This study followed the guidelines set forth by the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was under approval from the Sanford Research Institutional Animal Care and Use Committee (Protocol #170-06-23B). All animals were housed in a temperature-controlled, light–dark cycled facility with free access to water and chow. Female Sprague–Dawley rats (Envigo, Indianapolis, IN) received control (TD2018 Teklad, Envigo; 18% fat, 24% protein, 58% carbohydrates) or HFD (TD95217 custom diet Teklad, Envigo; 40% fat, 19% protein, 41% carbohydrates) for at least 28 days prior to breeding to simulate a dietary “lifestyle.” Diets were selected to equate commonly attainable low-fat diets (18% of calories as fat) or HFD (40% of calories as fat) with more saturated and monounsaturated fat content. Omega 6:3 ratios were similar between diets. Female rats were bred with healthy male rats and fed a control diet and monitored by daily vaginal swab for spermatozoa. When spermatozoa were first present, timed pregnancy started as embryonic day 0 (E0). After confirming the pregnancy with an ultrasound, dams received an intraperitoneal injection of either citrate-buffered saline (Thomas Scientific, Swedesboro, NJ) diluent or 65 mg/kg streptozotocin (Sigma-Aldrich, Inc., St. Louis, MO, USA) to induce late gestation diabetes on E14. With a goal to keep blood glucose levels at 200–400 mg/dL, dams were partially treated with sliding scale insulin (regular and glargine, Eli Lilly and Co., Indianapolis, IN) two times per day. Whole blood sampling from a tail nick was done to measure glucose at least twice daily and ketones (βHOB) daily (Precision Xtra glucometer and ketone meter, Abbott Laboratories, Abbott Park, IL, USA). Dams with blood glucose < 200 mg/dL within 48 h after streptozotocin were excluded from the study. Although this model induces maternal diabetes by streptozotocin-mediated pancreatic damage, we have consistently shown that the developing offspring, our experimental subjects, are exposed to maternal hyperglycemia, hyperlipidemia, and fetal hyperinsulinemia in the last 1/3 of pregnancy [9 (link),11 (link),12 (link),13 (link),56 (link)]. Dams were allowed to deliver spontaneously in order to yield offspring of both sexes from two distinct groups: controls and combination exposed (diabetes + HFD). On postnatal day 1 (P1), offspring hearts (n = 10–12 litter per group and each group comprised 5–6 male and female hearts) were collected under 5% isoflurane anesthesia and immediately used for the isolation of NRCM or snap frozen in liquid nitrogen and stored at −80 °C until analysis. Maternal and offspring characteristics are given in Supplementary Tables S1 and S2.
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