Figure 1: Change in Diffusion capacity of the lungs for carbon monoxide (DLCO, Panel A), Pulmonary blood flow (Qc, Panel B), Gas exchange surrogate of pulmonary capacitance (GxCap, Panel C) and stroke volume and oxygen pulse (SV and O2 Pulse, Panel D) from rest through unloaded and three 3 minute increasing workloads of submaximal exercise and recovery. Comparing individuals with Group 1 PAH (grey triangles and dashed line) to healthy controls (black circles and solid line). Oxygen pulse is represented by open grey triangles for PAH and open black circles for healthy controls. There was a significant workload x condition interaction effect for GxCap, p=0.007.

Figure 2 The Relationship of Cardiac Output to Alveolar-capillary Expansion in Response to Submaximal Exercise
Change in ratio of diffusion capacity of the lungs for carbon monoxide to pulmonary blood flow (DLCO/Qc) from rest through unloaded and 3 3 minute increasing workloads of submaximal exercise and recovery. Comparing individuals with Group 1 PAH (grey triangles and dashed line) to healthy controls (black circles and solid line). Workload x condition interaction effect p=0.02 for any of these variables. Black rectangle highlight the change from baseline to unloaded cycling (i.e., the initiation of exercise), for further analysis of this period see data in Table 4 and Figure 3.

Figure 3: Factors influencing Alveolar-capillary Expansion upon Exercise Initiation
The relationship between the change in ratio of diffusion capacity of the lungs for carbon monoxide to pulmonary blood flow (DLCO/Qc) and the change in gas exchange surrogate of pulmonary capacitance (GxCap, Panel A) and the change in pulmonary blood flow (Qc, Panel B) from rest to unloaded cycling. Individuals with PAH represented by grey triangles and grey trend line, and healthy controls represented by black circles and black trend line. Pearson correlation results and p-values for the entire group, and PAH and Healthy conditions separately are provided on the panels.

Table 1: Subject Population	Healthy	PAH
n	16	16
Gender female	9 (56%)	9 (56%)
Age (years)	55±16	54±17
Height (cm)	169±11	170±10
Weight (kg)	75±14	79±21
BMI (kg/m²)	26±4	27±5

RVSP (mmHg) 54±22

Peak TRV (m/s) 3.5±0.7

TAPSE (mm) 20±3

Functional Class (range 1-2) 1.5±0.5

Group 1 : 7 idiopathic Medications:

2 portopulmonary 5 single therapy (ambrisentan or PDE5) 2 scleroderma 7 dual therapy (ERA & PDE5 or SGC)

2 lupus 4 triple therapy (PDE5, ERA and PGI2 or IP)

1 familial

1 congenital HD

RVSP= right ventricular systolic pressure; TRV=tricuspid regurgitation velocity; TAPSE=tricuspid annular plane systolic excursion by M-mode; PDE5=phosphodiesterase inhibitor; ERA= endothelin receptor antagonist; PGI2 = prostacyclin IV; IP = prostacyclin receptor agonist; HD= heart disease

Table 2 Submaximal Exercise Final Stage	Healthy	PAH
Peak HR (bpm)	115±18	120±16
Percent of Predicted Max HR (%)	71±15	72±12
VO₂ peak (mL/min/kg)	14.7±5.2	11.9±2.7
ΔVO₂ from rest to the final stage (mL/min/kg)	10.8±4.7	7.8±2.4
Wattage final stage	71±16	53±17*
VO₂/W final stage	0.20±0.07	0.24±0.07
RER at peak	1.01±0.13	1.13±0.17
RPE	11.0±2.1	13.6±2.3
Desaturation (%)	0.9±1.5	2.6±3.8

VO2 peak= peak oxygen consumption; RER= respiratory exchange ratio; RPE= rating of perceived exertion. * p< 0.01 adjusted value for multiple comparisons in independent samples t-test Healthy vs. PAH

Baseline

Stage 1 (unloaded)

Stage 2

Stage 3

Stage 4

Recovery

Healthy

PAH

Healthy

PAH

Healthy

PAH

Healthy

PAH

Healthy

PAH

Healthy

PAH

VO₂ (mL/min/Kg)

3.9±1.1

4.5±1.7

8.2±2.4

8.0±2.1

9.8±2.6

9.7±1.8

12.0±3.7

11.0±2.3

14.6±5.3

12.1±3.0

10.2±3.5

10.3±3.2

Vt (mL)

755±436

831±258

1002±342

1014±284

1104±310

1242±304

1272±379

1396±333

1468±469

1466±367

1233±417

1362±308

RR (bpm)

17±4

19±6

24±7

25±6

25±3

27±6

26±5

28±4

28±7

31±5

28±5

30±6

VE (L/min)

11.8±5.4

15.4±7.8

22.3±6.9

24.5±7.5

26.2±6.7

32.3±7.1

31.0±6.8

38.8±10.0

40.0±11.0

45.1±12.4

33.8±11.7

40.5±12.1

VE/VCO₂

45±8

50±10

46±24

48±7

41±9

45±6

40±17

44±7

36±10

45±10

40±11

45±9

PETCO₂(mmHg)

33±6

29±6

33±4

29±5

34±4

30±6

35±4

31±68

36±4

31±7

35±4

31±7

PECO₂ (mmHg)

20±4

19±4

22±4

18±3

23±4

21±4

24±5

22±5

26±6

22±5

24±5

21±5

PECO₂/PETCO₂

0.62±0.07

0.66±0.09

0.67±0.09

0.68±0.06

0.68±0.10

0.71±0.06

0.69±0.11

0.71±0.07

0.70±0.13

0.71±0.09

0.69±0.12

0.71±0.09

SpO₂ (%)

100±0

98±2

99±1

97±3

99±1

97±3

99±2

96±4

99±2

96±4

99±1

98±3

ΔSpO2 (%)

-0.4±0.9

-0.8±2.0

-0.5±0.8

-1.4±2.2

-0.8±1.4

-1.8±2.6

-0.9±1.5

-2.6±3.8

-0.3±0.5

-0.7±2.6

SBP (mmHg)

122±10

119±12

130±17

131±16

139±19

136±21

146±22

149±22

162±27

159±29

133±16

135±23

DBP (mmHg)

76±5

72±9

78±6

74±9

77±6

75±10

79±9

77±10

80±10

80±11

76±6

72±10

HR (bpm)

69±10

78±10

83±17

93±11

90±15

100±12

100±15

110±13

115±17

119±16

85±17

94±15

DM (L/min/mmHg)

24.9±11.4

21.1±9.4

27.4±11.2

21.8±9.4

27.4±11.4

23.3±9.3

28.1±12.0

24.5±10.0

31.0±13.1

24.6±10.2

28.4±12.1

23.1±12.1

Vc (mL)

89.8±28.6

82.7±58.2

100.3±33.7

100.3±75.1

110.8±57.8

106.4±84.5

112.4±34.9

120.8±99.3

118.6±34.8

113.8±64.3

95.7±30.4

101.7±89.2

DM/Vc

0.30±0.13

0.31±0.13

0.29±0.11

0.28±0.13

0.29±0.11

0.28±0.11

0.27±0.11

0.26±0.12

0.28±0.12

0.26±0.13

0.31±0.11

0.31±0.15

Table 3 Change in Gas Exchange, Hemodynamic Parameters and Components of DLCO in Response to Submaximal Exercise

VO2= oxygen consumption; Vt= tidal volume; RR= respiratory rate; VE= minute ventilation; PETCO2= end-tidal CO2; PECO2= mixed expired CO2; SpO2= peripheral oxygen saturation; DM= alveolar capillary membrane conductance; Vc= pulmonary capillary blood volume; DM/Vc= functional unit of diffusion

Change upon initiation of exercise	Healthy	PAH	P-value
ΔDLCO (rest to stage 1)	1.9±2.4	1.2±1.4	0.31
ΔQc (rest to stage 1)	2.4±0.7	1.5±1.4	0.04
ΔSV (rest to stage 1)	20±13	7±15	0.02
ΔHR (rest to stage 1)	15±10	14±10	0.70
ΔGxCap (rest to stage 1)	103±61	78±53	0.22
ΔDLCO/Qc (rest to stage 1)	-1.17±0.74	-0.52±0.80	0.03

Δ=change from rest to stage 1; DLCO= diffusion capacity of the lungs for carbon monoxide; Qc= pulmonary blood flow; SV= stroke volume; GxCap= gas exchange derived pulmonary capacitance; DLCO/Qc= ratio of DLCO to Qc. P-value independent samples t-test Healthy vs. PAH

Table 5 Pre to Post ambrisentan Change upon initiation of exercise	Patient 1: De novo Med		Patient 2: Second Med
Addition of ambrisentan 12 weeks	Pre	Post	Pre	Post
ΔDLCO (mmHg)	-0.39	3.97	0.5	0.95
ΔQc (L/min)	-0.46	2.9	1.56	4.14
ΔSV (ml/beat)	-13.6	29.4	-0.5	29.7
ΔGxCap	54	98	63	62
ΔDLCO/Qc	0.25	-0.88	-0.64	-0.93
Peak Desaturation (%)	-3	0	-2	0