BIO20121370 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Fhod3 expression in the heart and body of wild-type mice. Lysates from the heart or the rest of the body of Fhod3^+/− (+/−) and Fhod3^−/− (−/−) embryos at E10.5 (5 µg of protein) were analyzed by immunoblot with anti-Fhod3 and anti-GAPDH antibodies.

• Supplemental Figure S2 -

  Fig. S2. Fhod3 localization in the embryonic heart. Confocal fluorescence micrographs of hearts of wild-type embryos. Sections of embryonic hearts at E 11.5 and E13.5 were subjected to immunofluorescent staining for anti-α-actinin (green) and anti-Fhod3-(C-20) (red) antibodies. Bars, 2 µm.

• Supplemental Figure S3 -

  Fig. S3. Electron micrographs of thin sections of wild-type (Fhod3^+/+) and Fhod3 null (Fhod3^−/−) hearts. Large images showing the cell-cell contact region at E9.5 are shown. In Fhod3^+/+ hearts, cyan arrowheads indicate fascia adherens, and magenta arrowheads indicate Z-lines. In Fhod3^−/− hearts, a cyan arrowhead indicates an immature fascia adherens-like structure, and a green arrowhead indicates an irregular shaped mitochondrion. Bar, 500 nm.

• Supplemental Figure S4 -

  Fig. S4. Cardiac expression of Fhod3 in the transgenic mice expressing Fhod3IA. The indicated amount of cardiac lysates from transgenic mice (Tg (IA)) or non-transgenic mice (Non-Tg) were analyzed by immunoblot with anti-Fhod3 antibodies.

• Supplemental Figure S5 -

  Fig. S5. Cardiac functions after surgical transverse aortic constriction (TAC). (A) Schematic representation of the experimental design for testing cardiac functions after surgical TAC. (B) Organ weights after surgical TAC. BW, body weight; HW, heart weight; LuW, lung weight; LivW, liver weight; and KidW, kidney weight. Error bars indicate s.e.m.; n = 4 (sham-operated Fhod3^+/+ and sham-operated Fhod3^+/−) and n = 7 (TAC-operated Fhod3^+/+ and TAC-operated Fhod3^+/−). (C) Cardiac parameters measured by echocardiography. HR, heart rate; IVSd, intervenricular septum diastolic diameter; LVPWd, left ventricular posterior wall diastolic diameter; LVIDd, left ventricular internal diameters at end-diastole; LVIDs, left ventricular internal diameters at end-systole; EF,ejection fraction; and FS, fractional shortening. Error bars indicate s.e.m.; n = 4 (sham-operated Fhod3^+/+ and sham-operated Fhod3^+/−) and n = 7 (TAC-operated Fhod3^+/+ and TAC-operated Fhod3^+/−). (D) Cardiac parameters measured by Millar Catheter. HR, heart rate; ESP, end systolic pressure; EDP, end diastolic pressure; dP/dt max, maximal rate of pressure development; dP/dt min, maximal rate of decay of pressure; and tau, monoexponential time constant of relaxation. Error bars indicate s.e.m.; n = 4 (sham-operated Fhod3^+/+, sham-operated Fhod3^+/−, and TAC-operated Fhod3^+/+) and n = 5 (TAC-operated Fhod3^+/−). * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

• Movie 1 -

  Movie 1. Real-time recording of the heartbeat of wild-type embryos at E8.5 (front view). Real-time movie of the heart of a wild-type embryo at E8.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.

• Movie 2 -

  Movie 2. Real-time recording of the heartbeat of Fhod3^−/− embryos at E8.5 (front view). Real-time movie of the heart of a Fhod3^−/− embryo at E8.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.

• Movie 3 -

  Movie 3. Real-time recording of the heartbeat of wild-type embryos at E9.5 (lateral view). Real-time movie of the heart of a wild-type embryo at E9.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.

• Movie 4 -

  Movie 4. Real-time recording of the heartbeat of Fhod3^−/− embryos at E9.5 (lateral view). Real-time movie of the heart of a Fhod3^−/− embryo at E9.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.

• Movie 5 -

  Movie 5. Real-time recording of the heartbeat of wild-type embryos at E10.5 (lateral view). Real-time movie of the heart of a wild-type embryo at E10.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.

• Movie 6 -

  Movie 6. Real-time recording of the heartbeat of Fhod3^−/− embryos at E10.5 (lateral view). Real-time movie of the heart of a Fhod3^−/− embryo at E10.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.

• Movie 7 -

  Movie 7. Real-time recording of the heartbeat of Fhod3^−/− embryos at E10.5 (lateral view). Real-time movie of the heart of a Fhod3^−/− embryo at E10.5 was taken with a stereomicroscope (SZX16; Olympus) coupled to a digital camera (DP21; Olympus) at a rate of 15 frames per second.