Human heart samples
Human myocardial tissue samples were acquired from patients in the Utah Cardiac Recovery Program at the University of Utah under an approved IRB protocol (IRB-00030622). All samples were collected with informed consent from patients, organ donors or their guardians. Samples were collected from consenting patients with advanced chronic heart failure with reduced ejection fraction at the time of heart transplantation. Nonfailing donors who were ineligible for heart transplantation for noncardiac reasons (such as donor-recipient size mismatch or infection) were used as controls. Myocardial tissue was collected in the operating room and immediately frozen in liquid nitrogen, then stored at −80 °C until further use.
Animal care
All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah. Mice were housed at 22-23°C using a 12 h light:12 h dark cycle. Mice were maintained on an irradiated diet (Teklad Diet 2920x) with ad libitum access to water at all times. Food was only removed in experiments beginning at the start of the light cycle.
Adult heart-specific MPC1-knockout (Mpc1fl/fl; αMHCSeaCreSea) mice were generated as described previously51. Mct1fl/fl mice were generated as previously described52. Heart-specific Cre recombinase was introduced by crossing mice with αMHCSeaCreSea mouse (strain 005657 from Jackson Laboratories). 8 weeks Mct1fl/fl:αMHCSeaCreSea+/− And Mct1fl/fl; αMHCMerCreMer−/− littermates received an intraperitoneal injection of 40 mg kg−1 tamoxifen for 3 consecutive days to give an adult Mct1iCKO And Mct1fl/fl (control) littermates.
Heart-specific MCT4 inactivation in adults (Mct4fl/fl; αMHC-MerCreMer) mice were generated as follows. Three embryonic carrier clones Slc16a3 tm1a (encoding MCT4) were obtained from the European Conditional Mouse Mutagenesis Program/European Mouse Mutant Archive (EUCOMM/EMMA; http://www.infrafrontier.eu/). All clones were from the JM8A3.N1 cell line (C57Bl/6NTac background strain with agouti coat color distribution). These embryonic stem cell clones were injected into the blastocoel of 3.5-day-old mouse blastocysts (host strain C57Bl/6 Tirec-Brd). Injected embryos were transferred into the uterine horns of a pseudo-pregnant recipient at the appropriate time C57BL/6-Tirec-Brd females. Chimeras with the highest embryonic stem cell contribution were crossed with C57Bl/6J partners to test for germline transmission of the targeted allele. Pups were screened for the presence of germline-transmitted tm1a and those positive were then propagated (first tm1a knockout allele, reporter-marked insertion with conditional potential). Mice carrying the appropriate flox alleles were crossed with αMHCSeaCreSea mouse as described above. Experiments on all mice began 4 weeks after injection (at 12 weeks of age).
Echocardiographic assessment
Mice were anesthetized with 1.5% isoflurane (Vet One, NDC 13985-046-60). Echocardiographic images were captured sequentially using the Vevo imaging system (VisualSonics). 2D long-axis and short-axis views were obtained and analyzed with Vevo Strain software (v3.1.1). All measurements were performed using three consecutive cardiac cycles. Limb leads were fixed using conductive cream to facilitate electrocardiogram (ECG) recording.
Osmotic mini pump model
Twelve-week-old C57BL/6 mice were divided into four groups, weighed, and examined before surgery by echocardiography to establish baseline cardiac parameters. Surgical instruments were sterilized using a glass bead sterilizer before surgery and aseptic technique was used during the procedure. The osmotic minipumps (Alzet, model 2006) were filled with angiotensin II (1.5 μg g day−1) and phenylephrine (50 μg g day−1) in 0.9% NaCl at 0.15 µl h−1 for 42 days (Model 2006) according to the manufacturer’s instructions. The pumps were surgically implanted subcutaneously in the back through a small incision, under isoflurane (1.5% in O2) anesthesia. Once the pump was implanted and the incision closed, the mice were placed in a clean, warm cage and monitored for signs of distress. Then, the mice were weighed and examined weekly by echocardiography. After 42 days of treatment with AngII/PE, mice were euthanized by cervical dislocation and blood and heart tissues were collected for further analysis.
TAC experiences
Baseline echocardiograms were acquired and TAC was performed surgically on men and women. Mct1fl/fl And Mct1iCKO mice aged 12 to 14 weeks, as previously described53.54. Briefly, before surgery, a subcutaneous dose of Buprenorphine SR extended release (0.15 mg kg−1) was administered for analgesic purposes. Mice were anesthetized with 2–3% isoflurane in oxygen, administered via a nose connected to a VetFlo vaporizer. The aortic arch was visualized and tightened using a titanium clip. The surgical incision was closed using layered 6-0 sutures. During recovery, regular-release buprenorphine was administered as needed for up to 48 hours. Unclipped Mct1fl/fl And Mct1iCKO mice (sham) served as controls, undergoing anesthesia and a small incision without aortic constriction or cardiac injury. After surgery, the mice received weekly echocardiograms for six consecutive weeks to assess cardiac function and structure.
Ischemia-reperfusion experiments
The experimental protocol was carried out on 20 to 30 g, aged 12 to 14 weeks. Mct1iCKO And Mct1fl/fl mice, 4 weeks after tamoxifen injection and before the appearance of symptoms of heart failure. Mice were anesthetized with 2–3% isoflurane, intubated using a 22 G IV catheter, and ventilated at 100 breaths per minute with a small rodent respirator. Mice were placed supine on a heating pad at 38°C and chest hair was removed with depilatory cream, which was rinsed with water or saline to avoid skin irritation. The skin was then prepared with betadine and alcohol. A thoracotomy was performed to access the heart and a retractor was used to improve visibility. The pericardium was carefully removed, thus avoiding myocardial damage. Under a dissecting microscope, the left anterior descending (LAD) coronary artery was located and a 6-0 silk suture was placed underneath. A double loose knot was tied with a loop of 2 to 3 mm diameter, through which a piece of 2 to 3 mm PE-10 tube was placed. The tube, previously soaked in 100% ethanol and rinsed with sterile water, was used to tighten the loop around the artery, thereby achieving coronary occlusion for 30 min. The success of LAD ligation was confirmed by a paler color on the anterior wall of the LV. After 30 min, the knot was untied and the tubing removed, confirming reperfusion by the return of a red color to the LV wall, which continued for 2 h.
In acute experiments, the coronary artery was briefly re-obstructed after injury and Evans blue dye was injected into the left atrium to mark the area at risk. Hearts were excised, perfused with saline, weighed, and sectioned into 1-mm slices using a vibratome, or separated into ischemic and nonischemic samples and frozen in liquid nitrogen. Slices were incubated in 1% triphenyltetrazolium chloride at 38°C for 20 min to stain viable myocardium and then fixed in 10% formalin to distinguish viable from necrotic tissue. Tissue sections were photographed the next day and myocardial recovery was quantified using ImageJ by blinded investigators. For chronic experiments, mice underwent ischemia for 30 min, after which the ligature was removed and the incisions were closed with sutures. Mice were placed in a warm cage, monitored for distress, weighed, and screened weekly by echocardiography.
Exercise Capacity Test
Mice aged 12 to 14 weeks (Mct1fl/fl And Mct1iCKO) practiced a 5-day treadmill acclimatization protocol before any maximal effort. This provided familiarity with the procedure, but produced no training effects on the mice.55. Briefly, after a day of rest, mice were weighed and the maximal treadmill test was performed. The test began with a warm-up period of 10 min, treadmill speed of 10 m min−1and a slope of 10%. After that the speed was increased by 5 m min−1 every 2 min until the mouse resists a physical stimulus (touching the tail or the back of the mouse with a bristle brush) to run for more than 20 s or sit on the back of the treadmill motionless belt five times in 30 s. If either occurred, the time was recorded, the test was completed, and the mouse was returned to its cage to recover. The maximum speed of the treadmill does not exceed 30 m min−1 throughout the stress test. Total work (in kg m−1) and time to exhaustion (in minutes) were then calculated for each mouse. Blood lactate (in mmol·l−1) was measured at 10, 20 and 30 m min−1and a score of 10% to assess the systemic effects of lactate production in the Mct1iCKO compared to the Mct1fl/fl controls.
Jugular vein catheterization surgery
Sterile mouse jugular vein catheters were purchased from SAI Infusion Technologies and a single-channel vascular access button from Instech Laboratories. Mice were fasted before surgery, shaved preoperatively, and administered bupivacaine before anesthesia. A dissecting microscope (Leica S9i Stereo) was used during catheter placement. Mice were provided with standard recovery media and allowed at least 5 days of recovery before further use in experiments. Mice with altered vital signs, activity, or reflexes were euthanized and were not included in experimental studies.
Stable isotope infusions
Surgically catheterized 12-week-old mice were maintained under a normal light cycle (06:00–18:00). On the day of the perfusion experiment, mice were transferred to new cages without food at 6:00 a.m. (start of their sleep cycle) and perfused for 2–3 hours (metabolite specific) starting around 12:00 p.m. to reach serum steady state based on previously published studies. The infusion device (Instech…
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