Comorbidity-Specific Molecular Programs of Early Diastolic Dysfunction Identify Glucocorticoid Receptor Activation as a Causal Driver in Obesity
Comorbidity-Specific Molecular Programs of Early Diastolic Dysfunction Identify Glucocorticoid Receptor Activation as a Causal Driver in Obesity
Subsets of animals were sacrificed at predefined time points. HG mice and a subset of untreated control mice were sacrificed at 25 weeks of age, as this stage corresponds to the onset of diastolic dysfunction in this model12. OB, SAH, and CIH, along with age-matched CTL mice, were sacrificed at 50 weeks, when these models begin to show diastolic function impairments. Additional groups of OB, SAH, CIH, and HG mice, together with their respective age-matched CTL groups, were monitored until they developed HFpEF or died naturally. HFpEF was defined as the presence of diastolic dysfunction assessed by echocardiography and lung congestion detected by ultrasound (prominent B-lines in at least half of one hemithorax and/or moderate pleural effusion), in the presence of preserved ejection fraction (EF). Mice fitting the definition of HFpEF were euthanized Nuclei isolation was performed following Cui et al. protocol16, with slight modifications. Briefly, frozen left ventricles were homogenized in lysis buffer with a mechanical probe and then further disrupted using a Dounce homogenizer. The homogenate was sequentially filtered through progressively finer strainers to remove tissue debris, and nuclei were collected and purified through a sucrose-based density separation. Isolated nuclei were then stained with DAPI and subjected to flow cytometry sorting to select DAPI-positive nuclei. Sorted nuclei were collected, resuspended in a suitable buffer, and their integrity was assessed.  Samples with >90% intact cells were considered acceptable for downstream single-nucleus RNA sequencing. Nuclei were adjusted to a final concentration of ~1,000 nuclei/µL and their concentration and integrity were assessed using the Countess 3 cell counter (Thermo Fisher Scientific).  A total of 288 C57BL/6 male and female mice, aged 8–10 weeks at baseline, were used in this study. They were assigned to one of the following experimental groups using simple randomization: obesity (OB), systemic arterial hypertension (SAH), chronic intermittent hypoxia (CIH), or chronic hyperglycemia (HG). Untreated animals served as controls (CTL group). Following our previous protocol12, OB mice were fed a high fat diet (HFD) [45 kcal% (24 g%) palm oil-based fat, 35 kcal% (41 g%) carbohydrate, 20 kcal% (24 g%) protein; based on OpenSource Diets No. D12451, Research Diet Services, Wijk bij Duurstede, The Netherlands]. Blood glucose (BG) levels were monitored monthly. For the SAH model, mice were fed a high-salt diet (HS, 8% NaCl; based on OpenSource Diets No. D12451, Research Diet Ser- vices, Wijk bij Duurstede, The Netherlands). A noninvasive tail cuff was used to assess blood pressure levels in conscious mice, with monthly assessment performed to verify the establishment of hypertension. CIH mice were exposed to chronic hypoxia (FiO2 10%) during 8 hours in their light cycle, 5 days per week. Throughout the hypoxic exposure, animals had free access to standard chow diet and water. For the HG model, mice were injected with streptozotocin (STZ, 50mg/kg, 0.05 mol/L in citrate buffer, pH 4.5, Sigma, St. Louis, USA) i.p for five consecutive days. BG levels were monitored before STZ injection and every 4-8 weeks to confirm the induction of hyperglycaemia. Body weight (BW) was monitored monthly across all models.  Each nuclei suspension was then loaded into a single port of a Chromium Next GEM Chip G (10x Genomics), targeting the recovery of approximately 10,000 nuclei per sample. Single nuclei were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics), Dual-indexed snRNA-seq libraries were generated with the Chromium Next GEM Single Cell 3′ GEM Library and Gel Bead Kit v3.1, following the manufacturer's protocol. Library fragment size was assessed with a High Sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies), and concentrations were quantified using the Qubit fluorometer (Thermo Fisher Scientific). Individual libraries from each sample were diluted to a final concentration of 10 nM and subsequently pooled