1. Dobutamine Stress Testing as a Diagnostic Tool for Evaluation of Myocardial Contractile Reserve in Asymptomatic or Mildly Symptomatic Patients with Dilated Cardiomyopathy Masakazu Kobayashi Hideo Izawa, Xian Wu Cheng, Hiroyuki Asano, Akihiro Hirashiki, Kazumasa Unno, Satoru Ohshima, Takashi Yamada, Yosuke Murase, Tomoko S. Kato, Koji Obata, Akiko Noda, Takao Nishizawa, Satoshi Isobe, Kohzo Nagata, Tatsuaki Matsubara, Toyoaki Murohara, and Mitsuhiro Yokota

2. Objectives We examined the potential of dobutamine stress testing for evaluation of myocardial contractile reserve in asymptomatic or mildly symptomatic patients with DCM.

3. Methods Dobutamine stress testing Data acquisition Hemodynamic measurements were made at baseline, and at the end of each 10-minute infusion period. DOB 10 min baseline 5 μg/kg/min 10 μg/kg/min baseline DOB 5 DOB 10 Protocol

4. ⊿ LV dP/dt max (%) % Increase in LVdP/dt max during DOB infusion (10 μg/min/kg) LV dP/dt max DOB10 – LV dP/dt max baseline LV dP/dt max baseline = ×100 Assessment of myocardial contractile reserve

5. Group Classification Group Ⅰ ⊿ LVdP/dt max > 100% Group Ⅱ ⊿ LVdP/dt max ≦ 100% ⊿ LVdP/dt max (%) Group Ⅱ b LVEF ≦ 25% Group Ⅱ a LVEF > 25% All patients in Group Ⅰ had LVEF > 25% 100 0 baseline DOB5 DOB10 (n=18)(n=17)(n=11)

6. ⅠⅡaⅡa ⅡbⅡb Age (y) 51 ± 952 ± 1350 ± 15 Sex (M/F) 12 / 613 / 49 / 2 NYHA functional class I/II 10 / 88 / 92 / 9 LVEDD (mm) 59 ± 460 ± 9 72 ± 13 * † LVESD (mm) 47 ± 550 ± 10 65 ± 13 * † LVEF (%) 41 ± 739 ± 6 21 ± 2 * † LVEDP (mmHg) 17 ± 515 ± 718 ± 9 PAWP (mmHg) 11 ± 4 17 ± 8 * † C I (L/min/m 2 ) 3.1 ± 0.53.0 ± 0.72.7 ± 0.5 Patients Characteristics mean ± SD, * P<0.05 vs Group Ⅰ, † P<0.05 vs Group Ⅱ a

7. ⅠⅡaⅡa ⅡbⅡb Plasma BNP (pg/mL) 59 ± 76140 ± 147 421 ± 442 *† Plasma NE (pg/mL) 403 ± 143693 ± 293 *655 ± 267 * Delayed H/M 1.9 ± 0.21.6 ± 0.3 *1.5 ± 0.3 * Washout Rate (%) 24.7 ± 12.635.6 ± 16.6242.8 ± 15.4 * Patients Characteristics mean ± SD, * P<0.05 vs Group Ⅰ, † P<0.05 vs Group Ⅱ a

8. Ⅰ Ⅱa Ⅱa Ⅱb Ⅱb HR baseline (bpm) 69 ± 776 ± 12 86 ± 13 * HR DOB10 (bpm) 98 ± 1498 ± 2099 ± 16 LVSP baseline (mmHg) 122 ± 15113 ± 16112 ± 26 LVSP DOB10 (mmHg) 133 ± 16123 ± 16124 ± 27 LV dP/dt max baseline (mmHg/s) 1131 ± 1781053 ± 1901048 ± 224 LV dP/dt max DOB10 (mmHg/s) 2741 ± 417 1692 ± 300 * 1544 ± 342 * T 1/2 baseline (mmHg/s) 41 ± 643 ± 8 44 ± 5 T 1/2 DOB10 (mmHg/s) 25 ± 5 29 ± 11 34 ± 7 * Hemodynamic response to dobutamine mean ± SD, * P<0.05 vs Group Ⅰ

9. Ⅰ Ⅱa Ⅱa Ⅱb Ⅱb β 1 -AR1.39 ± 0.68 0.71 ± 0.19 * 0.66 ± 0.29 * β 2 -AR1.29 ± 0.920.95 ± 0.180.91 ± 0.40 GRK21.54 ± 0.631.53 ± 0.261.59 ± 0.58 Gs  1.18 ± 0.400.94 ± 0.171.04 ± 0.34 Gi2  0.78 ± 0.350.77 ± 0.150.85 ± 0.25 SERCA2a0.60 ± 0.29 0.36 ± 0.08 * 0.37 ± 0.12 * PLB0.82 ± 0.28 0.56 ± 0.12 * 0.36 ± 0.16 * RyR20.74 ± 0.420.56 ± 0.170.69 ± 0.23 Calsequestrin1.34 ± 0.581.16 ± 0.251.30 ± 0.44 Na 2+ -Ca 2+ exchanger 1.69 ± 0.761.14 ± 0.141.46 ± 0.84 mean ± SD, * P<0.05 vs Group Ⅰ RT-PCR analysis of the abundance of mRNAs for contractile regulating proteins ( /GAPDH mRNA)

10. 0 50 100 150 200 250 2520 30 4050 Δ LV dP/dt max (%) LVEF (%) Relation between baseline LVEF and ⊿ LVdP/dt max

11. Plasma NE (pg/mL) 500 1000 1500 Group I Group IIaGroup IIb P < 0.05 0 Comparison of the plasma NE levels

12. Delayed H/M ratio 1.5 2.0 2.5 1.0 Group I Group IIaGroup IIb P < 0.05 Comparison of the delayed H/M ratio in 123 I-MIBG scintigraphy

13. A representative case of Group I

14. A representative case of Group IIa

15. Ca 2+ SERCA2a mRNA PKA Troponin RyR SR PLB mRNA Ca 2+ Ca channelGRK AC β-AR β1-AR mRNA cAMPATP norepinephrine P P P P FKBP12.6 Gsα In our study, the expression of β 1 -AR, PLB, SERCA2a were reduced in the patients with impaired contractile reserve determined by dobutamine stress testing.

16. Dobutamine stress testing is a useful diagnostic tool for identifying reduced adrenergic myocardial contractile reserve related to altered expression of β 1 -adrenergic receptor, SERCA2a, and phospholamban genes even in asymptomatic or mildly symptomatic patients with DCM. Conclusions