ONLINE DATA SUPPLEMENT

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1 Mitigation of Ventilator-Induced Diaphragm Atrophy by Transvenous Phrenic Nerve Stimulation Steven C. Reynolds, Ramasamy Meyyappan, Viral Thakkar, Bao D. Tran, Marc- André Nolette, Gautam Sadarangani, Rodrigo A. Sandoval, Laura Bruulsema, Brett Hannigan, Jason W. Li, Elizabeth Rohrs, Jason Zurba, and Joaquín Andrés Hoffer ONLINE DATA SUPPLEMENT

2 Online Table E1 Comparative Pressure Measurements Breath Type V T (ml) Peak (cmh 2 O) P AW % PTP (cmh 2 O.s) Contribution (from P AW ) Peak (cmh 2 O) P es P tp P ga P di PTP (cmh 2 O.s) Peak (cmh 2 O) PTP (cmh 2 O.s) Peak (cmh 2 O) PTP (cmh 2 O.s) Peak (cmh 2 O) PTP (cmh 2 O.s) Unpaced ± ± ± ± ± ± ± ± ± ± ± 0.1 Paced ± ± ± ± ± ± ± ± ± ± ± ± 1.6 Data from ventilated-paced subjects (N=6) reported by breath type as Mean ± SD of breath pressures during a 1-hour period at 25 hours post-intubation. V T - Tidal Volume, P AW - Airway Pressure, P es - Esophageal Pressure, P tp - Transpulmonary Pressure, P ga - Gastric Pressure, P di - Transdiaphragmatic Pressure, PTP - Pressure Time Product. For paced breaths, negative sign in the P es Peak and PTP measures denotes the direction of pressure change. For unpaced breaths, the negative sign in the P di Peak and PTP values denote absence of inspiratory effort. For paced breaths, P es PTP is calculated as the area below the baseline P es which is taken as the value just prior to start of the breath cycle. E2

3 Online Table E2 - Comparative clinical parameters Ventilated-Not Paced Median (IQR) Ventilated-Paced Median (IQR) p- value(2- tailed) ass (kg) ( ) ( ) 0.73 idal Volume (ml) ( ) ( ) 0.70 ormalized Tidal Volume ml/kg) 8.68 ( ) 8.74 ( ) 1.00 inute Ventilation (L/min) 9.93 ( ) ( ) 0.62 espiratory Rate (bpm) ( ) ( ) 0.49 H 7.48 ( ) 7.47 ( ) 0.70 ao 2 (mmhg) ( ) ( ) 0.39 F i O ( ) 0.28 ( ) 0.81 ao 2 /F i O ( ) ( ) 0.70 aco 2 (mmhg) ( ) ( ) 0.59 S a O 2 (%) ( ) ( ) 1.00 HCO 3 (mm) ( ) ( ) 0.82 eart Rate (beat/min) ( ) ( ) 0.24 ean Blood Pressure (mmhg) ( ) ( ) 0.70 E3

4 Online Table E3 Anesthesia Parameters Ventilated-Not Paced Ventilated-Paced p-value Median (IQR) Median (IQR) (2-tailed) Atropine (µg/kg/hr) 0.51 ( ) 0.93 ( ) 0.26 Ketamine (mg/kg/hr) 2.53 ( ) 1.43 ( ) 0.49 Midazolam (mg/kg/hr) 0.58 ( ) 0.53 ( ) 0.59 Fentanyl (µg/kg/hr) 3.90 ( ) 3.60 ( ) 0.64 Propofol (mg/kg/hr) 7.15 ( ) 7.37 ( ) 0.94 Online Table E4 - Pulse stimulation parameters during pacing Stimulation Current (ma) Pulse Width (µs) Paced Subject Phrenic Nerve Minimum Maximum Minimum Maximum 1 Left Right Left Right Left Right Left Right Left Right Left Right E4

5 Online Table E5 Myofiber Cross Sectional Areas Cross Sectional Area Normalized Cross Sectional by Weight and Raw Cross Area Normalized by Sarcomere Length Subject ID Sectional Area (µm²) Weight (µm²/kg) (µm²/kg) Left Right Left Right Left Right Control Left Right Left Right Left Right Median (IQR) 1338 ( ) 23.1 ( ) 24.6 ( ) Left Ventilated-Not Paced Right Left Right Left Right Left E5

6 Right Left Right Left Right Median (IQR) 954 ( ) 18.1 ( ) 17.9 ( ) Left Right Left Right Left Ventilated-Paced Right Left Right Left Right Left Right Median (IQR) 1202 ( ) 22.6 ( ) 24.9 ( ) E6

7 Online Figure E1 The diaphragm contribution during each paced breath was calculated from the normalized difference in inspiratory airway pressure-time product during the previous ventilator-only (unpaced) breath and the ventilated + paced breath, expressed as % reduction with respect to the pressure-time product during the unpaced breath. E7

8 Online Figure E2 The cross-sectional area was measured by manually tracing the area for each muscle fiber using ImageJ 1.48v software. These raw measurements were then normalized by the sarcomere length to account for distortions during sample collection. Sarcomere lengths were measured from longitudinal sections under a Leitz Wetzlab SM-LUX light microscope and a computer a Tucsen camera with TSView7 software. Finally, the cross sectional areas were normalized by the weight of the subjects. E8

9 Figure E1 E9

10 Figure E2 E10

ONLINE DATA SUPPLEMENT. First 24 hours: All patients with ARDS criteria were ventilated during 24 hours with low V T (6-8 ml/kg

ONLINE DATA SUPPLEMENT. First 24 hours: All patients with ARDS criteria were ventilated during 24 hours with low V T (6-8 ml/kg APPENDIX 1 Appendix 1. Complete respiratory protocol. First 24 hours: All patients with ARDS criteria were ventilated during 24 hours with low V T (6-8 ml/kg predicted body weight (PBW)) (NEJM 2000; 342