Tubulin expression and modulation in heart failure with preserved ejection fraction (HFpEF)

ZSF1 mice as a HFpEF . animal model

Animal care and procedures were performed in accordance with ARRIVE Guidelines and relevant Animal Care Guidelines and Regulations and were approved by the Local Animal Research Council, University of Leipzig and Landesbehörde Sachsen (TVV 30/18, TVV 40/19).

ZSF1-hybrid female ZSF1 mice were used between a Zucker female with adipose diabetes, and male mice with hypertensive heart failure were used as animal model (ZSF1-Lepr)football rabbitcp/ Carl, Charles River, Indianapolis, USA). We analyzed twelve 20-week-old O-ZSF1 (obese) and L-ZSF1 (lean) mice respectively, six O-ZSF1 mice and five 32-week-old L-ZSF1 mice. All animals were kept in identical conditions under a 12:12 h light/dark cycle with food and water provided with fame. The standard food rich in energy and protein content was delivered by Ssniff (Soest, Germany). Body weight and food intake were recorded each week. Non-invasive echocardiography (Vivid-J, GE Healthcare, Chicago, USA and Prospect Sharp T1, Scintica Instrumentation Inc., Maastricht, The Netherlands) was used to confirm the presence of HFpEF. Animals were sacrificed by attrition.

Sample preparation

Myocardial tissue was weighed prior to separation into the LV, RV, and septum. Samples were immediately frozen in liquid N2 Store at -80°C until further use. Frozen samples were crushed and used for protein and RNA analysis. RNA isolation was performed with up to 30 mg of tissue using the RNeasy Kit and QIAShredder (Qiagen, Hilden, Germany) according to the manufacturer’s recommendation. RNA quality was determined using a Fraqment Analyzer (Agilent, Santa Clara, USA) and evaluated using PROsize 2.0 software. RNA quality numbers ranged from eight to ten (ten indicates the highest quality RNA). RNA concentration was determined optically, and 200 ng of RNA was transcribed using an Omniscript RT kit with oligo dT Primer (Qiagen, Hilden, Germany). For protein analysis 10-20 mg of the frozen sample was analyzed in RIPA buffer (10 mM HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) containing a mixture of protease and phosphatase inhibitors (Serva, Heidelberg, Germany) and sonication. Protein concentration was analyzed using the BCA method (bicinchoninic acid assay, Pierce, Bonn, Germany). Blood is drawn at the sacrifice of the direct puncture to the heart before it stops beating.

Western blot analysis

To determine protein expression, 12% SDS-polyacrylamide gels were loaded with 20 μg of protein before being transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% milk (blotting grade, Roth, Germany), incubated overnight at 4 °C with primary antibodies and rinsed with 1 × TTBS 3 × 10 min. Peroxidase-conjugated secondary antibodies were incubated in 1% milk for 1 h and specific bands were visualized by enzymatic chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc., Bonn, Germany).

Densitometers were measured for each selected band using a 1D semi-automated scanning software package (Vision-Capt, Vilber Lourmat, Eberhardzell, Germany). Each range is selected manually and the density measurement is determined automatically [see supplement for original Western Blots (Supplementarys Fig. 3A–E, 4A–E) and example of ROI determination (Supplementary Fig. 2)]. Amounts of α-tubulin (Abcam ab7291, 1:10000), metyrosine tubulin (Sigma T9028, 1:400), de-tyrosine tubulin (Millipore ab3201, 1:500), TTL (Proteintech 13618-1-AP, 1:1000) and VASH1 (Abcam ab199732, 1:1000). To normalize α-tubulin (50 kDa), tyrosine (55 kDa) and dissolved tubulin (55 kDa), we used glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 37 kDa) (HyTest 5G4, 1:30000) and to normalize TTL (43 kDa) and VASH1 (41 kDa) we used acetylated histone 2B (H2B, 17 kDa) (Elabscience E-AB-66503, 1:500). We used peroxidase-conjugated secondary antibodies (Sigma POD 9044 anti-mouse, 1:5000 or Millipore AP187P anti-rabbit, 1:10,000; depending on the host species of the primary antibody). All protein amounts were normalized to the mean level in the L-ZSF1 control group.

Quantitative real time-PCR

Quantitative Realtime-PCR was performed to detect the gene expression of Tuba4a, Vash1, Ttl and Npbb in O-ZSF1 and L-ZSF1 mice. We used Takyon NoRox Sybr Mastermix Blue (Eurogentec, Lüttich, Belgium) on a BioRad CFX System (BioRad, Hercules, USA). Exon-spanning primers are designed with an annealing temperature of 60 °C (sequences in Table 2). To estimate the copy number and determine the interaction efficiency, we used standard curves. All samples were measured in triplicate and samples with standard deviation >0.250 were excluded. Hypoxanthine phosphoribosyltransferase 1 (Hprt1) was tested, identified as a stable housekeeping gene, and used for target gene normalization. Furthermore, expression of target genes in O-ZSF1 was normalized to L-ZSF1 mice.

Table 2 primers used for real-time quantitative PCR.

Investigation of NT-proBNP

N-terminal pro Brain Natriuretic Peptide (NT-proBNP) in serum was measured using ELISA assays according to the manufacturer’s recommendations (abx576280, Hölzel Diagnostika, Cologne, Germany and abx256287, Hölzel Diagnostika, Cologne, Germany). Because the manufacturers are different, the two assays cannot be compared directly. Due to sample limitations, it was not possible to repeat the measurements for all animals using the same assay.

statistical analysis

Data are presented as means ± standard deviation. P values ​​were calculated using Student’s t-test (for normal distribution) and non-parametric Mann-Whitney-U test. Figures were made using GraphPad Prism 8 (GraphPad Software, San Diego, USA). s– Value < 0.05 was considered statistically significant and s– A value < 0.1 was considered as a trend.