Mouse line generation and maintenance

Male and female C57BL/6J GPR37L1^wt/wt, GPR37L1^flx/flx, GPR37L1^wt/KO, GPR37L1^KO/KO, and GPR37L1^lacZ/wt were generated using the European Conditional Mouse Mutagenesis Program (EUCOMM) method for conditional gene inactivation using Cre/loxP and Flp1/FRT gene trapping [9] described as GOI#1 in a previous report [10]. Mice were group-housed in a temperature-controlled environment with a 12-h light-dark cycle (lights on at 0700). Standard chow and water were available ad libitum. All animal work was performed in accordance with the Australia Code for the Care and Use of Animals for Scientific Purposes, 8th Edition (2013), and was approved by the relevant Animal Ethics Committees (Garvan Institute for Medical Research/St Vincent’s Hospital, project number AEC 13/30; University of Melbourne, project number 1212573). All animals were entered into the study in a randomized order.

Quantitative PCR

Tissue was homogenized in TRIzol (15,596,026, Thermo Scientific, USA) using a PRO200 homogenizer (Bio-Gen, USA), and RNA was extracted using manufacturer’s protocol (Thermo Scientific). DNA contamination was removed from extracted RNA with TURBO DNAse kit treatment (AM1907, Invitrogen, USA) before cDNA synthesis was performed using SuperScript® III First-Strand Synthesis Supermix as per manufacturer’s protocol (11752050, Invitrogen). cDNA was stored at − 20 °C until use. Quantitative PCR (qPCR) was performed on cDNA using TaqMan Gene Expression Master Mix (4369016, Applied Biosystems, USA) and TaqMan probes (Applied Biosystems). All qPCR was performed using a LightCycler 480 (Roche, Switzerland) (cycling conditions: preincubation at 95 °C for 10 min; 55 amplification cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s; final cooling at 50 °C for 10 s). Probe Mm00661872_m1 was used to detect GPR37L1 in mouse. The following probes were used to detect components of the fetal gene program: ANP (Mm01255747_g1), BNP (Mm01255770_g1), β-MHC (Mm00600555_m1), and α-SkA (Mm00808218_g1). ΔCt values were calculated in reference to HPRT (mouse, Mm03024075_m1) or when GPR37L1 transcript abundance was determined in EUCOMM mice; ΔCt values were calculated in reference to β2M (mouse, Mm00437762_m1). Relative mRNA abundance was determined using the 2^−ΔΔCt method [11].

Tissues for pilot assay of GPR37L1 transcription

In reference to the experiment shown in Additional file 1: Figure S1, surplus tissue (left and right ventricles, left and right atria, aorta, kidney, lung, liver, and brain) from sham-operated wild-type C57BL/6J mice (12 weeks old, male), as harvested for a previous study [12], was used to generate cDNA to quantify GPR37L1 by qPCR.

Tissue fixation for β-galactosidase imaging

Tissue fixation was performed as adapted from a previous method [13]. At ≥ 12 weeks of age, GPR37L1^wt/wt and GPR37L1^lacZ/wt mice of either sex were anesthetized with 5% isofluorane in oxygen (flow rate 1 L/min) delivered via nose cone. The thoracic cavity was opened, and the LV was punctured with a needle that was then used to perfuse the heart with phosphate-buffered saline (PBS) (1 min; flow rate 20 mL/min), achieving sacrifice by exsanguination. Immediately after, tissues were fixed by further 2 min perfusion with 4% paraformaldehyde (PFA) in PBS (flow rate 20 mL/min). Following fixation, the brain, heart, and kidney were collected and further fixed in 4% PFA/PBS for 48 h at room temperature (RT). After post-fixation, tissue was stored in sucrose cryoprotectant buffer (5 mmol/L Trizma base, 77 mmol/L NaCl, 4 mmol/L Na₂HPO₄, 1.3 mmol/L NaH₂PO₄, 20% w/v sucrose) at 4 °C for ≥ 48 h. Fixed and cryoprotected tissue was embedded in Tissue Freezing Medium (TFM-5, Triangle Biomedical, USA) and frozen. Frozen tissue was cut into 12 μm sections using a CM1950 Cryostat (Leica Biosystems, Germany) and mounted on Superfrost Plus Slides (Thermofisher Scientific).

β-galactosidase immunofluorescent staining

The protocol was performed at RT unless otherwise specified. Slides containing mounted tissue sections from the above procedure were washed for 10 min, three times, in immunobuffer [TPBS (10 mmol/L Tris, 8 mmol/L Na₂HPO₄, 2.6 mmol/L NaH₂PO₄, 0.9% NaCl, 0.05% thimerosal) with 0.3% Triton X-100]. Non-specific antibody binding was blocked by incubating each section in immunobuffer containing 10% v/v normal donkey serum (NDS) (017-000-121, Jackson ImmunoResearch Laboratories, USA) for 30 min, in a humidifying chamber. Primary antibodies [1:250 chicken anti-β-galactosidase (AB9361, Abcam, Cambridge, UK) for all tissue, 1:500 rabbit anti-GFAP (Z0334, Agilent Technologies, USA) for brain only] were diluted in immunobuffer with 10% v/v NDS and applied to each section for 16 h, in a humidifying chamber at 4 °C. Slides were then washed for 10 min, three times, in TPBS. For the remainder of the protocol, slides were shielded from ambient light. Secondary antibodies [1:500 donkey anti-chicken Cy3 (703-165-155, Jackson ImmunoResearch Laboratories) for all tissue, 1:500 donkey anti-rabbit Alexa Fluor 647 (711-605-152, Jackson ImmunoResearch) for brain only], DAPI (1:5000 4′,6-diamidino-2-phenylindole dihydrochloride; D9542, Sigma Aldrich, USA) nuclear stain, and wheat germ agglutinin (WGA) membrane stain (for heart and kidney only, 1:500 Alexa Fluor 488 WGA conjugate; W11261, Invitrogen) were diluted in immunobuffer containing 10% v/v NDS and applied to each section for 1 h, in a humidifying chamber. Slides were then washed for 10 min in TPBS. To reduce autofluorescence, each section was then treated with 0.1% w/v Sudan Black B (S2380, Sigma Aldrich) in 70% v/v ethanol for 1 h, in a humidifying chamber. Excess dye was then removed by washing for 10 min, three times, in TPBS. Slides were then dried for 16 h in a humidifying chamber before being sealed with glass coverslips mounted using 2.5% w/v DABCO (1,4-diazabicyclo-[2,2,2]-octane; D2522, Sigma Aldrich), 10% w/v polyvinyl alcohol (P8136, Sigma Aldrich), 25% w/v glycerol (G5516, Sigma Aldrich), and 100 mmol/L Tris buffer (pH 8.7), as adapted from a previous method [14]. Slides were imaged on a LSM 7 Duo confocal microscope (Zeiss, Germany).

Immunoblotting and densitometry

Tissue homogenates and crude membranes were prepared as previously described [15, 16]. SDS-PAGE and immunoblotting was performed as previously described [15]. GPR37L1 was detected using goat anti-GPR37L1 (C-12) antibody (1:1000; sc-164532, Santa Cruz Biotechnology, USA) that was previously confirmed to be GPR37L1-specific using knockout tissue [15] and then rabbit anti-goat antibody (1:7500; 61-1620, Invitrogen). β-galactosidase was detected using rabbit anti-β-galactosidase antibody (1:5000; A-11132; Invitrogen) and then donkey anti-rabbit antibody (1:15000, NA934, GE Healthcare, Australia). GAPDH was detected using rabbit anti-GAPDH antibody (1:10000; 14C10, Cell Signaling, USA) and then donkey anti-rabbit antibody (1:20000, NA934, GE Healthcare). SuperSignal™ West Pico (34080, Invitrogen) or Clarity™ (1705060, Bio-Rad, USA) were used as chemiluminescent substrates. Densitometry was performed using ImageJ software (https://imagej.nih.gov/ij/) on non-saturated chemiluminescent exposures. Pixel density of background was measured and subtracted from subsequent measurements taken on the same image. Pixel density of GPR37L1 was measured in reference to the predominant, cleaved receptor species [15] and adjusted to equivalent protein abundance using each sample’s own GAPDH pixel density score.

Hemodynamic measurement and analysis

At age 10–12 weeks, mice were anesthetized with 1–2% isofluorane (Zoetis, USA) in oxygen (flow rate 0.5 L/min), delivered via nose cone regulated using a mechanical ventilator (150 strokes/min, 200 μl stroke volume). Hemodynamics in the aorta and LV were recorded with a high-fidelity pressure transducer (Millar Instruments, USA) following its insertion into the right carotid artery as described previously [17, 18]. Heart rate was maintained at approximately 500 beats per minute (bpm) by manual adjustment of the isofluorane concentration. Data were recorded and analyzed using AcqKnowledge software (version 3.9.0 for Windows, BIOPAC Systems, USA) by an observer blinded to genotype (and to treatment where applicable). Hemodynamics on osmotic mini pump animals were recorded using a Transonic Scisense (FTH-1211B-001, USA) high-fidelity pressure transducer.

Radiotelemetry blood pressure measurement

At 12–14 weeks of age, mice were anesthetized with 2% isofluorane (Abbott Laboratories, USA), with the rate adjusted to provide a deep surgical plane. PA-C10 telemeters (Data Sciences International, USA) were implanted as previously described [19], with the pressure-sensing catheter inserted into the left carotid artery. Analgesia was administered before and after surgery (carprofen, 0.5 mg/100 g, IP; Norbrook, UK). Hydration was maintained by administration of 0.5 mL 0.9% NaCl (IP), given at the time of operation and again 24 h later. Approximately 2 weeks after telemeter implantation, blood pressure monitoring was performed twice daily in the home cage, between 09:00 and 12:00 (light phase) and between 21:00 and 24:00 (dark phase) for 9 days. The average of the 9 days of measurements in each phase was taken to give a single value per animal per phase. [N.B. radiotelemetry blood pressure monitoring was only performed during initial phenotyping experiments in Fig. 2c, d, as indicated].

Angiotensin II infusion

Osmotic mini pumps (product 1007D, Alzet) were filled with either vehicle (0.15 mol/L NaCl, 1 mmol/L acetic acid) or AngII (A9525, Sigma Aldrich) at a concentration adjusted to allow 2 mg/kg/day release of AngII for 7 days. After filling, pumps were primed by incubation in 0.9% NaCl for 16 h at 37 °C. Mice (10–12 weeks old) were anesthetized [inhaled 3–4% isofluorane (Zoetis) in oxygen (flow rate 0.8–1.0 L/min) delivered by nose cone] and pumps implanted subcutaneously, as per the manufacturer's instructions. Following pump implantation, topical analgesia was administered in the form of bupivacaine (AstraZeneca, UK), and systemic analgesia was administered (SC) in the form of buprenorphine (0.075 mg/kg; Reckitt Benckiser, UK), before completion of surgery and recovery from anesthesia. Animals were allowed to recover from anesthesia on a heated pad, before being single-housed for the remainder of the study. [N.B. blood pressure following the 7 days of infusion was measured by anesthetized catheterization, as indicated].

Morphometry

Tissues were retrieved after sacrifice, briefly rinsed of blood in 0.9% NaCl, and then blotted dry. Tissue weights were measured immediately and expressed as a ratio of each animal’s own tibia length, measured with caliMAX fine calipers (WIHA, Germany).

Cardiomyocyte density

Cardiomyocyte density was determined using an adaptation from a previous method [18]. Briefly, LV tissue was fixed in 4% PFA/PBS for 24 h at RT before post-fixation, sectioning, and processing as described above for immunofluorescent staining and confocal microscopy. Cell membranes were visualized using 1:500 Alexa Fluor 488 WGA conjugate. Using ImageJ, an observer blinded to sex, genotype, and treatment counted cardiomyocytes from two representative areas of each LV and averaged them to give one value per animal. Cardiomyocyte counts were expressed as cells per square millimeter. Cardiomyocyte density was used instead of cardiomyocyte cross-sectional area as we found density to show less inter-observer variability (data not shown).

Fibrosis staining

LV tissue was fixed as described for cardiomyocyte density. Cytoplasm (red) and collagen (blue) were stained using Masson’s trichrome stain as per the manufacturer’s protocol (HT15, Sigma Aldrich). Coverslips were mounted onto slides using DEPEX mounting media (VWR International, USA). Sections were visualized with bright-field microscopy on a DM6000 power mosaic microscope (Leica Biosystems), and images were retained in RGB format. The degree of fibrosis was quantified using ImageJ by an observer blinded to sex, genotype, and treatment and expressed as a percentage of area occupied by blue-hued pixels relative to total cross-sectional tissue area, as adapted from previous methods [20].

RNA sequencing analysis

Human postmortem brain RNA sequencing data was obtained from the Genotype-Tissue Expression (GTEx) Consortium [21]. GTEx raw sequence counts were examined for differentially expressed genes using the edgeR statistical package [22], where samples were grouped by genders for each brain tissue sample.

Statistical analysis

All data are presented as mean ± s.e.m. The number of independent observations (n) in animal/human data represents a single animal/donor. Where multiple measurements were taken per animal, measurements were averaged to give a single observation (n) per animal. Where two untreated genotypes (per sex) were analyzed, a two-tailed Student’s t test was used, or a one-tailed Mann-Whitney test, as indicated. Where > 2 untreated genotypes (per sex) were analyzed, a one-way ANOVA was used with Dunnett’s multiple comparisons tests, or Kruskal-Wallis test with Dunn’s multiple comparisons test where ANOVA assumptions were violated. Where two genotypes (per sex), with and without treatment, were analyzed, a two-way ANOVA was used with Tukey’s multiple comparisons test. For all tests, p ≤ 0.05 was considered significant.

GPR37L1 protein is abundant in the central nervous system but not detectable in the heart or kidney

Given the reported role of GPR37L1 in the cardiovascular system [8], we investigated GPR37L1 transcription by qPCR in several mouse tissues of interest, including the heart and kidney. We noted that GPR37L1 mRNA appeared at late cycle numbers in both tissues, suggesting very low relative expression (Additional file 1: Figure S1). To clarify the expression profile of GPR37L1, we used EUCOMM ES cells [9] to generate GPR37L1^lacZ/wt mice (expressing one allele of β-galactosidase in place of GPR37L1) as we have described [10]. Consistent with previous reports of GPR37L1 localization [4–6, 23], β-galactosidase expression was most abundant in the brain, particularly in the cerebellar Bergmann glia (Fig. 1a, Additional file 1: Figure S2A). Astrocyte β-galactosidase co-localization was observed throughout the brain but was most evident in the hippocampus (Additional file 1: Figure S2B), while other regions such as the brain stem (Fig. 1a) and neocortex (Additional file 1: Figure S2C) contained GFAP-negative β-galactosidase-positive cells that were consistent with the reported expression of GPR37L1 in cells of oligodendrocyte lineage [23, 24]. In several key cardiovascular centers of the CNS including the caudal and rostral ventrolateral medulla (CVLM, RVLM), the nucleus of the solitary tract (NTS), and the A5 nucleus, diffuse β-galactosidase staining was observed close to, but not within, tyrosine hydroxylase-positive catecholaminergic neurons (Additional file 1: Figure S3). In contrast, we were unable to detect GPR37L1 β-galactosidase reporter immunofluorescence in peripheral tissues. Although we measured low levels of GPR37L1 mRNA in the mouse heart (Additional file 1: Figure S1), we could not detect β-galactosidase by immunofluorescence (Fig. 1b, Additional file 1: Figure S4A) or Western blot (Additional file 1: Figure S4B), nor wild-type GPR37L1 using a validated [15] anti-GPR37L1 antibody (Additional file 1: Figure S4C-E). Similarly, we did not detect β-galactosidase in the renal medulla (Fig. 1c) or cortex (Additional file 1: Figure S5A), nor could it be detected in GPR37L1^lacZ/wt kidney homogenates (Additional file 1: Figure S5B). Endogenous GPR37L1 was not detected in GPR37L1^wt/wt kidney (Additional file 1: Figure S5C). Because only GPR37L1 mRNA, but not protein, was found in the heart and kidney, we hypothesize that GPR37L1 exerts its cardiovascular effect via the CNS.

GPR37L1 regulates blood pressure in female mice

To investigate the role of GPR37L1 in the cardiovascular system, we further generated the GPR37L1^flx/flx and GPR37L1^KO/KO mice [9, 10]. Receptor ablation in both genders was verified in the cerebellum (mRNA and protein, respectively; Fig. 1d–f), and in the LV (mRNA; Additional file 1: Figure S4F). GPR37L1 expression was unchanged in GPR37L1^flx/flx as compared to GPR37L1^wt/wt mice (generated from GPR37L1^wt/KO breeding pairs) (Fig. 1d, Additional file 1: Figure S6A and B). Thus, GPR37L1^wt/wt mice were used as controls for the remainder of our studies.

Baseline cardiovascular measures were first assessed in anesthetized females by micromanometry (Fig. 2a) at a constant heart rate of ~ 500 bpm (Fig. 2b, Additional file 1: Table S1). Female GPR37L1^wt/KO and GPR37L1^KO/KO mice displayed significantly elevated aortic systolic pressure compared to controls (Fig. 2a, Additional file 1: Table S1). Furthermore, GPR37L1^KO/KO females showed increased aortic diastolic, mean arterial, and pulse pressures (Fig. 2a, Additional file 1: Table S1). No genotype differences in LV systolic pressure, end-diastolic pressure, and dP/dT max or dP/dT min were observed. To confirm that this phenotype was preserved in conscious animals, radiotelemetry BP monitoring was performed on a separate cohort of conscious GPR37L1^wt/wt and GPR37L1^KO/KO females. Again, GPR37L1^KO/KO females had elevated systolic and diastolic pressure, during both light and dark phases of the diurnal cycle, relative to GPR37L1^wt/wt controls (Fig. 2c).

To investigate if GPR37L1 deletion is deleterious in the setting of hypertensive stress, female GPR37L1^wt/wt and GPR37L1^KO/KO mice were infused for 7 days with AngII (2 mg/kg/day) or vehicle, before anesthetized hemodynamic measurement by pressure-transducing catheter and tissue collection. AngII infusion caused greater increases in systolic and mean arterial pressure in the GPR37L1^wt/wt females (Table 1), presumably because GPR37L1^KO/KO females have higher baseline systolic pressure (one-tailed Mann-Whitney test, p ≤ 0.05), consistent with our previous, independent cohort (Fig. 2). However, BP after 7 days of AngII did not differ between genotypes (Table 1), and both developed equivalent gross, cellular, and molecular LVH (Fig. 3a, b, Additional file 1: Figure S7, Figure S8), without progression to overt heart failure (see relative lung and right ventricle weights; Fig. 3c, Table 1). Although GPR37L1^wt/wt females developed cardiac fibrosis in response to AngII treatment, this was not observed in GPR37L1^KO/KO females (Fig. 4).

		Female
	Genotype	wt/wt			KO/KO
	Treatment	Vehicle	AngII	Δ	Vehicle	AngII	Δ
Aorta	HR (bpm)	515.6 ± 8.0	512.3 ± 7.1	− 3.3	504.4 ± 6.8	503.6 ± 3.6	− 0.8
	SP (mmHg)	109.3 ± 3.3	135.5 ± 4.1*	26.2	119.7 ± 6.3	130.9 ± 3.7	11.2
	DP (mmHg)	78.1 ± 2.3	83.7 ± 2.5	5.6	83.1 ± 2.8	86.8 ± 2.4	3.7
	MAP (mmHg)	88.5 ± 2.6	101 ± 2.8*	12.5	95.3 ± 3.9	101.5 ± 2.7	6.2
	PP (mmHg)	31.2 ± 1.2	51.8 ± 3.0*	20.6	36.6 ± 3.6	44.0 ± 2.2	7.4
LV	HR (bpm)	500.9 ± 8.2	500.4 ± 2.2	− 0.5	514.0 ± 9.6	508.9 ± 4.8	− 5.1
	SP (mmHg)	106.5 ± 3.1	128.8 ± 1.5*	22.3	112.9 ± 4.7	126.4 ± 2.6*	13.5
	EDP (mmHg)	6.4 ± 0.5	7.0 ± 1.1	0.6	5.3 ± 0.3	8.7 ± 1.1*	3.4
	dP/dT max (mmHg/s)	9799 ± 860.0	10,998 ± 426.2	1199	11,343 ± 745	12,455 ± 383.9	1112
	dP/dT min (mmHg/s)	− 9019 ± 853.7	− 9253 ± 584.0	− 234	− 9262 ± 470.2	− 9718 ± 330.8	− 456
Morphometry	Body weight (g)	21.2 ± 0.3	22.3 ± 0.3	1.1	23.3 ± 0.5	22.3 ± 0.3	−1.0
	Tibia length (mm)	16.1 ± 0.1	16.1 ± 0.1	0	16.4 ± 0.1	16.4 ± 0.1	0
	HW:TL (mg/mm)	6.5 ± 0.1	8.1 ± 0.2*	1.6	6.8 ± 0.1	8.2 ± 0.2*	1.4
	LVW:TL (mg/mm)	4.6 ± 0.1	6.0 ± 0.2*	1.4	4.9 ± 0.1	6.2 ± 0.1*	1.3
	RVW:TL (mg/mm)	1.4 ± 0.0	1.4 ± 0.0	0	1.4 ± 0.0	1.4 ± 0.0	0
	Lung:TL (mg/mm)	9.9 ± 0.4	9.5 ± 0.3	− 0.4	9.3 ± 0.5	9.7 ± 0.4	0.4

The two-way ANOVA results are displayed in Additional file 1: Table S3. The italics and the asterisk (*) indicate a significant (p ≤ 0.05) difference between AngII and vehicle-treated cohorts, within each genotype (Tukey’s multiple comparisons test). The italics and the dagger (†) indicate a significant (p ≤ 0.05) difference between AngII-treated animals of different genotypes (Tukey’s multiple comparisons test). Hemodynamic data, n ≥ 8; morphometry data, n ≥ 18

HR heart rate, SP systolic pressure, DP diastolic pressure, MAP mean arterial pressure, PP pulse pressure, ED end-diastolic pressure, HW heart weight, LVW left ventricle weight, TL tibia length, LV left ventricle, RVW right ventricle weight

GPR37L1 protects against AngII cardiovascular stress in male mice

Baseline cardiovascular measures were also assessed in males. GPR37L1^wt/wt, GPR37L1^wt/KO and GPR37L1^KO/KO males showed no differences in any hemodynamic parameter by micromanometry (Fig. 2a, Additional file 1: Table S1). BP radiotelemetry was performed on a separate cohort of conscious male GPR37L1^wt/wt and GPR37L1^KO/KO animals. Consistent with previous reports [25], males had higher systolic and diastolic BP than females (Fig. 2c) and lower heart rates (Fig. 2d). The expected circadian dip was seen in both sexes (Fig. 2c).

As for the females, GPR37L1^KO/KO males appeared to be protected from myocardial fibrosis (Fig. 4a, c). However, the more marked hypertrophy and therefore increased tissue area in the GPR37L1^KO/KO males may have obscured the extent of absolute fibrosis which was not grossly different between genotypes (Fig. 4a).