or Gapdh can be subject to regulation under various pathological conditions [34], we screened microarray data for stably expressed genes. Nudc showed lowest expression differences under all conditions compared by microarray analysis which was further validated by qPCR and was, therefore, used as reference gene. Due to variable expression Hprt and Gapdh had to be excluded.

Specific gene expression was first normalized to Nudc and then compared with control groups using both the comparative Cq method [25] as well as X(0) method. There the exponentially related Ct values are converted into linearly related X(0) values, where X(0) represent the amount of starting material in a qPCR experiment [48, 57]. Relative measurement for the following transcripts was performed: Il6, Il1b, Cxcl5, Cxcr2, Slc2a4, Ppargc1a, PDK4, Angptl4, TGFb2 (KiCqStart® SYBR® Green Primers, Sigma (#KSPQ12012)), for primer sequence see Supplemental Table S2.

To compare different qPCR plates a consistent calibrator was included on every plate. The calibrator was made of mRNA from hearts of different animals as well as different conditions. The mRNA was pooled and transcribed into cDNA, to guarantee that the same cDNA was used for each plate.

Transmission electron microscopy

Mouse hearts were prepared immediately after sacrifice by cervical dislocation and pre-perfused by aortic cannulation and retrograde perfusion with ~ 10 ml of Krebs–Henseleit-Buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4∙7H2O; 1.2 mM KH2PO4; 25 mM NaHCO3; 8,32 mM glucose; 1 mM lactate; 0.1 mM pyruvate; pH 7.4). After perfusion with 20 ml fixation buffer (0.1 M sodium cacodylate buffer pH 7.4; 1.2% glutaraldehyde, 1% paraformaldehyde), hearts were cut at respective regions to obtain tissue blocks (size 1 × 1 × 2 mm) and stored in storage buffer (0.1 M sodium cacodylate buffer pH 7.4; 1% glutaraldehyde). Samples were stained with 1% osmium tetroxide for 50 min and with 1% uranyl acetate/1% phosphotungstic acid for 1 h. A graded acetone series was used for dehydration before samples were embedded in spur epoxy resin at 65 °C for 24 h. Ultrathin sections were prepared using a microtome and images were acquired using a standard transmission electron microscope (Hitachi, H600) at 75 V equipped with Bioscan model 792 Gatan camera.

Atglistatin treatment

The lipolysis inhibition study was performed using Atglistatin, a small molecule inhibitor of adipose triglyceride lipase (ATGL) [30]. Atglistatin (Sigma-Aldrich, TargetMol) was administered via food at a dose of 0.4 mg/g CHOW food [52], 2 days prior to AngII mini-osmotic pump implantation and continued for 48 h thereafter. Mice were starved for 4–5 h prior to sacrifice. Plasma glycerol was measured using a commercial kit (Sigma-Aldrich, #MAK117), and performed according to the manufacturer's recommendation.

Propranolol treatment

Propranolol (Sigma-Aldrich) was solved in phosphate buffered saline (pH 5) and administered via drinking water (10 mg/kg/day) for 24 h and afterward for 48 h together with AngII via osmotic minipumps (10 mg/kg/day). Efficacy of propranolol was proven by a reduction in heart rate compared to vehicle-treated animals after dobutamine stimulation (1 mg/kg BW) (Supplemental Fig. S5A).

Flow cytometric analysis of hearts

Cells were isolated from the heart by a protocol adapted from [18, 43]. Hearts were isolated and flushed with PBS/heparin to remove the blood. Atria and valves were removed and hearts were cut in ~ 1 mm pieces using a tissue chopper (McIlwain tissue chopper, Cavey Laboratory Engineering Co. Ltd.). Tissue was incubated at 37 °C for 45 min in 450 U/mL collagenase type I (Worthington #LS004197) and 60 U/mL DNAse I (Roche Diagnostics #10104159001) in HBSS (Gibco Life Technologies #14025). After 15- and 30-min incubation tissue clusters were triturated by pipetting 12 times using a 10 mL serological pipette. At the end of the 45 min incubation period final trituration was performed by pipetting 30 times with a 1 mL pipette and the cells were centrifuged at 300×g for 10 min. The pellet was resuspended in FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) and filtered over 100 and 40 μm filters. Cardiomyocytes were removed by 1 min centrifugation at 50×g. The supernatant was centrifuged 10 min at 300×g and the pellet was dissolved in PBS and incubated with Fc-block (Biolegend, #101302) and, when applicable, Fixable viability dye eFluor 780 (eBioscience #65-0865-14). After washing, cells were stained in FACS buffer with the antibodies listed in Supplemental Table S3. All cells were washed prior to acquisition on BD FACSCanto II. Data were analysed using BD FACSDiva software version 8.0.2. A gating scheme is depicted in Supplemental Fig. S4.

Granulocyte depletion

Neutrophil granulocytes were depleted by i.p. injection of 500 μg anti-Ly6G-antibody, clone 1A8 (BioXCell, #BE0075-1) and corresponding isotype control (InVivoMab Rat IgG2a, clone 2A3, BioXCell, #BE0089) 16 h prior to osmotic mini pump implantation.

For flow cytometric analysis of circulating neutrophils EDTA anticoagulated blood was collected 48 h after implantation of osmotic mini pumps and stained followed by lysis of red blood cells. Cells were fixed in 1% neutral buffered paraformaldehyde for 20 min at room temperature, according to a protocol by [6]. To prevent unspecific binding, cells were incubated with anti-CD16/32 antibody (clone 93; Biolegend, San Diego, CA, USA) before antibody staining.

CD11b-PE (M1/70, BD Biosciences, San Jose, CA, USA) and anti-Ly-6C-AlexaFluor® 488 (HK1.4) were used to detect neutrophils and monocytes [21]. Respective isotype control IgGs were used to set all flow cytometric gates. Measurement and data analysis were performed using a Gallios™ Flow Cytometer, and Kaluza® Flow Analysis Software (both Beckman Coulter Inc., Krefeld, Germany).

Statistics

All data are presented as mean ± standard deviation. Data of each parameter were directly compared between control and iCMp38αKO mice. Statistical analysis was performed using Graph Pad Prism 5 and 8. Repeated measurements with two variables (time and genotype) were analyzed by two-way ANOVA or mixed-effects model in case of missing values followed by Bonferroni’s multiple comparisons test to compare the genotypes at each timepoint. Two groups were compared with unpaired, two tailed t test. For comparison of more than two groups, one-way ANOVA followed by Tukey’s multiple comparisons test was used as not otherwise specified. The present study must be classified as an exploratory study and all calculated p values are descriptive.

The following groups were compared when treatment was applied (Atglistatin, Propranolol, anti-Ly6G): ctrl. vehicle/IT treated vs. KO vehicle/IT treated, ctrl vehicle/IT treated vs. ctrl treated, ctrl treated vs. KO treated, KO vehicle/IT treated vs. KO treated. Inter-group comparisons were specified post-hoc. Correlation between cardiac neutrophil levels and ejection fraction (EF) was tested by Pearson correlation.

The sample sizes for the functional analysis were based on a power calculation and modified in line with the 3R principles and according to the experimental needs.

Statistical outliers were identified via ROUT test, Q = 1%. Based on this analysis two animals were excluded from the analysis in the following experiments: granulocyte depletion: KO, IT treated for parameter ejection fraction. Atglistatin treatment: KO, Atglistatin treated for parameter neutrophil number.