Mapping cardiac remodeling in chronic Heart disease

we performed single nuclear RNA-sequencing of left ventricular nuclei from two murine models of cardio-renal disease using SV129 mice. We validated data using inducible genetic fate tracing combined with single cell RNA-sequencing as well as immunostaining and cell-culture models Male 129S2/SV mice (n=7) were obtained from Envigo and used 6-8 weeks old. Mice were group-housed and provided with ad lib access to water and standard chow, as well as environmental enrichment. A 12-hour light-dark cycle was maintained. During the study, mice were weighed weekly and had their condition recorded. ARRIVE guidelines were adhered to. Animals were randomised to receive sham or subtotal nephrectomy surgery (STNx) using a random number generator website (random.org) For sham surgery, animals had bilateral flank incisions and both kidneys isolated and manipulated. At 10 weeks animals had a timed overnight urine collection then were culled by an approved schedule 1 method and terminal blood sample obtained (Figure 1a). For all animals tibia length was recorded. Urine and serum were analysed by in-house biochemical analysis service (surf.ed.ac.uk/facilities/specialist-assay-service/). Six 129S2/SvPasOrlRJ wildtype male mice underwent ischemia reperfusion injury (IRI) plus uninephrectomy at the age of 11-12 weeks and were housed for 21 more weeks。 Mice were anesthetized with ketamine/xylazine (120/16 mg/kg body weight) and after disinfection, flanks were incised, the right kidney was clamped for 26min and the contralateral kidney was fully removed. Mice received buprenorphine for three more days (0.1mg/kg) to achieve analgesia. For genetic fate tracing of Gli1+ cells, we bred bigenic Gli1CreER;tdTomato using Gli1tm3(re/ERT2)Alj/J, JAX Stock #007913 and Rosa26tdTomato (i.e., B6-Cg-Gt(ROSA)26Sorttm(CAG-tdTomato)Hze/J JAX Stock # 007909) mice. 6-7 week old male mice (n=7) received 3x 2 mg tamoxifen in corn oil with 3% ethanol via intraperitoneal injection to induce recombination, and underwent the unilateral IRI plus contralateral nephrectomy surgery at 21 days after the last tamoxifen dose. Mice were killed 21 weeks after surgery and the left ventricle was snap frozen in liquid nitrogen and transferred to -80°C. Serum urea was measured by clinically laboratory routine (Vitros 250, Ortho Clinical Diagnostics, Institut für Versuschtierkunde, Aachen). Heart weights were recorded (Sartorius 2007 MP, Germany) and normalized to tibia length, measured by a caliper (Forum, Germany). Animal experiment protocols were approved by the LANUV-NRW, Germany and by the UK Home Office Regulations. All animal experiments were carried out in accordance with their guidelines.