SIRT5 is one of the seven members of the NAD+-dependent sirtuin family of protein deacylase that is mainly present in mitochondria and regulates metabolism. In heart, SIRT5 is highly expressed and responsible for succinylation on metabolic enzymes. Although the role of SIRT5 in maintaining cardiac homeostasis under physiological stress and few potential substrates of SIRT5 have been preliminarily revealed, the regulatory network and key cellular signaling pathways involving SIRT5 in myocardial hypertrophy remain largely unknown. Here, we used an established murine model of pressure overload-induced myocardial hypertrophy caused by transverse aortic constriction (TAC) to outline the network and pathway involving SIRT5 in cardiac stress responses. Remarkably, SIRT5 KO mice had enhanced myocardial hypertrophy after TAC surgery compared with wild-type mice.
Overall design: The 5 weeks old conventional SIRT5 knockout (SIRT5 KO), basing on the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) methods, male mice (C57BL/6N) and wild-type (WT) male mice using the tamoxifen-inducible Cre-ERT2 system. Each mouse was intraperitoneally injected with 1 mg tamoxifen every 24 h for a total of 10 consecutive days. There is a 4-week waiting period between the final injection of tamoxifen and the establishment of TAC model. After the mice were weighed, TAC surgery was performed under general anesthesia intraperitoneally injected with 50 mg/kg pentobarbital sodium. Mice were fixed on a constant-temperature operating table in the supine position for a trans-sternal thoracotomy. After the aortic arch appeared clearly, a 27-gauge needle was placed along the thoracic aorta, followed by a tight banding between the aorta and the needle with a 6-0 suture. Then, the needle was withdrawn and the chest was closed. The mice were monitored on the operating table until completely recovered. Sham-operated mice underwent the same procedure but without banding. Intraperitoneal injection of 1×104 U penicillin was performed to prevent infection for a total of 3 consecutive days after surgery. The left ventricular myocardial samples were collected from TAC-induced cardiac hypertrophy mice. 2.0g tissue per samples were used for total RNA extraction.Ribosomal RNA (rRNA) was removed from Total RNA using QIAseq FastSelect RNA Removal Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instructions. The enriched mRNAs and non-coding RNAs were fragmented into short fragments (approximately 300 bp) using a fragmentation buffer and reverse-transcribed to cDNA with random primers, and this first-strand cDNA was used as the template for second-strand cDNA synthesis. Then, the cDNA fragments were end-repaired and ligated to Illumina sequencing adapters. The second-strand cDNA was digested using Uracil-DNA Glycosylase (UDG). The digested products were size selected (approximately 450 bp), PCR amplified, and quality assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). After cDNA library construction, a paired-end sequencing was performed using Illumina Hiseq 4000 systems (Illumina, San Diego, CA, USA). The raw data were filtered, and the clean data were mapped to the reference genome Mus_musculus. GRCm38. RNA sequencing libraries were constructed using Illumina TruSeq RNA Preparation kit and Illumina Ribo-Zero GoldrRNA Removal Kit according to the manufacturer’s instructions, and subsequently sequencing was performed via Illumina NextSeq 500 with the 300bp paired-end reads. The filtered RNA reads alignment and map on reference genome (GRCm38) via HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml). Consequently, the raw read counts were quantified by HTSeq software (https://htseq.readthedocs.io,version 0.6.1) based on BAM files. Fragments Per Kilo bases per Million fragments (FPKM), quantified by Stringtie (https://ccb.jhu.edu/software/stringtie/, version 1.2.0) software, was applied normalizing for gene expressional differential analysis.
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