Materials and Methods s8

Abstract

Cardiomyocytes (CMC) adapt to physical training by increasing cell size and maintaining cell function, whereas during pressure overload CMC size and function do not increase accordingly. We have previously shown that transgenic mice overexpressing the E40K mutant of Akt (E40K-Akt) present with increased CMC size and improved cardiac function in vivo, and increased inotropism and lusitropism at the cellular level.

In this report, we determined the effects of E40K-Akt overexpression on cardiac function after pressure overload induced by transverse aortic constriction (TAC). After TAC, mice overexpressing

E40K-Akt (Tg) had a better cardiac function compared to wild type (WT) littermates. In fact, percent fractional shortening (FS%) and dP/dtmax were significantly higher in Tg vs WT mice at 1 and 8 weeks after TAC (at 8 weeks after TAC: FS%: 41.5 vs 26.6, P<0.05; dP/dTmax: 6851+1972 vs

2442+536 mmHg, P<0.05). Differences in “stress” gene expression, evaluated by quantitative real time PCR, were also evident between Tg and WT animals. Histological analyses showed significantly less fibrosis in Tg vs WT mice; moreover, after TAC, the levels of VEGF protein remained constant in Tg while in WT mice, it was increased at 1 week and decreased at 8 weeks.

More strikingly, apoptosis was considerably less in the myocardium of Tg mice when compared to

WT animals.

Thus, E40K-Akt overexpression maintains cardiac function after TAC. This effect is associated with decreased myocardial apoptosis. These data strongly suggest a protective role of Akt in the myocardium during conditions of stress such as pressure overload.

Keywords: hypertrophy, heart failure, signal transduction, apoptosis, angiogenesis Materials and Methods

Transgenic Mice. The generation of transgenic (Tg) mice with cardiac-specific overexpression of

1 constitutively active Akt (E40K-Akt) has already been described . Wild type (WT) littermate mice were used as controls. The animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.

Transverse Aortic Constriction (TAC). Male mice between 9 and 12 weeks of age were anesthetized with a ketamine (100mg/Kg)-xylazine (2.5mg/Kg) mixture administered i.p., and connected to a rodent ventilator after tracheal intubation1. The chest cavity was opened with scissors by a small incision at the level of the second intercostal space. After isolation of the aortic arch, a 7–0 silk suture was placed around the aorta and a 27-27, 5G needle. The needle was immediately removed to produce an aorta with a stenotic lumen. The chest cavity was then closed with one 6–0 nylon suture and all layers of muscle and skin closed with 6–0 continuous absorbable and nylon sutures, respectively. Sham-treated animals, which underwent surgery without the final tightening of the constrictive suture, were used as controls.

Echocardiographic Assessment of Left Ventricular (LV) Function. Transthoracic echocardiography was performed to evaluate LV function noninvasively in isoflurane-anesthetized closed-chest mice using a SONOS 5500 echocardiographer (Agilent Technology) fitted with an 8-15 MHz linear array transducer. LV end systolic and end diastolic dimensions, and wall thicknesses were evaluated by 2-

D M-mode as previously described2. Echocardiography was performed at 0, 1 and 8 weeks after

Hemodynamic Assessment of Cardiac Function. Mice were anesthetized with ketamine-xylazine as above, then intubated and ventilated. The carotid artery was cannulated to measure arterial pressure.

A high fidelity 1.4F micromanometer catheter (Millar Instruments, Houston TX) was inserted into the LV through the right carotid artery and secured in position. Heart rate, aortic pressure, LV systolic and diastolic pressures, and the maximal and minimal first derivative of LV pressure

(dP/dtmax and dP/dtmin) were measured. Ten sequential beats were averaged for each measurement. After performing hemodynamic measurements (conduced at 0, 1 and 8 weeks), mice were sacrificed by cervical dislocation and hearts explanted.

Histology:

Capillary Density. Capillary density was determined in LV sections with transversely-sectioned

CMCs stained against isolectin B4 (Vector Laboratories, Ltd., Peterborough, U.K.) and counterstained with wheat germ agglutinin (Vector Laboratories, Ltd., Peterborough, U.K.) and

Hoechst 33258 (Sigma, St. Louis, MO), as described previously3. For each mouse, high power fields (400X; from LV base, middle part and apex with transversely sectioned myocytes; n>4 per genotype) were digitally recorded (Quantimet 500MC, Leica, Cambridge,U.K.) to calculate the number of capillaries per 50 to 100 CMCs.

Evaluation of Apoptosis by TUNEL. Histological samples were processed with a TUNEL kit (Roche

Diagnostics, Manheim, Germany) according to manufacturer’s instructions. Nuclei were counterstained with DAPI. Sequential sections from the same sample were imaged under a standard fluorescence optical microscope at a 400X magnification factor in the green emission band. Images were digitally processed using the automatic segmentation feature of the Image-J analysis software to obtain the number of TUNEL-positive nuclei over the total number of nuclei per field. Ten serial sections per heart were analyzed and the values from 3 hearts were averaged per time-point.

Masson’s Trichrome: staining was performed as described1.

Western Blots: Preparation of Cytosolic Extracts: Frozen LVs were homogenized in cold lysis buffer (30mM Tris-HCl, pH 7.5, 150 mM KCl, 300 mM sucrose and 10 mM NaF) and cleared for

20 min at 8000xg at 4C. The supernatant was collected and ultracentrifuged for 30min at 100,000xg at 4C and the resulting supernatant, considered the cytosolic fraction, loaded onto 10% SDS-PAGE for detection of phospho-AKT (S473), total AKT and VEGF (Cell Signaling Technology, Waltham,

MA, USA). Secondary antibody horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse and ECL were obtained from Amersham Biosciences (Little Chalfont, UK). RNA Extraction and Real-Time PCR. Total RNA was extracted from hearts of mice (n = 5 per group) with Trizol (Invitrogen). The RT-PCR reaction was performed using Superscript III Reverse

Transcriptase (Invitrogen) and Random Primers (Invitrogen) with 1µg total RNA in an ABI PRISM

7000 instrument (Applied Biosystems) using TaqMan oligonucleotides (Assay on Demand, Applied

Biosystems) for β-isoform of myosin heavy chain, atrial natriuretic factor or skeletal actin. Delta-Ct values were normalized with the values obtained for amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Statistical Analysis: Data are reported as mean ± S.D. Statistical analysis was carried out using paired Student’s t test and assumed to be significant at P< 0.05.

Figure 1s. Fibrosis induced by TAC in E40K-AKT and WT mice: Representative photographs of histological sections from WT and Tg mice hearts.

Long-term exposure to TAC induces necrosis and subendocardial fibrosis. To determine the effects of E40K-Akt overexpression on stromal tissue deposition, sections of myocardium from WT and

Tg animals were stained with Masson’s trichrome (Figure 1a to f). Sham-operated mice had normal tissue, with no collagen deposition (Figure 1a and d). Only minimal fibrosis around arterioles was observed in WT mice 1 week after TAC (arrowheads), while no evident changes were observed in other areas (Figure 1b) or in samples from Tg mice (Figure 1e). As expected, scar tissue was prominent in myocardium from WT mice 8 weeks after TAC (Figure 1c): typical to the pressure overload model, lesions were scattered in multiple foci rather that being uniformly distributed. In contrast, the myocardial tissue of Tg mice never presented with focal lesions (Figure 1f).

Masson’s trichrome stains collagen in green; scale bar is 100m . Upper row: a) WT sham; b) WT after 1 week of TAC and c) WT after 8 weeks of TAC. Bottom row: d) E40K-Akt Tg sham; e)

E40K-Akt Tg after 1 week TAC and f) E40K-Akt Tg after 8 weeks TAC. SHAM 1 Week 8 Weeks

a) b) WT c)

d) e) f) E40K-Akt

WT (n=6) WT (n=14) WT (n=14) Tg (n=5) Tg (n=10) Tg (n=6) Sham 1wk 8wks Sham 1wk 8wks BW(g) 23.1+2.3 20.8+2.9 25.5+1.5 25.6+0.9 23.6+4.4 25.2+2.7 H/BW 0.44+0.06 0.62+0.05* 0.81+0.23 0.74+0.12† 1.18+0.32**‡ 1.11+0.24° HR, bpm 422+29 432+19 430+11 446+10 423+8 435+27 IVSd, mm 0.56+0.08 0.60+0.06* 0.70+0.09 0.73+0.05† 0.91+0.32* 0.88+0.18°° LVEDD, mm 2.48+0.18 2.60+0.19 4.02+0.44 3.10+0.53†† 2.49+0.02**‡‡ 3.04+0.14° LVPWd, mm 0.66+0.05 0.82+0.08** 0.73+0.12 0.81+0.06† 0.91+0.04* 0.84+0.02°° IVSs, mm 0.75+0.07 1.10+0.07* 1.04+0.12 1.24+0.11† 1.14+0.04 1.05+0.18 LVESD, mm 1.38+0.10 1.22+0.14 2.93+0.43 1.52+0.16 1.23+0.09** 1.78+0.07° LVPWs, mm 0.70+0.08 1.00+0.09* 0.92+0.12 1.11+0.47† 1.14+0.09** 1.12+0.13°° FS (%) 44.1+1.0 52.3+4.2* 26.6+4.7 50.1+3.4† 49.6+2.1 39.4+5.2° VCF, circ/s 6.2+0.9 7.7+1.1** 3.7+0.8 7.5+0.9 9.1+1.1** 8.1+2.3° Table 1. Echocardiographic and hemodynamic assessment in WT and Tg mice in basal conditions and after TAC. Values are expressed as mean ± SD. BW indicates body weight; HW indicates heart weight; LVEDD, left ventricle end-diastolic diameter; LVESD, left ventricle end-systolic diameter; IVSd, interventricular septum thickness in diastole; LVPWd/s, left ventricle posterior wall thickness in diastole/systole; FS, fractional shortening; VCF, velocity of circumferential fiber shortening calculated as FS divided by ejection time multiplied by square root of RR interval. */**P<0.001/P<0.01 for TAC vs respective sham WT or sham E40K-Akt Tg. †/††P<0.001/P<0.01 for sham WT mice vs E40K-Akt Tg mice. ‡/‡‡ P<0.001/P<0.05 for after 1 week TAC E40K-Akt Tg mice vs after 1 weeks TAC WT mice. °/°°P<0.001/ P<0.05 for after 8 Wks TAC E40K-Akt Tg mice vs after 8 Weeks TAC WT mice.

Wt (n=6) Wt (n=9) Wt (n=11) Tg (n=13) Tg (n=6) Tg (n=6) Sham 1wk 8wks Sham 1wk 8wks BW (g) 22.9+1.4 20.6+2.5 24.4+1.2 26.1 + 3.6† 24.9+4.0 25.2+2.6 LV (mg) 76+5 105+18* 138+40 140+30† 170+12‡ 196+55°° RV (mg) 14+3 16+3 20+6 33+7† 39+6** 38+7° LA (mg) 2+0 12+2 3+1 16+7†† 19+3** 21+5° RA (mg) 3+0 17+19** 7+8 28+6†† 35+9** 28+13° H (mg) 96+8 151+44* 170+54 220+39† 264+27** 284+78°° LV/BW 0.33+0.03 0.51+0.06* 0.56+0.16 0.54+0.08† 0.70+0.13** 0.77+0.17°° HW/BW 0.42+0.04 0.73+0.21 0.69+0.21 0.83+0.09† 1.09+0.18* 1.11+0.24°

Table 2. Post-mortem heart weight data of WT and Tg mice. Values are expressed as mean ± SD. BW indicates body weight; LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; H, heart weight. */**P<0.001/P<0.01 for TAC vs respective sham WT or sham E40K-Akt Tg mice. †/††P<0.001/P<0.01 for sham E40K-Akt Tg mice vs WT mice. °/°°P<0.001/ P<0.05 for after 8 Weeks TAC E40K-Akt Tg mice vs after 8 Weeks TAC WT mice.

Bibliography

1. Condorelli G, et. al,. (1999) Circulation.;23:3071-8.

2. Tanaka N, et. al,. (1996). Circulation.;94:1109-17.

3. Hilfiker-Kleiner D, et. al,. (2005) Circulation.;112:1470-7.