Supplementary Data, Redfern et al, PNAS, 2000

7A. MethodsDoxycycline-mediated Gene Expression. tTA-dependent transactivation was suppressed by providing doxycycline (200 micrograms/ml or as specified) (Sigma, St. Louis, MO) in the drinking water. In all experiments, day 0 is the day when doxycycline was withdrawn. To attain expression levels at approximately 40% of maximal expression, we administered doxycycline at 2 micrograms/ml of drinking water (See Fig. 7H).

ECG Monitoring and Pertussis Toxin Treatment. Implantation of cardiac telemetry units (PhysioTel Implant, Model TA10EA-F20, Data Sciences International, St. Paul, MN) has been described [Redfern, C.H., Coward, P., Degtyarev, M.Y., Lee, E.K., Kwa, A., Hennighausen, L., Bujard, H., Fishman, G.I., Conklin, B.R. (1999) Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nat. Biotechnol. 17:165–169]. Heart rate and wave form in ambulatory mice were monitored continuously with Chart software (AD Instruments, Mountain View, CA). For al l pertussis toxin (PTX) experiments, PTX (30 ng/g body weight) was injected intraperitoneally. The durations of the first 250 consecutive QRS events each hour were measured for 8 hr before PTX injection and for 8 hr starting 36 hr after PTX injection (prolonged QRS is defined as >25 ms).

Spiradoline Administration. For all spiradoline experiments, H2O (10 microliters/g body weight) was injected intraperitoneally, and heart rate was monitored for 20 min. Next, spiradoline (1 mM, 10 microliters/g body weight; U-62066E, Upjohn Pharmaceuticals, Kalamazoo, MI) was injected intraperitoneally, and heart rate was again monitored for 20 min. Spiradoline-mediated bradycardia was calculated by averaging the heart rate between 3 and 20 min after spiradoline injection and dividing by the average heart rate between 3 and 20 min after H2O injection.

Histology. Hearts were collected from anesthetized animals, rinsed in PBS, arrested in diastole with a PBS solution containing 1 M KCl, and fixed overnight in neutral buffered formalin. The hearts were then embedded in paraffin and cut into 4-micrometer sections according to standard protocols. These sections were stained with hematoxylin and eosin (H&E) for light microscopy or with Direct Red 80 (Sirius Red) and Fast Green FCF (Sigma) for collagen analysis.

Echocardiography. Each mouse was anesthetized with avertin, shaved, and placed on a thermally controlled table. Echocardiography was performed with a 12-MHz transducer (Acuson Sequoia, San Jose, CA). The transducer was applied to the chest with a gel-filled standoff to obtain two-dimensional guided M-mode tracings of a cross section of the left ventricular minor axis at the tips of the papillary muscles. M-mode was used to calculate left ventricular end-systolic and end-diastolic dimensions and left ventricular fractional shortening (LVFS). Pulsed Doppler was used to calculate aortic peak flow velocity.

Analysis of Gene Expression. Data were collected from eight control mice (alphaMHC-tTA) and nine littermate mice expressing Ro1 for 8 wk (alpha MHC-tTA/tetO-Ro1) with a new set of arrays used for each mouse. Cross-sections of mouse ventricle (50 mg) were frozen at –90°C. For purification of poly(A) mRNA, frozen heart tissue was placed in OL1 buffer (Qiagen, Valencia, CA) with 3% 2-mercaptoethanol, homogenized with a tissue tearer (Biospec, Bartlesville, OK), passed through a shredder column (Qiagen), separated from protein, resuspended with Oligotex (Qiagen), eluted, precipitated with ethanol, dried in a vacuum, and resuspended in deionized H2O. Next, double-stranded cDNA was generated from the purified poly(A) mRNA (SuperScript Kit, Gibco BRL,

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Grand Island, NY) and subjected to in vitro transcription to form cRNA (Megascript Kit, Ambion, Austin, TX), incorporating biotinylated CTP and UTP nucleotides (Enzo Diagnostics, Farmingdale, NY).

Biotinylated mouse heart cRNA (10 micrograms) was fragmented into lengths of 50–100 nucleotides (Mg2+ buffer, 94°C, 35 min) and mixed with a hybridization cocktail (bacterial control cRNAs for BioB, C, D, and Cre; control 948B biotinylated oligonucleotide [Affymetrix, Santa Clara, CA.]; herring sperm DNA, and 10 mM Tris-HCl, pH 7.6, 1 M NaCl, and 0.01% Triton X-100). The cocktail was hybridized to the DNA array for 18–24 hr at 45°C at 60 rpm. The DNA array was then rinsed 10 times, washed in hybridization buffer (0.1x) for 20 min at 50°C at 60 rpm, and stained with a streptavidin-phycoerythrin solution (25 micrograms/ml, hybridization buffer 1.0x, 1 mg/ml acetylated bovine serum albumin at room temperature at 60 rpm in the dark). The DNA array was washed another 10 times.

DNA arrays were excited with an argon-ion laser; the emission was detected with a photomultiplier tube and digitized after confocal scanning. Separate DNA arrays were used for each mouse heart (eight controls, nine with Ro1 expressed for 8 wk). GeneChip 3.1 automated analysis software (Affymetrix) was used to collect hybridization patterns and intensity information for each sample and to analyze the data. The hybridization intensities were scaled by setting the total fluorescence intensity of a DNA array to a fixed value of 150 for all the genes except the top and bottom 2% of the genes. Gene expression was calculated in absolute hybridization intensity units.

Statistics. For comparisons of mouse pathology, physiology, and gene expression, the statistical significance of differences between control and experimental mice were assessed with a two-tailed, unpaired t test. In the echocardiography experiments, a two-tailed, paired t test was used to compare the same mice at two time points.

Methods for Quantitative Microscopy (Fig. 7D). Quantitative analysis was performed with a color video camera (CCD Iris, Sony, Tokyo, Japan) attached both to the ocular of a light microscope (Zeiss, Thornwood, NY) and to a video-based Image-1/AT image-analyzer system (Universal Imaging, West Chester, PA). Cross sections of hearts at the mid-ventricular level were analyzed for collagen staining. Each cross section was analyzed at four different areas in the left ventricle, starting at the midpoint of the interventricular septum and at 90° intervals from that point. Each analyzed area covered the entire thickness of the left ventricle. For each area of each section, the total amounts of red and green staining were measured. The red-to-green ratio was then calculated and averaged for each mouse.

Methods for Measuring Muscle Strip Mechanics. Mice were anesthetized with sodium pentobarbitone (5 mg, intraperitoneal) and heparinized (100 U, Sigma). Hearts were removed to a dissection dish and briefly perfused through the aorta with a modified Krebs-Henseleit solution [120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 20 mM NaHCO3, 10 mM glucose, and CaCl2 (as indicated)] that was gassed with 95% O2/5% CO2 at 22°C. During perfusion and dissection, the KCl concentration was raised to 15 mM, and the CaCl2 concentration to 0.1 mM. The right ventricle (RV) was opened, and a small cube of the septal wall containing a thin papillary muscle was removed. A remnant of the tricuspid valve was left attached to the other end of the muscle. The muscle dimensions were measured with an ocular micrometer in the dissecting microscope. The muscle width and thickness were measured halfway along its length. The papillary muscle was

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mounted to the experimental apparatus by placing the cube of ventricle in a platinum-wire basket, which was attached by the valve remnant to a stainless steel pin mounted on a micormanipulator.

The papillary muscle was placed in a chamber and superfused with Krebs-Henseleit solution at 22˚C. The Ca2+ concentration of the superfusate was gradually raised to 1mM. Muscles were repetitively stimulated at a frequency of 1 Hz (4-ms stimuli at 150% of the threshold voltage). Force measurements were digitized, stored, and normalized to the cross-sectional area of the muscle (assuming an elliptical cross section).

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Fig. 7B. Ventricular conduction delay in a mouse expressing Ro1

A single mouse (#4304) was monitored continuously by ECG telemetry over a 1-wk period. A variety of abnormal ECG findings were noted; however, the one common feature was a prolonged QRS complex. The grid intervals are 40 ms, and all the QRS complexes shown below are >25 ms. Since only two leads can be used in mice via telemetry, a definitive ECG reading is difficult. The differing wave forms over time could reflect movement of the leads or the mouse during the study. Readings for the three representative strips are presented below.

a. Atrial rhythm with ventricular condition delay

b. Atrial rhythm with ventricular condition delay (different axis)

c. Atrial rhythm with premature atrial beats every third beat and ventricular condition delay

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In four MH-tTA/tetO-Ro1 mice displaying wide QRS complexes for more that 24 hr, PTX injection abolished arrhythmia within 72 hr. Effects of PTX are long lasting, consistent with ADP-ribosylation of Gi, resulting in continuous inactivation of Gi signaling until the protein can be regenerated.

Time after initial injection (hours)-12 720-6 3024126

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Fig. 7C. PTX abolishes wide QRS complexes.

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Each time point represents the duration of Ro1 expression in mouse heart. Collagen deposition increased with the duration of Ro1 expression. Suppression of Ro1 expression for 4 wk after 8 wk of expression (8 wk + reversal) did not reverse collagen deposition. Alpha MHC-tTA/tetO-Ro1 mice on doxycycline (doxy) (0 wk), like the alpha MHC-tTA control mice (n = 15, data not shown), showed minimal evidence of collagen. Collagen deposition in mice (alpha MHC-tTA/tetO-Ro1) raised on low-level doxycycline (2 micrograms/ml, 40% of maximal Ro1 expression) was indistinguishable from that in alpha MHC-tTA/tetO-Ro1 mice with complete receptor suppression (0 wk, doxy 200 micrograms/ml) (0 wk, n = 5; 2 wk, n = 7; 4 wk, n = 6; 8 wk, n = 7; 8 wk + reversal, n = 7; low-level doxy, n = 7; *P < 0.01 vs. 0 wk; †P < 0.01 vs. previous time point).

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Fig. 7D. Quantification of Collagen Formation in Ro1-expressing Mice

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Fig. 7E and F. Expression of Ro1 in Mouse Heart Decreases the Force and Speed of Myocardial Contraction

(E) Ro1 expression for 8 wk decreased the absolute force (g/mm2) of myocardial contractility by 50% (Ro1, alpha MHC-tTA/tetO-Ro1, n = 4; control, alpha MHC-tTA, n = 4).

(F) Representative twitch force records from control and Ro1-expressing mice. Force records have been scaled to the same peak value. Ro1 expression slowed contraction and relaxation compared with control. For all experiments, there was a significant (*P < 0.01) increase in t1/2, the time from peak force to 50% decline of force (Inset).

Supplementary Data, Redfern et al, PNAS, 2000 Page 7

b. Echocardiogram of an Ro1-expressing mouse (alpha MHC-tTA/tetO-Ro1) that developed edema (weight gain from 30 to 55 g in 1 wk) and had decreased activity. This echocardiogram shows an enlarged systolic and diastolic LV chamber size, indicating a dilated cardiomyopathy.

a. Echocardiogram of a control mouse (alpha MHC-tTA).

Fig. 7G. Echocardiograms of Control and Ro1-expressing Mice

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Doxycycline dose (µg/ml)20 10

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Ro1 was originally designed to allow drug-induced activation of Gi signaling in vivo. Because of the basal signaling effects associated with maximal Ro1 expression in the heart, we sought to reduce Ro1 expression while maintaining drug-induced activation of the Gi pathway. At low levels (2 micrograms/ml), doxycycline reduced Ro1 expression to 40% of maximum (estimated by quantitative immuno-precipitation and western blot analysis; data not shown). At this reduced level of receptor expression, all mice (28/28) survived more than 16 wk, and there was no evidence of cardiac pathology or fibrosis at 8 wk (see Fig. 7D, column at right). However, administration of the Ro1 agonist spiradoline continued to cause a significant bradycardia (see above), demonstrating the presence of a functional receptor. These data suggest that a decreased level of Ro1 expression maintains drug-induced Gi signaling but prevents pathologic changes associated with maximal expression of the receptor. Thus, at reduced expression levels, Ro1 acts as a RASSL without signs of constitutive signaling. In Fig. 7H, heart rate was assessed between 3 and 20 min after intraperitoneal infusion of spiradoline, averaged, and divided by the average heart rate between 3 and 20 min after an H2O injection.

Supplementary Data, Redfern et al, PNAS, 2000 Page 9

Figure 7H. Drug-induced Ro1 Signaling with Reduced Expression Levels