By Filip Konecny Mouse transverse aortic constriction (TAC) and TAC hemodynamic assessment

Short TAC procedural notes All adult size FVB/NJ mice were anesthetized using 5% isoflurane with using oxygen as a driving gas at a flow rate ~ 2 l/min and were placed in the dorsal position on heating pad and the limbs were fixed with tape during procedure. Syringe-made close fitting nose cone was used to deliver mixture of isoflurane in 100% oxygen (Fig 1 A)., Animals were maintained in surgical plane of anesthesia using 2% isoflurane and Temgesic (Buprenorphine) at dose 0.05-0.1 mg/kg delivered peri-operatively by s.q. injection. Depilatory cream (Nair) was used to remove hair from surgical area (Fig 1B) and incision site was disinfected using povidone-iodine (Betadine) and 100% alcohol (cleaning/prep step not shown). To prevent dehydration, mice will be given a pre-warmed saline bolus (1.5ml/kg/1h) intraperitoneally peri- operatively A 2 cm skin cut in the area of the suprasternal notch was performed using blade size 12 (area at 1C). Then using blunt forceps a partial thoracotomy in the area of the level of the suprasternal notch was made, carefully not entering the sternum and (or) pleural cavity but aiming to enter the area of transverse aortic arch; past the brachiocephalic trunk that was visible on the left (Fig 1C, D).

Short TAC procedural notes Ribs might be retracted and thymus can be carefully separated allowing direct visualization of the transverse aorta paying attention not to enter into pleural space while further dissecting the area, avoiding need of mechanical ventilation (Fig 1D). A 7-0 silk suture was used to pass under and around the transverse aorta passing the brachiocephalic trunk and position the silk just before left common carotid artery (Fig 1E, F). A bend polished 27-gauge needle (OD=0.4128mm) with needle conus was placed over the transverse aorta and a double knot was secured to the diameter of a 27-gauge needle yielding approximately 70-80% constriction (step not shown). The needle was then retracted and 7-0 silk suture was cut short (step not shown). A successful suture band was snug while blood flow to the brain and body was maintained (Fig 1F, G). The sternum, thoracic musculature and the skin incision was closed by standard techniques with absorbable suture and auto clips (step not shown). Animal was subcutaneously injected with Temgesic (Buprenorphine) at dose 0.05-0.1 mg/kg and placed under the heating lamp to recover in clean cage. In addition, wet food was placed in the recovery cage. All procedure described in this protocol followed current Canadian Animal Care Guidelines and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

A HR ESP EDP dPmax dPmin ESV EDV SV CO EF SW Tau Weiss bpm mmHg mmHg/sec uL ul/min % mJoules msec Mean 668.338 77.296 1.431 11032.57 -8212.67 12.228 35.628 23.4 15829.46 65.673 0.273 4.316 SD 16.675 1.085 0.599 55.074 115.24 0.478 0.593 0.708 1120.86 1.381 0.009 0.133 Invasive open chest PV loop data from mice (t.b.w. mean 26g). Red PV loop and tabulated data (in red) are collected from control while blue data were collected from post-TAC animal. Very right image is visual combination of red and blue PV loops data. Animals were volume ventilated and at time of recording under 1% of Isoflurane in 100% oxygen anesthesia with animal placed on a warming pad (38°C) in a supine position. HR ESP EDP dPmax dPmin ESV EDV SV CO EF SW Tau Weiss bpm mmHg mmHg/sec uL ul/min % mJoules msec Mean 625.048 102.343 4.572 9219.592 -8157.635 33.005 55.655 22.65 14155.61 40.7 0.342 6.136 SD 4.907 4.951 0.281 125.132 151.407 1.032 1.073 0.971 602.773 1.548 0.009 0.092

Short procedural notes of assessment of hemodynamic changes post-TAC (pressure overload) using invasive PV catheter [open chest approach] Mice (t.b.w. mean 26g) were transorally intubated using a 21-gauge polyethylene catheter. Volume assisted ventilation with tidal volume of 0.5-1 ml per gram of mouse body weight, with ~128 ventilation cycles per minute. Post-induction Isoflurane anesthesia was be adjusted to 2% with animal placed on a warming pad (38°C) in a supine position, with the upper and lower extremities attached to the table with surgical tape, then maintained on 2% of Isoflurane by using volume ventilation. V shape skin incision was made in the lower thorax/upper abdomen area over the xyphoid. Skin was separated the from the chest wall by blunt lateral dissections. Abdominal wall was opened in the proximity of the sternal manubrium. Diaphragm was cut to expose the heart apex, avoiding any incisions around sternum to limit bleeding. Cardiac apex was gently maneuvered by Q-tips to assess and find the best catheter insertion site. A 29 G (OD 0.337 mm) needle was used to make an LV apical stab. After successful stab, blood was found in the needle conus. At this stage 29G needle was carefully withdrawn and at the same time 1.2F pressure-volume catheter was inserted until all electrodes of the catheter were fully in the LV chamber in order to measure and record pressure-volume parameters. After period of stabilization baseline recordings were obtained. Mouse ventilator was temporary shut to record 10 seconds of pressure-volume steady state data. All procedure described in this protocol followed current Canadian Animal Care Guidelines and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.