Quaking Inhibits Doxorubicin-Mediated Cardiotoxicity Through Regulation of Cardiac Circular RNA Expression

Supplemental Digital Content is available in the text.


Cell size measurement
Primary neonatal rat cardiomyocytes were seeded in 48 well plates, fixed with paraformaldehyde, permeabilized with 0.1% Triton-X-100 and stained with sarcomeric alpha-actinin (Sigma-Aldrich #A7811) and dapi. Images were taken with Nikon Eclipse Ti microscope and images were analyzed with NIS Elements. For analysis in each case, ten different images were analyzed from different regions and average value was taken. Three data points representing three individual experiments are shown.
Paraffin embedded or cryo preserved heart sections were stained with Alexa Fluor 488 labelled wheat germ agglutinin (Invitrogen) and dapi. Images were taken with Nikon Eclipse Ti microscope and images were analyzed with NIS Elements. For each heart six-ten different images were taken from different regions of the heart section and nearly two hundred to six hundred cells (depending upon the availability) area were measured and average value was taken for each heart.

Immunostaining
Neonatal rat cardiomyocytes or HL-1 cells were fixed with 4% paraformaldehyde for ten minutes and permeabilzed with 0.1% Triton-X-100 for 10 minutes. Blocking was done for thirty minutes with donkey serum and then incubated with QKI antibody (Sigma-Aldrich) (1:100) at 4°C overnight. Next day, cells were incubated with secondary antibody Anti-Rabbit-Alexa 594/488 (Invitrogen) and dapi for 30 minutes. Images were taken with Nikon Eclipse Ti microscope and images were analyzed with NIS Elements.
Similarly, cryosections of hearts were also stained for QKI.

Electron microscopy
All tissues were immersion-fixed in 150 mM HEPES, pH 7.35, containing 1.5% formaldehyde and 1.5% glutaraldehyde. Embedding and section preparation was done as before 4 at Institute of Functional and Applied Anatomy, Hannover Medical School. Images were at Transmission Electron Microscopy Morgagni 268 located in Central Electron Microscopy Facility, Hannover Medical School.

RNA Isolation & PCR
RNA isolation from cell culture and heart tissue was done using Trifast (Peqlab) as per the manufacturer's instructions. Isolated RNA (500ng-1000ng) was reversed transcribed with random primer using iScript Select cDNA synthesis kit (Biorad). Real-Time quantative PCR was done with iQ SYBR Green mix (Biorad) on C1000 Touch Thermocycler (Biorad) using specific primer pairs listed in Supplementary Table 4. For amplification of circular RNAs, divergent primers were used while normal linear transcripts were amplified by convergent primers as usual. For RNase R resistant assay RNA was incubated with RNase R (Biozym Scientific) at 37°C for 10 minutes and heat activated at 95°C for three minutes. RNA was then reverse transcribed and amplified by specific PCR primers as mentioned before. Circular RNAs screening were performed in pLV Empty, pLV Qki5, Cri Empty, Cri Qki 3+4 and Cri Qki 5+6 cell lines in triplicates. Validation was performed as n=3 individual experiments with three replicates each time.

Lentiviral overexpression cell lines
Qki5, Qki6 and Qki7 cDNA were synthesized as gene string from Thermo Fisher Scientific and cloned in pLV lentiviral backbone. Lentiviral overexpression of Qki5, Qki6 and Qki7 in HL-1 cells was mediated by transduction of lentivirus from pLV plasmid carrying cDNA sequence of Qki5, Qki6, Qki7 and empty control. pLV plasmid were provided by Prof. Axel Sambach, Hannover Medical School. For lentiviral production, HEK293T were transfected with pLV plasmid together with helper plasmids (vsvg, gagpol, rev and NovB2) using CaCl2. Medium was changed after eight hours of transfection. Viral supernatant was collected forty eight to seventy two hours later. HL-1 cells were transduced with lentivirus and after three days puromycin selection was initiated to remove nontransduced cells. After puromyocin selection, overexpression was confirmed by western blot.
Similarly, exonic sequence of Ttn 105-111 circular RNA was synthesized as gene string from Thermo Fisher Scientific and cloned in circular RNA forming plasmid pCDNA3.1(+) ZKSCAN1 MCS exon vector (Addgene) with EcoRV and SacII. Then the insert together with repeat element responsible for circularization was released with digestion using KpnI and XhoI and cloned into pLV (lentiviral) plasmid. Virus production and permanent cell line were made similar to Qki.

Western Blotting
Cell pellet were lysed in 1X Cell lysis buffer (Cell Signaling) and isolated protein was measured by Bradford (Biorad) for quantification. 15 -30 μg of protein was loaded for each sample on SDSpolyacrylamide gel to resolve the protein. Proteins were transferred from SDS PAGE gel to polyvinylidene fluoride membrane in Mini PROTEAN Tetra cell (Biorad). Specific proteins were identified by following antibodies: Quaking (Sigma #HPA019123) and Vinculin (Sigma #V9131). HRP conjugated secondary antibody (Cell Signalling) was used for detection of bands. Band intensity was calculated by Image J software.

Human Cardiac tissue samples
Human patient data was provided by Prof. Hendrik Milting. Heart tissue samples were obtained at Herz-und Diabeteszentrum NRW, Ruhr-Universität Bochum, Bad Oeynhausen. Patients gave informed consent for the use of their explanted hearts (ethical committee by the Medical Faculty of the Ruhr-University Bochum, suboffice Bad Oeynhausen). Failing group included ten patients each with ischemic cardiomyopathy and dilated cardiomyopathy, while non-failing included ten healthy donor hearts. Details of the patients are provided in Supplementary Table 4.

mRNA profiling
Microarray was performed on n=3 samples from each vehicle control and doxorubicin. The Microarray utilized represents a refined version of the Whole Mouse Genome Oligo Microarray 4x44K v2 (Design ID 026655, Agilent Technologies), called '048306On1M' (Design ID 066423) developed at the Research Core Unit Transcriptomics (RCUT) of Hannover Medical School. Microarray design was created at Agilent's eArray portal using a 1x1M design format for mRNA expression as template. All non-control probes of design ID 026655 have been selected to be printed four times within a region comprising a total of 181560 Features (170 columns x 1068 rows). Four of such regions were placed within one 1M region giving rise to four microarray fields per slide to be hybridized individually (Customer Specified Feature Layout). Control probes required for proper Feature Extraction software operation were determined and placed automatically by eArray using recommended default settings.
Prior to the reverse transcription reaction, 0.5μl of a 1:1000 dilution of Agilent's 'One-Color spike-in Kit stock solution' (#5188-5282, Agilent Technologies) were added to each 250ng of total RNA sample.
cRNA fragmentation, hybridization and washing steps were carried-out as recommended in the 'One-Color Microarray-Based Gene Expression Analysis Protocol V5.7', except that 800ng of each fluorescently labeled cRNA population were used for hybridization.
Extracted signal intensities were filtered with cut-off of 2000 to remove low expressed mRNAs and global normalization was performed. Candidates with significant fold change of 1.5 were selected and heatmap was generated with Clustvis online tool.

Adeno-associated virus production
HEK 293T cells were transfected with AAV-Empty or AAV-Qki5 plasmid together with pDG (a kind gift from Prof. Roger Hajjar, Mount Siani Hospital, New York) (pDG2 for AAV2 and pDG9 for AAV9) with polyethylenimine. Medium was changed next day and cells were left for seventy two hours. Supernatant was collected and mixed with 40% Polyethylene Glycol 8000 solution for an hour and later incubated overnight at 4°C. Cells were harvested and lysed in presence of benzonase (Novagen). Supernatant was centrifuged at 2800xg for 15 minutes to collect precipitated virus and mixed with the cell lysate. Cell lysate was ultracentrifuged on iodixanol (OptiPrep, Progen) gradient in ultracentrifuge (Beckman Coulter) at 63000 rpm for one hour in Ti 70 rotor. AAVs were extracted from 40% fraction and concentrated using Amicon Ultra 15 (Millipore) columns. AAvs were titrated with PCR primer amplifying CMV (Cytomegalovirus) promoter.