ONLINE SUPPLEMENTAL MATERIALS

Treatment of catecholaminergic polymorphic ventricular tachycardia in mice using novel RyR2

modifying drugs

Na Li, Qiongling Wang, Martha Sibrian-Vazquez, Robert Klipp, Julia O. Reynolds, Tarah A. Word,

Larry Scott Jr., Guy Salama, Robert Strongin, Jonathan J. Abramson, Xander H.T. Wehrens

Detailed methods

Synthesis of compounds. Unless otherwise indicated, all commercially available starting materials

were used directly without further purification. Silica gel Sorbent Technologies 32-63 m was used for

flash column chromatography. 1H- and 13C NMR were obtained on either ARX-400 Advance Bruker

spectrometer. Chemical shifts () are given in ppm relative to CDCl3 (7.26 ppm, 1H, 77.16 ppm 13C)

unless otherwise indicated. MS (HRMS, ESI) spectra were obtained at the PSU Bioanalytical Mass

Spectrometry Facility on a ThermoElectron LTQ-Orbitrap high-resolution mass spectrometer with a

dedicated Accela HPLC system.

Synthesis of 2-(dimethylamino)ethyl 4-(butylamino)-5-chloro-2-methoxybenzoate, EL1. 4-

amino-5-chloro-3-methyl-benzoic acid (0.258 g, 1.24 mmols) and butyraldehyde (0.135 g, 1.860

mmols) are dissolved in 45 ml of anhydrous dichloroethane. Sodium triacetoxyborohydride (0.415 g,

1.860 mmols) is added in one portion; the mixture stirred for 10 min, then acetic acid (0.075 g, 1.24

1

mmols) is added. The mixture is stirred at room temperature 24h. The mixture is diluted with 150 mL

of EtOAc; 150 ml of saturated solution of NaHCO3 are added, then the mixture is stirred for 15 min.

Phases are separated, the organic phase is dried over Na2SO4, filtered and the solvent evaporated

under vacuum to leave a precipitate that corresponds to the target product 2 (208 mg; 65% yield). 1H

NMR (400 MHz, CDCl3) δ 10.39 (s, 1H), 8.02 (s, 1H), 6.12 (s, 1H), 4.85 (s, 1H), 4.04 (s, 3H), 3.22 (d,

J = 4.0 Hz, 2H), 1.70 (dd, J = 8.5, 6.3 Hz, 2H), 1.48 (dd, J = 15.1, 7.4 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H).

13C NMR (101 MHz, CDCl3) δ 165.11, 158.94, 149.22, 133.69, 112.26, 105.60, 92.90, 56.81, 43.26,

31.12, 20.32, 13.95. Compound 2 (0.5 g, 1.94 mmols), EDCI (0.413 g, 2.134 mmols) and DMAP

(0.024 g, 0.194 mmols) are dissolved in 2 ml of anhydrous DMF. After 10 min, 2-

(dimethylamino)ethanol (0.191 g, 2.134 mmols) is added in one portion. The mixture is stirred at

room temperature for 48h. The mixture is quenched by the addition of 150 mL of saturated NaHCO3.

The mixture is extracted with EtOAc (3 x 100 ml). The organic extracts are pooled, dried over

Na2SO4, filtered and the solvent evaporated under vacuum. The target compound is isolated by flash

column chromatography on silica gel using DCM:MeOH 92:8 for elution. Yield 0.471 g (74%). 1H NMR

(400 MHz, CDCl3) δ 7.85 (s, 1H), 6.13 (s, 1H), 4.70 (t, J = 5.0 Hz, 1H), 4.39 (t, J = 5.9 Hz, 2H), 3.91

(s, 3H), 3.22 (td, J = 7.1, 5.3 Hz, 2H), 2.76 (t, J = 5.9 Hz, 2H), 2.38 (d, J = 6.1 Hz, 6H), 1.79 – 1.60 (m,

2H), 1.48 (dq, J = 14.5, 7.3 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.75,

161.18, 148.60, 132.78, 109.91, 107.10, 94.03, 62.04, 57.89, 56.20, 45.77, 43.16, 31.22, 20.32,

13.96. HR ESI [M + H]+ m/z 329.1626; calc. for C16H26ClN2O3, 329.1637.

Synthesis of 2-(dibutylamino)ethyl 4-(butylamino)-2-methoxybenzoate, EL2. 4-amino-2-

methoxybenzoic acid, 3 (2.062 g, 11.96 mmols) and butyraldehyde (1.307 g, 17.95 mmols) are

dissolved in 40 ml of anhydrous dichloroethane. Sodium triacetoxyborohydride (4.0 g, 17.95 mmols) 2

is added in one portion followed by the addition of acetic acid (0.726 g, 11.96 mmols). The mixture is

stirred at room temperature 24h. The mixture is diluted with 150 mL of EtOAc, 150 ml of saturated

solution of NaHCO3 are added and the mixture is stirred for 15 min. Phases are separated; the

organic phase is dried over Na2SO4, filtered and the solvent evaporated under vacuum. The target

compound 4, is isolated by flash column chromatography on silica gel using DCM:MeOH 96:4 for

elution. Yield 2.24 g, 84%. 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 8.7 Hz, 1H), 6.27 (dd, J = 8.7, 2.1

Hz, 1H), 6.09 (d, J = 2.1 Hz, 1H), 4.00 (d, J = 2.5 Hz, 3H), 3.17 (t, J = 7.1 Hz, 2H), 1.62 (ddd, J = 12.4,

8.4, 6.5 Hz, 2H), 1.43 (dq, J = 14.4, 7.3 Hz, 2H), 0.96 (dd, J = 8.2, 6.5 Hz, 3H). 13C NMR (101 MHz,

CDCl3) δ 166.57, 160.59, 154.75, 135.82, 106.66, 105.93, 94.51, 56.78, 43.60, 31.81, 20.73, 14.36.

Compound 4 (0.5 g, 2.239 mmols), EDCI (0.477 g, 2.463 mmols) and DMAP (0.028 g, 0.224 mmols)

are dissolved in 2.4 ml of anhydrous DMF. After 10 min, 2-(dibutylamino)ethanol (0.429 g, 2.463

mmols) is added in one portion. The mixture is stirred at room temperature for 72h. The mixture is

quenched by the addition of 150 ml of saturated NaHCO3. The mixture is extracted with EtOAc (3 x

100 ml). The organic extracts are pooled, dried over Na2SO4, filtered and the solvent evaporated

under vacuum. The target compound is isolated by flash column chromatography on silica gel using

DCM:MeOH 95:5 for elution. 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.6 Hz, 1H), 6.13 (dd, J = 8.7,

2.1 Hz, 1H), 6.06 (d, J = 2.1 Hz, 1H), 4.34 (s, 2H), 4.10 (s, 1H), 3.86 (s, 3H), 3.15 (dd, J = 12.5, 7.0

Hz, 2H), 2.88 (d, J = 5.2 Hz, 2H), 2.58 (s, 4H), 1.60 (dd, J = 14.8, 7.3 Hz, 2H), 1.46 (ddd, J = 22.5,

13.1, 7.2 Hz, 6H), 1.30 (dq, J = 14.7, 7.3 Hz, 4H), 0.96 (t, J = 7.3 Hz, 3H), 0.89 (t, J = 7.3 Hz, 6H). 13C

NMR (101 MHz, CDCl3) δ 165.56, 162.30, 153.61, 134.21, 104.14, 95.17, 55.80, 54.47, 52.21, 43.18,

31.54, 20.67, 20.33, 14.12, 13.97. HR ESI [M + H]+ m/z 379.2950; calc for C22H38N2O3, 379.2966.

3

Synthesis of 2-(dibutylamino)ethyl 4-(butylamino)-5-chloro-2-methoxybenzoate, EL3.

Compound 2 (0.1 g, 0.388 mmols), EDCI (0.083 g, 0.427 mmols) and DMAP (0.0047 g, 0.039 mmols)

are dissolved in 1 ml of anhydrous DMF. After 10 min, 2-(dibutylamino)ethanol (0.075 g, 0.427

mmols) is added in one portion. The mixture is stirred at room temperature for 48 h. The mixture is

quenched by the addition of 150 mL of saturated NaHCO3. The mixture is extracted with EtOAc (3 x

100 ml); the organic extracts are pooled, dried over Na2SO4, filtered and the solvent evaporated

under vacuum. The target compound is isolated by flash column chromatography on silica gel using

DCM:MeOH 9:1 for elution. Yield 0.072 g, (45%). 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 6.11 (s,

1H), 4.68 (s, 1H), 4.34 (s, 2H), 3.89 (s, 3H), 3.21 (td, J = 7.1, 5.4 Hz, 2H), 2.95 – 2.82 (m, 2H), 2.57

(s, 4H), 1.68 (dd, J = 14.7, 7.2 Hz, 2H), 1.55 – 1.39 (m, 6H), 1.32 (dt, J = 15.0, 7.3 Hz, 4H), 0.99 (t, J

= 7.3 Hz, 3H), 0.90 (t, J = 7.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 164.59, 161.27, 148.67, 132.70,

109.92, 94.01, 56.23, 54.40, 52.03, 43.17, 31.23, 20.65, 20.33, 14.09, 13.97. HR ESI [M + H]+ m/z

413.2576; calc. for C22H38ClN2O3, 413.2576.

Synthesis of 2-(diethylamino)ethyl 4-(butylamino)-3-methoxybenzoate, EL4. Compound EL6

(0.5 g, 2.239 mmols), EDCI (0.477 g, 2.463 mmols) and DMAP (0.028 g, 0.224 mmols) are dissolved

in 2.3 ml of anhydrous DMF. After 10 min, 2-(diethylamino)ethanol (0.290 g, 2.463 mmols) is added

in one portion. The mixture is stirred at room temperature for 72h, then quenched by the addition of

150 mL of saturated NaHCO3. The mixture is extracted with EtOAc (3 x 100 ml). The organic

extracts are pooled, dried over Na2SO4, filtered and the solvent evaporated under vacuum. The

target compound is isolated by flash column chromatography on silica gel using DCM:MeOH 95:5 for

elution. Yield 0.063 g (8.8 %). 1H NMR (400 MHz, CDCl3) δ 7.62 (dd, J = 8.3, 1.8 Hz, 1H), 7.39 (d, J =

4

1.8 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 4.67 (s, 1H), 4.42 (t, J = 6.1 Hz, 2H), 3.87 (s, 3H), 3.18 (t, J =

6.8 Hz, 2H), 2.95 (t, J = 6.1 Hz, 2H), 2.75 (q, J = 7.1 Hz, 4H), 1.65 (dt, J = 19.8, 7.3 Hz, 2H), 1.44 (dq,

J = 14.5, 7.3 Hz, 2H), 1.17 – 1.09 (m, 6H), 0.96 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ

167.08, 145.65, 142.91, 124.92, 116.72, 109.93, 107.73, 61.99, 55.69, 50.87, 47.82, 42.88, 31.49,

20.40, 14.00, 11.59. HR ESI [M + H]+ m/z 323.2336; calc. for C18H31N2O3, 323.2340.

Synthesis of 2-(dimethylamino)ethyl 4-(butylamino)-2-methoxybenzoate, EL5. Compound 4 (0.5

g, 2.239 mmols), EDCI (0.477 g, 2.463 mmols) and DMAP (0.028 g, 0.224 mmols) are dissolved in

2.4 ml of anhydrous DMF. After 10 min, 2-(dimethylamino)ethanol (0.221 g, 2.463 mmols) is added

in one portion. The mixture is stirred at room temperature for 72h, then quenched by the addition of

150 mL of saturated NaHCO3. The mixture is extracted with EtOAc (3 x 100 ml). The organic

extracts are pooled, dried over Na2SO4, filtered and the solvent evaporated under vacuum. The

target compound is isolated by flash column chromatography on silica gel using DCM:MeOH 85:15 for

elution. Yield 0.469 g, (71%). 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 8.6 Hz, 1H), 6.13 (dd, J = 8.7,

2.2 Hz, 1H), 6.05 (d, J = 2.1 Hz, 1H), 4.37 (t, J = 5.9 Hz, 2H), 4.09 (t, J = 5.1 Hz, 1H), 3.85 (s, 3H),

3.15 (td, J = 7.1, 5.6 Hz, 2H), 2.75 (t, J = 5.8 Hz, 2H), 2.36 (d, J = 9.5 Hz, 6H), 1.69 – 1.54 (m, 2H),

1.44 (dt, J = 14.8, 7.3 Hz, 2H), 0.96 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 165.61, 162.29,

153.62, 134.32, 107.28, 104.20, 95.19, 61.73, 57.92, 55.78, 45.75, 43.19, 31.54, 20.34, 13.98. HR

ESI [M + H]+ m/z 295.2012; calc. for C16H26N2O3, 295.2027.

5

Synthesis of 4-(butylamino)-3-methoxybenzoic acid, EL6. 4-amino-3-methoxybenzoic acid, 5

(2.105 g, 11.96 mmols) and butyraldehyde (1.307 g, 17.95 mmols) are dissolved in 40 ml of

anhydrous dichloroethane. Sodium triacetoxyborohydride (3.92 g, 17.95 mmols) is added in one

portion followed by the addition of acetic acid (0.792 g, 11.96 mmols). The mixture is stirred at room

temperature for 24h. The mixture is diluted with 150 ml of EtOAc; 150 ml of saturated solution of

NaHCO3 are added, then stirred for 15 min. Phases are separated, the organic phase is dried over

Na2SO4, filtered and the solvent evaporated under vacuum. The precipitate obtained corresponds to

the target product EL6. Yield 2.36 g (88%). 1H NMR (400 MHz, CDCl3) δ 7.73 (dd, J = 8.3, 1.8 Hz,

1H), 7.44 (d, J = 1.8 Hz, 1H), 6.55 (d, J = 8.4 Hz, 1H), 3.89 (d, J = 5.7 Hz, 3H), 3.20 (t, J = 7.1 Hz,

2H), 1.71 – 1.61 (m, 2H), 1.45 (dd, J = 15.1, 7.4 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz,

CDCl3) δ 172.83, 145.57, 143.52, 125.97, 115.85, 110.20, 55.69, 42.83, 31.47, 20.40, 13.99. HR ESI

[M]+ m/z 224.12895; calc for C12H18NO3, 224.12921.

Synthesis of 2-(dimethylamino)ethyl 4-(butylamino)-3-methylbenzoate, EL7. 4-amino-3-methyl-

benzoic acid, 6 (0.510 g, 3.31 mmols) and butyraldehyde (0.361 g, 4.96 mmols) are dissolved in 40 ml

of anhydrous dichloroethane. Sodium triacetoxyborohydride (1.107 g, 4.96 mmols) is added in one

portion followed by the addition of acetic acid (0.201 g, 3.31 mmols). The mixture is stirred at room

temperature for 24h. The mixture is diluted with 150 mL of EtOAc, 150 ml of saturated solution of

NaHCO3 are added; then is stirred for 15 min. Phases are separated, the organic phase is dried over

6

Na2SO4, filtered and the solvent evaporated under vacuum. The precipitate obtained corresponds to

the target compound 7 and is used without further purification. Yield 0.597 g (87%). 1H NMR (400

MHz, CDCl3) δ 7.91 (dd, J = 8.5, 2.0 Hz, 1H), 7.80 (d, J = 1.3 Hz, 1H), 6.58 (d, J = 8.6 Hz, 1H), 3.24

(t, J = 7.1 Hz, 2H), 2.15 (s, 3H), 1.74 – 1.59 (m, 2H), 1.46 (dd, J = 15.1, 7.4 Hz, 2H), 0.98 (t, J = 7.3

Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 172.65, 151.02, 132.28, 130.86, 120.64, 116.51, 108.34,

43.32, 31.56, 20.42, 17.34, 14.01. Compound 7 (0.5 g, 2.412 mmols), EDCI (0.514 g, 2.65 mmols)

and DMAP (0.030 g, 0.241 mmols) are dissolved in 2.5 ml of anhydrous DMF. After 10 min, 2-

(dimethylamino)ethanol (0.238 g, 2.65 mmols) is added in one portion. The mixture is stirred at room

temperature for 72h, then quenched by the addition of 150 mL of saturated NaHCO3. The mixture is

extracted with EtOAc (3 x 100 ml). The organic extracts are pooled, dried over Na2SO4, filtered and

the solvent evaporated under vacuum. The target compound is isolated by flash column

chromatography on silica gel using DCM:MeOH 93:7 for elution. Yield 0.512 g (76%). 1H NMR (400

MHz, CDCl3) δ 7.85 (dd, J = 8.5, 2.0 Hz, 1H), 7.75 (d, J = 1.3 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 4.43 (t,

J = 5.8 Hz, 2H), 3.93 (s, 1H), 3.23 (d, J = 5.0 Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 2.41 (s, 6H), 2.15 (s,

3H), 1.77 – 1.60 (m, 2H), 1.47 (dd, J = 15.1, 7.4 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz,

CDCl3) δ 166.97, 150.33, 131.59, 129.95, 120.49, 117.23, 108.18, 61.86, 57.83, 45.62, 43.20, 31.45,

20.28, 17.23, 13.89. HR ESI [M + H]+ m/z 279.2062; calc. for C16H27N2O2, 279.2078.

Synthesis of 2-(diethylamino)ethyl 4-(butylamino)-3-methylbenzoate, EL8. Compound 7 (0.5 g,

2.412 mmols), EDCI (0.514 g, 2.65 mmols) and DMAP (0.030 g, 0.241 mmols) are dissolved in 2.5 ml

of anhydrous DMF. After 10 min, 2-(diethylamino)ethanol (0.313 g, 2.65 mmols) is added in one

portion. The mixture is stirred at room temperature for 72h; then is quenched by the addition of 150

mL of saturated NaHCO3. The mixture is extracted with EtOAc (3 x 100 ml). The organic extracts 7

are pooled, dried over Na2SO4, filtered and the solvent evaporated under vacuum. The target

compound is isolated by flash column chromatography on silica gel using DCM:MeOH 94:6 for

elution. Yield 0.461 g (63%). 1H NMR (400 MHz, CDCl3) δ 7.81 (dd, J = 8.5, 2.0 Hz, 1H), 7.72 (d, J =

1.3 Hz, 1H), 6.55 (d, J = 8.6 Hz, 1H), 4.37 (t, J = 6.2 Hz, 2H), 3.91 (s, 1H), 3.21 (dd, J = 11.4, 7.1 Hz,

2H), 2.89 (t, J = 6.2 Hz, 2H), 2.69 (q, J = 7.2 Hz, 4H), 2.13 (s, 3H), 1.72 – 1.59 (m, 2H), 1.45 (dq, J =

14.5, 7.3 Hz, 2H), 1.10 (dd, J = 8.9, 5.4 Hz, 6H), 0.97 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3)

δ 167.11, 150.39, 131.69, 129.98, 120.60, 117.47, 108.29, 62.23, 51.07, 47.90, 43.32, 31.57, 20.40,

17.36, 14.00, 11.89. HR ESI [M + H]+ m/z 307.2380; calc. for C18H30N2O2, 307.23910

Synthesis of 2-(diethylamino)ethyl 4-(butylamino)-5-chloro-2-methoxybenzoate, EL9.

Compound 2 (0.5 g, 1.94 mmols), EDCI (0.413 g, 2.134 mmols) and DMAP (0.024 g, 0.194 mmols)

are dissolved in 2 ml of anhydrous DMF. After 10 min, 2-(diethylamino)ethanol (0.251 g, 2.134

mmols) is added in one portion. The mixture is stirred at room temperature for 48h. The reaction

mixture is quenched by the addition of 150 ml of saturated NaHCO3. The mixture is extracted with

EtOAc (3 x 100 ml). The organic extracts are pooled, dried over Na2SO4, filtered and the solvent

evaporated under vacuum. The target compound is isolated by flash column chromatography on

silica gel using DCM:MeOH 92:8 for elution. Yield 0.518 g (75%) 1H NMR (400 MHz, CDCl3) δ 7.82

(s, 1H), 6.11 (s, 1H), 4.68 (t, J = 5.0 Hz, 1H), 4.36 (t, J = 6.3 Hz, 2H), 3.89 (s, 3H), 3.20 (td, J = 7.1,

5.3 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.70 (q, J = 7.1 Hz, 4H), 1.75 – 1.62 (m, 2H), 1.46 (dq, J = 14.5,

7.3 Hz, 2H), 1.11 (t, J = 7.2 Hz, 6H), 0.98 (dd, J = 9.1, 5.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ

164.72, 161.18, 148.61, 132.77, 109.91, 107.07, 94.01, 62.03, 56.20, 51.00, 47.85, 43.16, 31.22,

20.32, 13.96, 11.74. HR ESI [M + H]+ m/z 357.1938; calc. for C18H30ClN2O3, 357.1950.

8

Isolation of ventricular myocytes. Hearts were removed from R176Q/+ and WT mice during

isoflurane anesthesia. After excising, the heart was rinsed in KB solution (90 mmol/L KCl, 30 mmol/L

K2HPO4, 5 mmol/L MgSO4, 5 mmol/L pyruvic acid, 5 mmol/L β-hydroxybutyric acid, 5 mmol/L

creatine, 20 mmol/L taurine, 10 mmol/L glucose, 0.5 mmol/L EGTA, 5 mmol/L HEPES, pH 7.2) and

cannulated through the aorta. The heart was perfused using a Langendorff setup with 0 Ca2+ Tyrode

solution (3 ~ 5 minutes, 37 °C), followed by Liberase (TH Research Grade, Roche Applied Science) in

0 Ca2+ Tyrode for 10-15 minutes at 37 °C. After digestion, the heart was perfused with 3 ml KB

solution to wash out remaining collagenase. The hearts were minced in KB solution, gently agitated,

then filtered through a 210μm polyethylene mesh. After settling, ventricular myocytes were washed

once with KB solution, and stored in KB solution at room temperature before use.

Confocal imaging of Ca2+ handling. Ventricular myocytes were isolated by a modified collagenase

method, and incubated with Ca2+ indicator Fluo-4-acetoxymethyl ester (Fluo-4-AM; 2 mol/L, Fluo-4

AM, Invitrogen) in KB solution for 30 minutes at room temperature as described.1 Cells were rinsed

with dye-free normal Tyrode solution (1.8 mmol/L Ca2+) for 20 minutes for de-esterification. Cells were

transferred to a chamber equipped with a pair of parallel platinum electrodes on a confocal

microscope (LSM 510, Carl Zeiss, Thornwood, NY) with a 40X oil immersion objective. Fluo-4 was

excited at 488 nm with emission collected through a 515 nm long pass filter. Fluorescence images

were recorded in line-scan mode with 1024 pixels per line at 500 Hz. After being paced at 1 Hz (5 ms

pulses, 10 V) for at least 2 minutes, only myocytes showing clear striation and normal contractility

were selected for further experiments. Once steady state Ca2+ transient was observed, pacing was

stopped and Ca2+ sparks were counted using SparkMaster.2

Programmed electrical stimulation. In vivo electrophysiology studies were performed in mice as

previously described.3 Briefly, atrial and ventricular intracardiac electrograms (ECGs) were recorded

using an 1.1F octapolar catheter (EPR-800, Millar Instruments, Houston, Texas) inserted via the right

9

jugular vein. Surface and intracardiac electrophysiology parameters were assessed simultaneously at

baseline using a computer-based data acquisition system (EMKA Technologies, Falls Church, VA).

ECG waveform results included a clearly defined P wave, denoting atrial depolarization, and a QRS

wave, which signified ventricular depolarization. PR, RR, and QTc (corrected QT interval) were

evaluated at baseline. Next, right ventricular pacing was performed using 2-ms current pulses

delivered by an external stimulator (STG-3008, Multi Channel Systems, Reutlingen, Germany) to

determine VT inducibility after injections of caffeine (2 mg/kg i.p.) and epinephrine (120 mg/kg i.p.).

The incidence of reproducible bidirectional and sustained VT (at least positive twice among three

tests) was determined. Standard pacing protocols were used to determine basic electrophysiologic

parameters such as effective refractory periods. Inducibility of ventricular tachycardia (VT) was

determined by using single extra stimuli protocols. Premature ventricular complexes (PVCs) were

defined as spontaneous abnormal ventricular contractions that occurred prior to programmed

stimulation protocols. These ventricular contractions were differentiated from aberrated atrial

premature beats and catheter ectopy based on intracardiac electrograms and surface morphology.

Non-sustained VT was defined as an episode of 4-9 beats of VT, whereas sustained VT was defined

as 10 or more consecutive beats of VT.4, 5 All occurrences were tested twice for reproducibility.

Echocardiography. To facilitate high-quality imaging, the chests of the mice were depilated using

Nair crème. Next, mice were placed supine on a heated ECG board to maintain the body temperature

within a narrow range of 36.5 ºC to 37.5 ºC. The heart rate was maintained above 400 beat per

minute in order to circumvent the confounding effects of hypothermia and bradycardia. Transthoracic

echocardiography was performed on mice anesthetized with inhaled 2% Isoflurane and using the

VisualSonics Vevo 2100 Imaging System with a high frequency (30 Mhz) probe. A combination of B-

mode short axis images and M-mode scans were collected as described.6.

10

11

Supplemental Figure 1. Tetracaine derivatives inhibit abnormal SR Ca2+ release events in

R176Q/+ myocytes.

(A) Representative confocal microscope recordings of Ca2+ sparks and (B) quantification of Ca2+ spark

frequency (CaSF) in ventricular myocytes of WT and R176Q/+ mice in the absence and presence of

isoproterenol (100 nmol/L). (C) The percent decrease in CaSF caused by tetracaine and 9 derivatives

(EL1-9) normalized to the vehicle-treated cells. *P<0.05.

12

Supplemental Figure 2. Chemical structure of tetracaine and the 9 tetracaine derivatives.

13

Supplemental Fig. 3. EL9 reduced CaSF in S2814D myocytes.

(A) Representative recordings of Ca2+ sparks in ventricular myocytes of WT and S2814D mice in the

presence of vehicle or EL9 (500 nmol/L). (B) Summary data showing that EL9 reduced CaSF in

S2814D myocytes compared to vehicle. *P<0.05, **P<0.01.

14

Supplemental Table S1. Effects of EL9 on Ca2+ spark parameters in WT and R176Q/+ myocytes.

Parameter WT +

Vehicle

WT + EL9 P

value

R176Q +

Vehicle

R176Q +

EL9

P

value

N (mice) 5 5 3 3

N (cells) 13 12 18 13

N (sparks) 180 175 1195 205

Ca spark

amplitude

(F/F0)

0.40±0.01 0.38±0.01 0.28 0.35±0.003 0.40±0.008 <0.001

FWHM (µm) 1.32±0.06 1.20±0.05 0.12 1.51±0.03 1.34±0.05 0.03

FDHM (ms) 22.87±1.18 26.31±1.33 0.054 29.06±0.67 21.86±1.25 <0.001

Tau(ms) 27.49±2.71 28.25±2.05 0.82 38.29±2.03 25.61±4.77 0.02

∆F/F0/∆t

max(ms-1)

41.89±1.27 38.43±1.35 0.06 36.07±0.46 43.56±1.32 <0.001

15

Supplemental Table S2. Absence of EL9 effects on ventricular function of WT mice.

Baseline

(n=4)

EL9

(n=4)

ESD (mm) 2.7 ± 0.1 2.7 ± 0.1

EDD (mm) 4.1 ± 0.1 4.1 ± 0.1

FS (%) 64.1 ± 1.5 62.2 ± 1.4

Cardiac output (mL/min) 23.6 ± 0.7 21.2 ± 0.3

LVAWs (mm) 1.0 ± 0.07 1.0 ± 0.1

LVAWd (mm) 0.77 ± 0.03 0.78 ± 0.04

LVPWs (mm) 1.1 ± 0.05 1.1 ± 0.04

LVPWd (mm) 0.81 ± 0.05 0.77 ± 0.04

EDD, end-diastolic diameter; ESD, end-systolic diameter; FS, fractional shortening; LVAWd, left-

ventricular end-diastolic anterior wall thickness; LVAWs, left-ventricular end-systolic anterior wall

thickness; LVPWd, left-ventricular end-diastolic posterior wall thickness; LVPWs, left-ventricular end-

systolic posterior wall thickness.

16

Supplemental Table S3. Effects of EL9 on electrophysiological parameters in WT and R176Q/+

mice.

WT (n=8) R176Q/+ (n=6)

Pre-EL9 Post-EL9 Pre-EL9 Post-EL9

RR (ms) 98.1 ± 2.4 95.4 ± 1.1 129.3 ± 7.4 117.5 ± 6.6

HR (bpm) 613.9 ± 14.4 629.7 ± 7.6 471.7 ± 27.4 518.9 ± 29.6

PR (ms) 34.8 ± 1.1 34.3 ± 0.6 42.7 ± 2.7 39.5 ± 1.6

QRS (ms) 8.8 ± 0.4 8.9 ± 0.5 9.3 ± 0.6 8.8 ± 0.3

QTc (ms) 28.2 ± 1.5 29.3 ± 1.1 20.2 ± 1.1 23.2 ± 1.1

Bpm, beats per minute, HR, heart rate.

17

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