Beta-3 adrenergic receptor overexpression reverses aortic stenosis–induced heart failure and restores balanced mitochondrial dynamics

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985).

Mice

All experimental and other scientific procedures with animals conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC, enforced in Spanish law under Real Decreto 53/2013. Animal protocols were approved by the local ethics committees and the Animal Protection Area of the Comunidad Autónoma de Madrid. C57BL/6 J wild-type (WT) mice and the following transgenic lines on the C57BL/6 J genetic background were used in this study, and all experiments were conducted with adult males.

R26-ADRB3 transgenic mice

To target the human β3AR transgene (ADRB3) into the Rosa26 (R26) locus in embryonic stem (ES) cells by homologous recombination, we generated a construct with a PGK-Neo cassette plus a transcriptional stop site flanked by loxP elements followed by ADRB3 cDNA and an IRES-EGFP construct, using a previously described strategy [61]. Upon Cre recombination, transgene expression will be controlled by the R26 promoter. Complete human ADRB3 cDNA was obtained from clone IMAGE. The sequence was PCR amplified with Phusion High-Fidelity DNA Polymerase (NEB) and primers containing EcoRI sites and was cloned into the EcoRI site of a pCDNA3.1-IRES-EGFP plasmid previously generated by cloning a SalI IRES-EGFP fragment into the XhoI site of pCDNA3.1. The resulting plasmid was digested with XbaI, treated with DNA Polymerase I, large Klenow fragment (NEB) to form blunt ends and digested with NheI to obtain a NheI-blunted XbaI ADRB3-IRES-EGFP fragment. The fragment was cloned into NheI-blunted NotI sites of pBigT. We obtained the PacI-AscI-cassette containing loxP-PGK-Neo-STOP-loxP-ADRB3-IRES-EGFP by digestion and cloned it into the PacI-AscI sites of modified pROSA26-1 plasmid. The final construct was linearized with XhoI and electroporated into G4 ES cells derived from a 129S6/SvEvTac x C57BL/6Ncr cross [71]. After G418 (200 µg/mL) selection for 7 days, 192 clones were picked. Homologous recombination was identified by Southern blot of DNA digested with EcoRV and hybridized with 5' and 3' probes. Four clones were positive, and we selected two to confirm karyotype. One clone was injected into B6CRL blastocysts to generate chimeras, which were analyzed for germ line transmission. The resulting human β3AR transgenic mouse line (ADRB3tg/tg) was crossed with C57BL/6 J mice to achieve a pure genetic background.

Transgenic mouse lines expressing human β3AR

Upon Cre recombination, the stop codon between the R26 promoter and the ADRB3-IRES-EGFP sequence is removed, and the R26 promoter drives ADRB3 expression. We used the cTnTCre/+ line, with cardiomyocyte-specific expression of Cre recombinase [34], to drive hβ3AR expression. Crossing of both lines generated c-hβ3tg mice (cTnTCre/+;ADRB3tg/tg) with cardiomyocyte-specific overexpression of β3AR (human β3AR expressed against a background of endogenous mouse β3AR expression). Controls were wild-type (WT) littermates (cTnT+/+;ADRB3tg/tg), with normal levels of mouse β3AR.

c-hβ3tg mice were further crossbred with a knockout line with targeted disruption of the mouse β3AR gene (adrb3),[69] generating c-hβ3tg mβ3KO mice (cTnTCre/+;ADRB3tg/tg;adrb3−/−), with sole expression of human β3AR in cardiomyocytes. Controls were mβ3KO littermates (cTnT+/+;ADRB3tg/tg;adrb3−/−), with no β3AR expression.

Binding assay

Snap frozen hearts were crushed and200-400 mg of tissue was mixed with 50 mM Tris–HCl (pH 7.5). The samples were homogenized using an ultrasonic cell disruptor (MicrosonTM ultrasonic cell disruptor) keeping the sample on ice and they were filtered using nylon mesh. Next, the samples were centrifuged at 4 °C for 15 min at 1000 g. Protein in the resulting supernatant was quantified using the Bradford method and 2 mg of protein was incubated in duplicate for 60 min at 37 °C with different concentrations of [3H]-CGP 12,177 (from 0.25 to 120 nM) (Perkin Elmer, Waltham, MA, USA) in 50 mM Tris–HCl (pH 7.5). Experiments were terminated by rapid filtration through fiberglass filters (Schleicher and Schuell, GF 52), presoaked in 0.3% polyethyleneimine, using a Brandel cell harvester (M24R). The filters were then washed three times with 4 ml of ice-cold 50 mM Tris–HCl buffer (pH 7.5), and the filter-bound radioactivity was determined by liquid scintillation counting (2480 WIZARD, PerkinElmer, Waltham, Massachusetts, USA). Nonspecific binding was measured in the presence of 1 mM propranolol (Sigma). Specific binding is defined as total binding minus nonspecific binding. The saturation data were analyzed by non-linear regression using Prism version 4.0 (GraphPad Software; San Diego, California, U.S.A) to determine the maximum number of binding sites (Bmax) expressed as fmol/mg of protein.

Western blot

Cells and tissue samples (0.1 mg) were lysed in RIPA buffer containing protease inhibitors (complete-Roche, Indianapolis, IN, USA) and phosphatase inhibitors (PhosSTOP-Roche, Indianapolis, IN, USA). The supernatant was separated by centrifugation at 12000 g for 15 min at 4 °C, and total protein concentration was detected with the BCA protein assay kit (Thermo Fisher, USA) using bovine serum albumin (BSA) as the standard. Equal amounts of protein (15ug) were separated by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer’s protocol (BioRad). After incubation with 5% of nonfat milk or BSA in TBST for 60 min, membranes were incubated overnight at 4 °C with primary antibodies against GFP (1:1000; Living Colors® Full-Length GFP Polyclonal Antibody, 632,592, Clontech), OPA1 (1:1000; Thermo Fisher, PA1-16,991), MFN2 (1:1000; Abcam, ab56889), Vinc (1:1000, Sigma, V4505), UCP2 (1:2000, Genetex, GTX132072) and GAPDH (1:10,000; Abcam, ab8245). Membranes were washed 3 times for 5 min each with TBST and incubated for 1 h with HRP-conjugated anti-mouse or anti-rabbit antibodies (1:5000). Bound antibody signals were developed with the ECL (Luminata) system. Quantitative densitometry analysis was performed using Fiji (ImageJ) software.

Histology and immunofluorescence

Heart specimens were fixed in 4% formaldehyde, dehydrated to xylene, and embedded in paraffin. After deparaffinization and rehydration, 5-μm sections were cut at 3 levels, mounted on glass slides, and stained with hematoxylin and eosin and with 1% Sirius red in picric acid (Sigma-Aldrich) to detect interstitial fibrosis. All sections were examined with a Nikon Eclipse Ni microscope and scanned with a NanoZoomer-RS scanner (Hammamatsu), and images were exported with NDP.view2. The percentage of fibrosis was quantified using Fiji (ImageJ) software in at least three sections per heart, and the mean was used for statistical analysis.

For immunofluorescence, hearts were fixed in 4% formaldehyde, dehydrated through 15% sucrose in PBS and then 30% sucrose overnight at 4 °C, and embedded in Tissue-Tek® OCT compound (SAKURA, Netherlands). Cryostat sections were blocked and permeabilized for 1 h at RT in PBS containing 0.3% Triton X-100 (90,002-93-1, Sigma), 5% BSA (A7906, Sigma), and 5% normal goat serum (055-000-001, Jackson InmunoResearch). Sections were then incubated overnight at 4 °C with anti-GFP (Living Colors® Full-Length GFP Polyclonal Antibody, 632,592, Clontech) diluted (1:500) in PBS containing 0.3% Triton X-100 and 2.5% normal goat serum. After washes, samples were incubated for 2 h at RT with a secondary antibody (Alexa Fluor, Invitrogen) and the nucleic acid stain Hoechst 33,342 (B2261, Sigma) and were mounted in Fluoromount G imaging medium (4958–02, Affymetrix eBioscience).

For cell immunofluorescence, adult mouse ventricular myocytes (AMVM) and neonatal rat ventricular myocytes (NRVM) were fixed with 4% paraformaldehyde in PBS for 10 min. Cells were then washed 1–3 times with PBS and blocked with 2% BSA (in PBS) for 1 h at RT. Samples were incubated overnight at 4 °C with primary antibodies (anti hβ3AR; A4854 Sigma and anti α-actinin; A7811 Sigma). After 1–3 washes with PBS, cells were incubated with Alexa Fluor secondary antibodies for 1 h at RT. Cells were then washed 1–3 times with PBS and incubated for 5 min with DAPI (1:10,000 in PBS) and washed again 1–3 times with PBS. AMVMs were additionally incubated with FITC-conjugated lectin for 1 h (L4895, Sigma) before a final wash in PBS. Stained cells were mounted in Fluoromount G imaging medium (4958–02, Affymetrix eBioscience).

Mouse left ventricular catheterization and pressure–volume loops

Ventricular catheterization was performed as previously described [54]. Mice were anesthetized (sevoflurane 1.5%) and intubated. A skin incision was made to visualize the diaphragm, which was heat cauterized to expose the heart apex. The pericardium was removed gently with forceps. Using a 25–30 gauge needle, a stab wound was made near the heart apex into the left ventricle (LV). The catheter tip (Transonic, NY, USA) was inserted retrogradely into the LV until the proximal electrode was just inside the ventricular wall. The catheter position was adjusted to obtain rectangular shaped pressure–volume (PV) loops. After allowing the signal to stabilize for 5 min, recordings were made of baseline PV loops, heart rate, maximal derivative of LV pressure (dP/dtmax), minimal derivative of LV pressure (dP/dtmin), left ventricular end-systolic pressure (LVESP), minimal derivative of LV pressure (dP/dtmin), and time constant of isovolumic relaxation (Tau). The same parameters were recorded after the injection a single dose of mirabegron (1 µg/kg) through the femoral vein. At the conclusion of the experiment, the catheter was removed by gently pulling it back through the stab wound, and the animal was euthanized.

Adeno-associated virus production and in vivo delivery

HEK293T cells were transfected using linear polyethylenimine hydrochloride with two plasmids: pDG-9 or pDG-6 plus an AAV transfer plasmid in which the transgene (ADRB3 or EGFP) is placed between 2 ITRs. pDG-9 encodes Rep78, Rep68, Rep52, and Rep40 (required for the AAV life cycle); serotype 9 VP1, VP2, and VP3 (capsid proteins); and adenoviral genes E4, E2a and VA (mediating AAV replication) and was used to generate AVV-9. Similarly, pDG-6 encodes the same genes except that VP1, VP2, and VP3 are from serotype 6, and this plasmid was used to generate AVV-6. An AAV transfer plasmid encoding ADRB3 under the control of the troponin T promoter was used to generate recombinant AAV (rAAV) for hβ3AR cardiac-specific expression (Fig. S5A). An AAV transfer plasmid encoding EGFP instead of ADRB3 as used to generate control rAAV (Fig. S5B). The AAV transfer plasmids also contained an IRES sequence followed by the luciferase gene after the transgene sequence (ADRB3 or EGFP). Luciferase activity was used as a reporter to check viral transduction in vivo.

Cells were harvested 3 days after transfection, lysed, frozen and thawed three times, and digested with benzonase (150 units/mL). The final supernatant containing the virus was then purified on an iodixanol gradient in an optiseal polypropylene tube (361,625, Beckman Coulter). The concentrated and purified viral fraction was collected between the 40% and 60% iodixanol layers after ultracentrifugation (350,333 g, 18 °C for 1 h).

Adult mice were anesthetized and maintained on 1–2% isoflurane. A skin incision was made on the medial face of the hindlimb, and the femoral vein was exposed. A dose of 3 × 1011 viral genomes (vg)/mouse in 50 µl saline was injected using a 31G insulin syringe, and the skin was closed with a 6/0 silk thread. AAV-9 was used for in vivo delivery.

In vivo and ex vivo imaging system for luminescence detection

To verify correct viral transfection, 875 µg of D-luciferin (Xenogen, Alameda, CA) was administered to mice in a volume of 50 µl by intraperitoneal injection. Three minutes later, animals were anesthetized and maintained on 1–1.2% isoflurane in oxygen. Six minutes after D-luciferin administration, all mice were imaged using a Xenogen IVIS100 imaging system. Emitted photons were collected and integrated over 2 min periods. Images were processed using Xenogen Living Image software. For ex vivo bioluminescence imaging, animals were killed, organs were removed and quickly dipped in D-luciferin (17.5 g/mL), and images were captured with a supercooled charge-coupled camera. Emitted photons were collected and integrated over 2 min periods. Results are expressed as mean luminescence intensities (photons/s/cm2/sr).

RNA extraction and cDNA preparation

Tissues were homogenized using TissueLyser (Qiagen), and total RNA was extracted with QIAzol reagent (Qiagen). The RNA pellet was dissolved in RNase-free water, and concentration was measured in a NanoDrop spectrophotometer (Wilmington). RNA (2 µg) was transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

ADRBs PCR

cDNA (100 ng) was amplified by PCR using DNA polymerase (Biotools, Spain). PCR products were separated on a 2% agarose gel containing ethidium bromide. Images were taken with a Molecular Imager® Gel Doc™ XR + System (BioRad). Primers were designed specifically to match only the human ADRB3 cDNA sequence and not the mouse sequence: (Forward primer: TGCCAATTCTGCCTTCAACC; Reverse primer: CAGGCCTAAGAAACTCCCCA). Primers used to evaluate endogenous mouse Adrb1 and Adrb2 transcript levels in the context of transgenic Adrb3overexpression were: Adrb1(Forward primer: TCATCGTGGTGGGTAACGTG; Reverse primer: ACCAGCAATCCCATGACCAG); Adrb2(Forward primer: TTCGAAAACCTATGGGAACG; Reverse primer: GGGATCCTCACACAGCAGTT). To compare the expression level of the hβ3AR compared to the endogenous m Adrb3 levels, we used the following primers: mouse Adrb3 (Forward primer: TGATGGCTATGAAGGTGCG; Reverse primer: AAAATCCCCAGAAGTCCTGC); human Adrb3(Forward primer: TGCCAATTCTTGCCTTCAACC; Reverse primer: CAGGCCTAAGAAACTCCCCA).

Adult mouse ventricular myocyte isolation

The protocol for AMVM isolation was as previously described [21]. Briefly, 10- to 12-week-old mice were heparinized (50 USP units) and anesthetized with a mixture of ketamine (140 mg/kg), xylazine (33 mg/kg), and atropine (9 mg/kg). Once pedal pinch reflexes were completely inhibited, animals were placed in a supine position, ventral thoracic regions were wiped with 70% alcohol, and animals were euthanized. The heart was quickly removed, cannulated through the ascending aorta, and mounted on a modified Langendorff perfusion apparatus. The heart was then retrogradely perfused (3 mL/min) for 5 min at RT with prefiltered Ca2+-free perfusion buffer (113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4-7H2O, 12 mM NaHCO3, 10 mM KHCO3, 0.032 mM Phenol Red, 0.922 mM Na-HEPES, 30 mM taurine, 5.5 mM glucose, and10 mM 2,3-butanedione-monoxime; pH 7.4). Enzyme digestion was performed for 20 min at 37ºC in digestion buffer [perfusion buffer containing 0.2 mg/mL Liberase™, 2.5% (5.5 mM) trypsin, 5 × 10–3 U/mL DNase, and 12.5 µM CaCl2]. At the end of the enzyme digestion, both ventricles were isolated and gently disaggregated in 5 mL digestion buffer. The resulting cell suspension was filtered through a 100 µm sterile mesh (SEFAR-Nitex) and transferred to a tube containing 10 mL stopping buffer-1 [perfusion buffer supplemented with 10% v/v fetal bovine serum (FBS) and 12.5 µM CaCl2]. After gravity sedimentation for 20 min, cardiomyocytes were resuspended in stopping buffer-2 (as stopping buffer-1 but with 5% v/v FBS) for another 20 min. Cardiomyocytes were reloaded with Ca2+ by 10 min incubations in stopping buffer-2 with five progressively increasing CaCl2 concentrations (62 µM, 112 µM, 212 µM, 500 µM, and 1 mM). Resuspension and decanting of cells at each step contributed to the purification of the cardiomyocyte suspension. The homogeneous suspension of rod-shaped cardiomyocytes was then resuspended in M199 supplemented with Earle’s salts and L-glutamine (5 mM), 1% penicillin–streptomycin (P/S), 0.1 × insulin–transferin–selenium-A, 2 g/L BSA, 25 µM blebbistatin, and 5% FBS. Cells were plated in single drops onto 22 mm2 glass coverslips precoated with 200 µL mouse laminin (10 mg/mL) in PBS for 1 h.

Adult mouse hearts perfusion

The protocol for mouse adult ventricular myocytes is described here [21]. Briefly, 10- to 12-week-old mice were euthanized with CO2. Once pedal pinch reflexes were completely inhibited, animals were placed in a supine position and ventral thoracic regions were wiped with 70% of ethanol. The heart was quickly removed, cannulated through ascending aorta, and mounted on a modified Langendorff perfusion apparatus. The heart was retrogradely perfused for 20 min at room temperature with Perfusion Buffer [NaCl (113 mmol/L); KCl (4.7 mmol/L); KH2PO4 (0.6 mmol/L); Na2HPO4 (0.6 mmol/L); MgSO4-7H2O (1.2 mmol/L); NaHCO3 (12 mmol/L); KHCO3 (10 mmol/L); Phenol Red (0.032 mmol/L); HEPES-Na Salt (0.922 mmol/L); taurine (30 mmol/L); glucose (5.5 mmol/L); 2,3-butanodione monoxime (10 mmol/L), pH 7.4]. Perfusion Buffer was supplemented with 3.8 × 10–9 M of Mirabegron (SML2480-Merck) and 0.45 mM of IBMX (P019-Quimigen). Heart was stored at -80ºC for processing.

cNMP quantification

Cyclic AMP and GMP levels were measured in ether-extracted samples by EIA using cAMP (501,040) and cGMP (581,021) kits from Cayman Chemical, performed according to the manufacturer’s instructions. Both cyclic AMP and GMP levels (pM) were then normalized by μg of heart tissue protein.

ATP quantification

ATP levels were measured in heart samples using an EnzyLight™ ATP Assay Kit (EATP-100, BioAssay Systems), performed according to the manufacturer’s instructions. ATP concentrations (μM) were then normalized by μg of heart tissue protein.

Neonatal rat ventricular myocyte isolation

The protocol for hypoxia/reoxygenation in NRVMs was as described previously [13]. Ventricular cardiomyocytes were isolated from 1- to 2-day-old neonatal rat hearts. Hearts were prewashed in ADS buffer (116 mM NaCl, 20 mM HEPES, 0.8 mM Na2HPO4, 5.6 mM glucose, 7 mM KCl, and 0.8 mM MgSO4·7H2O; pH 7.35) to remove blood and then placed in dishes containing 7 mL ADS. Hearts were then minced into small pieces with sterile razor blades, and the tissue suspensions were then transferred to flasks containing 7 mL of enzyme solution (ADS containing 0.6 mg /mL pancreatin, 8820 U /L collagenase II, and 50 mM CaCl2) and incubated for 10 min at 37 °C. The supernatant from this predigestion step was discarded, and the tissue pieces were incubated in 15 mL of digestion solution for 15-min periods at 37 °C. At the end of each incubation period, the supernatant was collected in 50 mL conical tubes containing 19 mL F-10 medium and 20% FBS preheated to 37 °C. Three-six fractions were collected and centrifuged at 1400 g for 10 min, the supernatant was discarded, and cells in each tube were washed with 5 mL FBS. The cells were then centrifuged at 1400 g for 10 min, and the supernatant was discarded. The resulting pellet containing NRVMs was resuspendend in HAM’s F10 complete medium supplemented with 10% horse serum (HS), 5% FBS, and 1% P/S, pH 7.4. The cell suspension was filtered through a 70 μm filter and preplated for 2 h on a Nunc Nunclon 100 mm cell culture dish (Thermo Fisher Scientific, Waltham, MA, USA) to separate fibroblasts from the myocyte fraction. The supernatant, containing mostly myocytes, was collected and plated on culture dishes in Hams F-10 complete medium. The fibroblasts attached to the Nunclon dishes were cultured in DMEM containing 1% P/S. Cells were maintained in medium without HS and supplemented only with 10% FBS and 1% P/S. Experimental treatments and controls were conducted in serum-free medium.

Neonatal rat ventricular myocyte transfection

Recombinant AAV6 were generated encoding the hβ3AR gene under the control of a truncated chicken cardiac troponin-T (cTnT) promoter and strengthened by the Cmr4 enhancer. The luciferase reporter gene placed after the hβ3AR gene was used to confirm expression and track transduced cells. The β3AR and luciferase genes were separated by an IRES sequence to prevent formation of a fusion protein that could alter β3AR function. A polyA sequence was added at the end to confer mRNA stability. The gene construct was flanked by ITR sequences for AAV machinery recognition. NRVMs were isolated and cultured for 24 h before being transfected with recombinant AAV-6 at the MOI indicated in each figure. A 10 K MOI was used in NRVM hypertrophy and chronotropy experiments. Transfections were performed in free-serum medium for 12 h, Ham’s F-10 complete medium was added, and transgene expression was allowed for 72 h before experiments.

The efficiency of AAV6 transduction of NRVMs was checked at 48 h and 72 h by monitoring luciferase activity in cells transduced with the AAV6-EGFP control virus. Luciferase reporter luminescence identified a 10 K MOI as the appropriate dose for subsequent experiments.

Neonatal rat ventricular myocyte luciferase assay

NRVMs (2 × 105 cells per well in a 24-well multi-well dish) were transduced with AAV-6 containing the luciferase gene. NRVMs were washed three times with ice cold PBS and collected in passive lysis buffer (Promega, Madison, WI, USA). Luciferase activity was measured with a luciferase assay system kit (Promega) and a plate reader (Infinite M1000 PRO-TECAN).

Neonatal rat ventricular myocyte hypertrophy

The protocol was as previously described [13]. After isolation, NRVMs (~ 3 × 105 cells per well) were plated in a six-well multi-well dish, transfected for 72 h with AAV6, and stimulated as indicated (isoproterenol, 10 μM; L-NAME, 100 μM; or both). NRVMs were then fixed in 3% PFA for 10 min, washed three times with ice cold PBS, and permeabilized with 0.2% Triton X-100. The cells were then incubated with 1% BSA for 30 min and incubated overnight at 4 °C with anti-α-sarcomeric actinin (α-SMA, A7811, Sigma-Aldrich) diluted 1:200 in 1% BSA. After washes, cells were incubated with FITC-conjugated secondary antibody anti-mouse (Sigma-Aldrich; 1:200). Cells were examined with a Nikon Eclipse Ni microscope, and images were acquired with a Nikon digital camera. For each sample, five to six fields (~ 50 cells per field) were acquired.

Mouse model of supravalvular aortic stenosis (AS) by transaortic constriction (TAC)

Male 8- to 12-week-old mice were intraperitoneally anesthetized with ketamine (60 mg/ kg), xylacine (20 mg/kg), and atropine (9 mg/kg). Once deeply asleep, animals were orally intubated under direct tracheal visualization using a blunted 22G cannula, and mechanical ventilation was maintained throughout the procedure (SAR-830. CWE Inc).

While models of valvular AS have been recently refined [62], we decided to use the more widely described model of supravalvular AS by TAC [64], since it resembles many of the features of AS-induced HF [29]. Partial thoracotomy to the second rib was performed under a surgical microscope, and the sternum was retracted with a chest retractor. Fine tip 45° angled forceps were used to gently separate the thymus and fat tissue from the aortic arch. After identification of the transverse aorta, a small piece of a 7.0 prolene suture was placed between the brachiocephalic and left carotid arteries. Two loose knots were tied around the transverse aorta, and a small piece of a blunt 27 gauge needle was placed parallel to the transverse aorta. The knots were quickly tied against the needle, and the needle was removed, leaving 0.36 mm diameter constriction. In sham-operated control mice, the entire procedure was identical except that the aortic ligation of the aorta was omitted. The chest retractor was removed, and the ventilator outflow was pinched off for 2 s to re-inflate the lungs. The rib cage was closed with a 6.0 silk suture using an interrupted suture pattern. The skin was closed with a 6.0 silk suture using a continuous suture pattern. Animals were allowed to recover in a warmed cage with a 98% oxygen supply.

Echocardiography

Echocardiographic evaluations of mice were performed by an experienced observer blinded to the study at baseline and at 1, 3, 4, 5, 8, 10, and 12 weeks post-TAC, depending on the experiment. Mice were lightly anesthetized with 0.5–2% isoflurane in oxygen, administered via a nose cone and isoflurane delivery adjusted to maintain a heart rate of 450 ± 50 bpm. Anesthetized mice were placed in a supine position on a heated platform, and warmed ultrasound gel was used to maintain normothermia. Mice were examined with a 30-MHz transthoracic echocardiography probe and a Vevo 2100 ultrasound system (VisualSonics, Toronto, Canada). A base-apex electrocardiogram (ECG) was continuously monitored through 4 leads placed on the platform and connected to the ultrasound machine. Images were transferred to a computer and were analyzed off-line using the Vevo 2100 Workstation software. For the assessment of LV systolic function, standard 2D parasternal long axis views were acquired at a frame rate > 230 frames/sec. End-systolic and end-diastolic LV volumes (LVESV and LVEDV) and LV ejection fraction (LVEF) were calculated using the area-length method. LV mass was calculated from short-axis M-mode views using end-diastolic left ventricular wall thickness.

In vivo treatment with L-NAME

Wild-type and c-hβ3tg male 8- to 12-week-old mice were treated for 15 days with the nitric oxide synthase inhibitor L-NAME (1 mg/mL). Systolic and diastolic arterial pressures were measured after 2-week of L-NAME treatment. Afterwards, mice were subjected to TAC surgery and followed up to monitor survival.

Lung water content

Lungs were first weighed and then dried for 7 days in a 60 °C oven. The mass of the dry lung was then measured, and water content was calculated as the difference in mass between the dry and wet lung, expressed as a percentage.

Positron emission tomography – computed tomography

All PET-CT studies were performed with a small-animal PET-CT device as previously described [72]. Briefly, animals were fasted overnight, and anatomic thorax CT scans were performed 1 h after [18F]FDG injections, followed by metabolic PET static acquisition for 15 min. Prefused and prereconstructed images were analyzed with Osirix (Aycam Medical Systems, LLC); we selected myocardium of the whole heart and calculated the mean myocardial standardized uptake value (SUV med) for each animal.

Transmission electron microscopy

Transmission electron microscopy (TEM) was performed as previously described [16]. Immediately after excision, LV samples were fixed in 4% formahaldehyde: 1% glutaraldehyde in cacodylate buffer, and postfixed in 1% osmium tetroxide. Tissues were then washed in PBS, dehydrated through graded alcohols followed by acetone, and then infiltrated with Durcupan ACM Fluka resin and polymerized at 60 °C for 48 h. Blocks were cut with a Leica ultracut UCT ultramicrotome (Leica, Heerbrugg, Switzerland), and Sects. (60–70 nm) were mounted onto 200-mesh grids. Sections were stained with a 2% solution of aqueous uranyl acetate for 10 min, followed by lead citrate staining for 10 min. Stained sections were viewed with a JEOL JEM-1010 transmission electron microscope (Tokyo, Japan) operating at 80 kV through 6000 × , 10,000 × , and 40,000 × objectives. Images were acquired with a GATAN Orius 200SC digital camera. Mitochondrial morphometry was analyzed using ImageJ (National Institutes of Health).

Seahorse

The bioenergetic response of AMVMs was measured with the Seahorse Bioscience XF96 Flux Analyzer, as previously described [72]. For glucose and palmitate tolerance experiments, cells were preincubated for 30 min in 160uL unbuffered DMEM supplemented with 4 mM glutamine, 1 mM pyruvate, 5 mM glucose, 0.5 mM L-carnitine, and BSA (0.17 mM)-conjugated palmitate (0.4 mM) for 30 min. The XF96 automated protocol consisted of a 10 min delay after microplate insertion, baseline OCR/ECAR measurements [3x (3 min mix, 3 min measure)], followed by injection of port A (20uL) containing the β3AR agonist BRL37344 (1 μM), and OCR/ECAR measurement [3x (3 min mix, 3 min measure)]. PortB, PortC, and Port D were injected and measured similarly to Port A. Final concentrations of glucose (10, 20, and 40 mM) were adapted to the final volume increase: 200uL after Port B injection, 220uL after Port C, and 240 after Port D. Final concentrations for palmitate characterization were 0.03 mM after Port B, 0.3 mM after Port C, and 3 mM after Port D. All values were first normalized to protein content in each well and then normalized to baseline values in order to compare 8 independent experiments.

Statistics

Experimental data are presented as mean ± standard error of the mean (SEM) and were analyzed with Prism software (Graph pad, Inc.). For normally distributed variables, comparisons between two groups were made by unpaired two-tailed Student t-test; for nonnormally distributed variables, the nonparametric Wilcoxon-Mann–Whitney test was used. Comparisons between more than two groups were made by two-way ANOVA with Tuckey’s post hoc test. Comparisons between more than two groups in response to increasing drug dose, substrate concentration, or time exposure were made by two-way ANOVA with Sidak's multiple comparisons test. Power calculations were used to obtain statistical significance at p-values below 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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