Chemogenetic activation of neurons in the rat medial preoptic area (MPA, A) induces synthetic torpor.

characterised by reduced core body temperature (B, F(3,24) = 106.4. p < 0.0001), bradycardia (C, F(3,24) = 9.637, p = 0.0002), and reduced oxygen consumption (D, MPAGFP t(7) = 0.5655, P = 0.5894, MPAGq t(7) = 4.305, P = 0.0035), For B and C, n = 6 controls and 10 synthetic torpor, mean value for 90-100 minutes after CNO 2mg/kg IP. For D, n = 8 MPAGFP and 8 MPAGq pre (50-60 minutes) vs post (120-130 minutes) CNO. Shaded area indicates S.E.M. * indicates p <0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Abbreviations: TB, core temperature; VO2, oxygen consumption; HR, heart rate; CNO, clozapine-N-oxide; ST, synthetic torpor; bpm, beats per minute.

Characterisation of neurons in the rat MPA responsible for inducing synthetic torpor.

A, viral vector transduction with mCherry in the MPA (red) and corresponding anatomical schematic. B, RNA in-situ hybridisation labelling vector transduced cells (mCherry, red), and those expressing Vglut2 (yellow) and/or Vgat (turquoise). C, RNA in-situ hybridisation labelling vector transduced cells (mCherry, red), and those expressing Ptger3 (purple), Lepr (green), and Opn5 (turquoise). White arrows indicate transduced cells that express Ptger3, Lepr, and Opn5. D, co-expression of Vglut2 and Vgat amongst transduced cells. E, expression of QPLOT markers amongst vector transduced cells (note all transduced cells expressed Ptger3). F, schematic showing extent of viral vector transduction common to all animals. Abbreviations: MPA, medial preoptic area; MnPO, median preoptic nucleus; VOLT, vascular organ of the lamina terminalis; 3V, 3rdventricle; AVPe, anteroventral periventricular nucleus; VMPO, ventromedial preoptic nucleus; och, optic chiasm; aca, anterior commissure (anterior part); Vglut2, vesicular glutamate transporter 2; Vgat, vesicular GABA transporter; Ptger3, prostaglandin EP3 receptor; Lepr, leptin receptor; Opn5, opsin-5.

Synthetic torpor protects the heart from ex vivo ischaemia-reperfusion injury.

Rats first underwent bilateral injection of vector (hM3DGq or GFP) into the MPA. After two weeks, they all received CNO, inducing synthetic torpor in rats that had received hM3DGq vector, followed by removal of the heart and ischaemia-reperfusion in the Langendorff preparation (A). Pre-conditioning with synthetic torpor resulted in significantly reduced infarct size as measured by 2,3,5-Triphenyltetrazolium Chloride (TTC) staining (B, C). Scale bar represents 1cm. This cardioprotective effect was not dependent on a reduction in core temperature as rats held at a thermoneutral ambient temperature following CNO injection were still protected (C, ANOVA F (3,35) = 6.542, p = 0.0013). Inducing synthetic torpor in a thermoneutral environment prevented the drop in core temperature (D, F(3,24) = 106.4. P < 0.0001), but not the bradycardia (E, F(3,24) = 9.637, p = 0.0002). Holm-Sidak correction for multiple comparisons. Data shown is median and IQR. * = p <0.05, ** = p < 0.01, **** = p < 0.00001, ns = p >0.05. Abbreviations: CNO, clozapine-N-oxide; RV, right ventricle; LV, left ventricle; TB, core temperature; HR, heart rate; bpm, beats per minute.

Phosphoproteome analysis of the heart identifies gene pathways associated with synthetic torpor.

,Volcano plot showing differentially expressed phosphopeptides after limma modelling (A). Heatmap shows the top 300 differentially expressed phosphosites between synthetic torpor (n = 5) and controls (n = 7) after limma modelling,rows (phosphosites) and columns (sample) clustered by similarity of expression so that phosphopeptides that covary cluster and samples that covary also cluster toether.(B). Principal component analysis confirms group separation and effective batch correction (C). Gene-level set enrichment analysis (GSEA) performed using a single representative phosphosite per gene (selected based on limma t statistic) identifies key pathways modulated by entry into synthetic torpor (D).

Site-level phosphoproteome analysis.

After mapping from rat to human phosphoproteins, post-translational set enrichment analysis (PTM-SEA) was performed on 1384 aligned phosphosites from 595 rat gene-human protein pairs. Kinase signatures are shown ranked by normalized enrichment score (NES); the top 15 by absolute NES are labelled for clarity. Colour represents −log10(raw p-value), and point size indicates pathway size.

Changes to phosphoproteome signature that drive cardioprotection in synthetic torpor.

Synthetic torpor induction by MPA neuronal activation induces changes to kinase signatures in cardiac tissue which are associated with cell survival/stress, cell cycle/death and cellular function. Activated kinases are shown in green and inhibited kinases in blue. Arrows indicate kinases that act downstream of the ones above. Abbreviations: CK2A2, casein kinase II alpha II subunit; AMPKA1, AMP-activated protein kinase alpha 1; MAPK10/JNK3, Mitogen-activated protein kinase 10/ c-Jun N terminal kinase; ACVR1, Activin A receptor type 1; MAPK12/13/14, Mitogen-activated protein kinase 12/13/14; MK2/3/5, Mitogen-activated protein kinase-activated protein kinase 2/3/5; AKT1, AKT serine/threonine kinase 1; p70S6K, 70 kDa ribosomal protein s6 kinase; CAMKIIA/G, calcium/calmodulin-dependent protein kinase II A/G; TP53, Tumour protein p53; PI3K, Phosphoinositide 3-kinase; mTOR, mechanistic target of rapamycin.

RNAscope probes used for ISH.

https://rddc.tsinghua-gd.org/

Pre-Langendorff in vivo physiological changes (A,B) and ex vivo heart rates (C).

Core temperature (A) and heart rate (B) in vivo prior to excision of heart for Langendorff preparation. A,B, n = 6 controls, 10 synthetic torpor, 6 thermoneutral synthetic torpor, & 6 thermoneutral controls. (C) No significant differences in ex vivo HR were observed between the four groups during the 30-minute equilibration period of the Langendorff assay (F(3,19) = 1.35, p = 0.29). Controls (n = 16), torpor (n = 12), thermoneutral synthetic torpor (n = 5), & thermoneutral controls (n = 6). Data shows mean and 95% confidence interval. Abbreviations: ST, synthetic torpor; HR, heart rate; bpm, beats per minute.

Phosphoproteomics work flow.

Proteomics QC metrics.

Heat map of 600 most variable phosphosites prior to limma modelling and batch correction (A) and principal component analysis of the same uncorrected log2 normalised phosphosites (B) both showing clear batch effect. High sample to sample correlation (all samples, top panel and within batch, bottom panel), indicating subtle biological effects and no outlier samples or process-specific effects (C). Sample-wise expression distributions assessed by computing the median and interquartile range (IQR) of log-transformed phosphoprotein intensities for each sample confirm no global shifts in signal intensity or variability prior to downstream modelling (D). Composite QC outlier scores confirms no outliers (E).