Smooth muscle protein-22-mediated deletion of Tsc1 results in cardiac hypertrophy that is mTORC1-mediated and reversed by rapamycin

Abstract

Constitutive activation of mammalian target of rapamycin complex 1 (mTORC1), a key kinase complex that regulates cell size and growth, is observed with inactivating mutations of either of the tuberous sclerosis complex (TSC) genes, Tsc1 and Tsc2. Tsc1 and Tsc2 are highly expressed in cardiovascular tissue but their functional role there is unknown. We generated a tissue-specific knock-out of Tsc1, using a conditional allele of Tsc1 and a cre recombinase allele regulated by the smooth muscle protein-22 (SM22) promoter (Tsc1c/cSM22cre+/-) to constitutively activate mTOR in cardiovascular tissue. Significant gene recombination (∼80%) occurred in the heart by embryonic day (E) 15, and reduction in Tsc1 expression with increased levels of phosphorylated S6 kinase (S6K) and S6 was observed, consistent with constitutive activation of mTORC1. Cardiac hypertrophy was evident by E15 with post-natal progression to heart weights of 142 ± 24 mg in Tsc1c/cSM22cre+/- mice versus 65 ± 14 mg in controls (P < 0.01). Median survival of Tsc1c/cSM22cre+/- mice was 24 days, with none surviving beyond 6 weeks. Pathologic and echocardiographic analysis revealed severe biventricular hypertrophy without evidence of fibrosis or myocyte disarray, and significant reduction in the left ventricular end-diastolic diameter (P < 0.001) and fractional index (P < 0.001). Inhibition of mTORC1 by rapamycin resulted in prolonged survival of Tsc1c/cSM22cre+/- mice, with regression of ventricular hypertrophy. These data support a critical role for the Tsc1/Tsc2-mTORC1-S6K axis in the normal development of cardiovascular tissue and also suggest possible therapeutic potential of rapamycin in cardiac disorders where pathologic mTORC1 activation occurs.

Figure 1.

Timing and extent of recombination in the Tsc1 gene in Tsc1c/cSM22cre^+/− mice. (Left panel) MLPA of DNA extracted from the hearts of Tsc1c/cSM22cre^+/− embryos (cc+) (n = 14) and Tsc1c/wSM22cre^+/− controls (c/w+) (n = 8) showing the percentage recombination of the conditional allele (c) is 75–80% at E15 through the DOB. Note the percent recombination reflects the amount of conditional allele that has been successfully recombined to the knockout allele and does not measure the wild-type allele (w), and is therefore equal in c/w+ and c/c+ embryos. Heart weight, embryo weight and percentage of heart weight relative to embryo weights are also shown. (Right panel) Immunoblot and densitometric analysis of lysates derived from the RV, LV and aorta (Ao) in 3-week-old mice. Tsc1c/cSM22cre^+/− mice (cc+) (n = 6) and littermate controls (ctrl; Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (n = 6) are compared. The proteins assessed are Tsc1 (hamartin), Tsc2 (tuberin), pS6K (phospho-S6 kinase), Thr 421/424, pS6 (phospho-S6), Ser 240/244, pERK (phospho-p42/44 ERK), Thr202/Tyr204, pAkt (phospho-Akt), Ser 473, pmTOR (phospho-mTOR), Ser 2448 and actin (loading control).

Figure 2.

Distribution of recombination due to the SM22cre allele. β-Galactosidase staining of cryosections of the aorta, heart, brain, kidney and lung from 2-week-old (n = 8) SMA22cre/RosaR26 β-gal mice. β-Galactosidase expression (blue) is extensive throughout the heart and vascular SMCs, and rare in other cell types.

Figure 3.

Reduced body weight and survival in Tsc1c/cSM22cre^+/− mice. (A) Representative images of body and heart size in Tsc1c/cSM22cre^+/− (cc+) and Tsc1c/wSM22cre^+/− control mice (Ctl). Body weight and heart weight from birth (0 week) through 5 weeks and body weight to tibial length ratio at 4 weeks are shown. (B) Kaplan–Meier curves showing survival of Tsc1c/cSM22cre^+/− mice (cc+) (n = 30) compared with littermate controls of mixed genotype (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (n = 30) (left panel) and segregated by gender (right panel). In younger mice, the gender was not clearly identified (N/A).

Figure 4.

Cardiac enlargement and reduced cardiac function in Tsc1c/cSM22cre^+/− mice. (A) Upper panel: Gross anatomy and H&E-stained axial sections from Tsc1c/cSM22cre^+/− (cc+) mice hearts and littermate controls (Control) (n = 8, each group). LV and RV are labeled; note both asymmetric and symmetric hypertrophic cardiac phenotypes and bulging of the interventricular septum into the RV cavity. In the asymmetric phenotype, the hypertrophic anterolateral wall (arrow) and thinner posterior wall (arrowhead) of the LV are indicated. Higher magnification (×4, ×20) views of the LV show loss of striations but no obvious fibrosis or infiltrate in Tsc1c/cSM22cre^+/− (cc+) mice. Lower panel: RV and LV+S weights in Tsc1c/cSM22cre^+/− (cc+) mice and their littermate controls (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctrl) over time (0–5 weeks) (n = 6 per group, each time point) are compared. Values are expressed as actual weight, with body weight as a denominator (RV/BW, LV + S/BW) and as a ratio relative to one another (RV/LV + S ratio). (B) Echocardiographic images (axial and M-mode) are shown with measurements of the anterior (A) and posterior (P) wall thickness as well as LVEDD, LVESD and fractional index. Tsc1c/cSM22cre^+/− mice (cc+) (n = 6) and their littermate controls of mixed genotype (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctrl) (n = 6) are compared.

Figure 5.

Cell growth and apoptosis, cell size and cell number in Tsc1c/cSM22cre^+/− mice. (A) Paraffin-embedded sections of the LV were stained with trichrome (collagen), PAS (glycogen), Ki67 (proliferation) and TUNEL (apoptosis). Positive (pos) control samples were used to confirm marker expression. Tsc1c/cSM22cre^+/− (cc+) mice (n = 8) and their littermate controls (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctrl) (n = 8) are compared. (B) Surface area of LV myocytes measured on ImageproPlus software is shown; littermate control (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctl) and Tsc1c/cSM22cre^+/− mice (cc+) were compared (n = 8, each group). Number of ventricular myocytes counted from hearts of Tsc1c/cSM22cre^+/− mice (cc+) and littermate controls (Tsc1c/wSM22cre^+/−) (ctl) is shown (n = 4, each group). Results are expressed as total number of myocytes counted in each sample and number of myocytes normalized to the original sample weight; statistical significance is indicated.

Figure 6.

Systemic and RV pressures in Tsc1c/cSM22cre^+/− mice. (A) Representative images in 4-week-old mice of H&E-stained aortas, tail cuff tracings (left panel), tail cuff systolic pressure (mmHg) (n = 24, each group) and contractile force generated from aortic rings (g) (n = 6, each group) (middle panel) and systolic pressure measured by carotid catheterization (mmHg) (n = 6, each group) from Tsc1c/cSM22cre^+/− mice (cc+) and their littermate controls (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctrl) are shown. Note that, rarely, vessels showed irregular smooth muscle layers (insert; ×100). (B) Representative images of Geisen-stained (elastin) lung tissue (n = 8, each group), RVSP (mmHg) (n = 16, each group) and wet:dry lung weight ratios (n = 16, each group) from Tsc1c/cSM22cre^+/− mice (cc+) and their littermate controls (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctrl) are shown. Distribution of the muscularization of the distal pulmonary arteries are compared between the indicated groups; results are expressed as a percentage of the total number of vessels counted.

Figure 7.

Renal pathology and function in Tsc1c/cSM22cre^+/− mice. Gross and histopathologic H&E-stained sections of kidneys showing cystadenomatous tumors in Tsc1c/cSM22cre^+/− (cc+) mice (n = 16) compared with littermate control (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (ctrl) mice (n= 16). BUN and creatinine levels measured in the serum of both groups (n = 8, each group) are also shown.

Figure 8.

Reversal of the phenotype with rapamycin. (A) Kaplan–Meier curves showing survival of Tsc1c/cSM22cre^+/− mice (cc+ without Rapamycin) and their littermate controls (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (Controls) given 4 mg/kg of subcutaneous rapamycin using three different treatment schedules (cc+ Rapamycin 5 times weekly starting at 2 weeks (n = 15); cc+ Rapamycin 3 times weekly starting at 2 weeks (n= 15); cc+ Rapamycin 3 times weekly starting at 3 weeks (n = 8) [late rescue]). The following measurements are compared in rapamycin (Rapa) and vehicle-treated Tsc1c/cSM22cre^+/− mice and littermate controls (ctrl) at ∼4 weeks: body weight (BW), RV/BW, LV+S/BW, RV/LV+S ratio, systolic pressure (mmHg) and RVSP (n = 15, each group). Statistical significance is indicated (*). (B) Images of gross hearts and heart weight: body weight ratio (HW:BW) in: 2-week- and 4-week-old untreated Tsc1c/cSM22cre^+/− mice (cc+) and Tsc1c/wSM22cre^+/− control mice (control); ∼6-week-old rapamycin-treated mice depicting the regressive effect of rapamycin with 4 weeks of therapy; 14-week-old mice following rapamycin withdrawal (at 6 weeks of age) depicting re-emergence of cardiac enlargement off therapy in Tsc1c/cSM22cre^+/− (cc+) mice. The lower graphs show heart weights in rapamycin-treated (Rap) and vehicle-treated (Veh) Tsc1c/cSM22cre^+/− (cc+) and Tsc1c/wSM22cre^+/− (ctrl) mice (0–6 weeks) where treatment was started in one cohort at 2 weeks and in another cohort at 3 weeks; and heart weight following rapamycin withdrawal. (C) Echocardiographic images (axial and M-mode) and measurements of the anterior and posterior wall thickness as well as LVEDD, LVESD and fractional index in rapamycin-treated (Rapa) Tsc1c/cSM22cre^+/− mice (cc+) and controls (ctrl) (n = 6, each group) showing no significant differences between the groups. (D) Western immunoblot of lysates derived from the LV in vehicle- and rapamycin-treated Tsc1c/wSM22cre^+/− control mice (c/w+) and Tsc1c/cSM22cre^+/− mice (cc+) (n = 6, each group). The proteins assessed are pS6 (phospho-S6), pAkt (phospho-Akt) and actin (loading control).

Figure 8.

Reversal of the phenotype with rapamycin. (A) Kaplan–Meier curves showing survival of Tsc1c/cSM22cre^+/− mice (cc+ without Rapamycin) and their littermate controls (Tsc1w/wSM22cre^+/+, Tsc1c/wSM22cre^+/−, Tsc1c/cSM22cre^−/−) (Controls) given 4 mg/kg of subcutaneous rapamycin using three different treatment schedules (cc+ Rapamycin 5 times weekly starting at 2 weeks (n = 15); cc+ Rapamycin 3 times weekly starting at 2 weeks (n= 15); cc+ Rapamycin 3 times weekly starting at 3 weeks (n = 8) [late rescue]). The following measurements are compared in rapamycin (Rapa) and vehicle-treated Tsc1c/cSM22cre^+/− mice and littermate controls (ctrl) at ∼4 weeks: body weight (BW), RV/BW, LV+S/BW, RV/LV+S ratio, systolic pressure (mmHg) and RVSP (n = 15, each group). Statistical significance is indicated (*). (B) Images of gross hearts and heart weight: body weight ratio (HW:BW) in: 2-week- and 4-week-old untreated Tsc1c/cSM22cre^+/− mice (cc+) and Tsc1c/wSM22cre^+/− control mice (control); ∼6-week-old rapamycin-treated mice depicting the regressive effect of rapamycin with 4 weeks of therapy; 14-week-old mice following rapamycin withdrawal (at 6 weeks of age) depicting re-emergence of cardiac enlargement off therapy in Tsc1c/cSM22cre^+/− (cc+) mice. The lower graphs show heart weights in rapamycin-treated (Rap) and vehicle-treated (Veh) Tsc1c/cSM22cre^+/− (cc+) and Tsc1c/wSM22cre^+/− (ctrl) mice (0–6 weeks) where treatment was started in one cohort at 2 weeks and in another cohort at 3 weeks; and heart weight following rapamycin withdrawal. (C) Echocardiographic images (axial and M-mode) and measurements of the anterior and posterior wall thickness as well as LVEDD, LVESD and fractional index in rapamycin-treated (Rapa) Tsc1c/cSM22cre^+/− mice (cc+) and controls (ctrl) (n = 6, each group) showing no significant differences between the groups. (D) Western immunoblot of lysates derived from the LV in vehicle- and rapamycin-treated Tsc1c/wSM22cre^+/− control mice (c/w+) and Tsc1c/cSM22cre^+/− mice (cc+) (n = 6, each group). The proteins assessed are pS6 (phospho-S6), pAkt (phospho-Akt) and actin (loading control).