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ScienceDirect Journal of Genetics and Genomics 41 (2014) 73e77
JGG LETTER TO THE EDITOR
Acquired Cardiomyopathy Caused by Cardiac Tsc1 Deficiency Cardiomyopathies are myocardial disorders with enlarged, thick, or rigid cardiac muscles. Because no effective treatment is available to prevent the deterioration of the myocardium function, the prognosis of cardiomyopathy is usually poor. Cardiomyopathies can be inherited or acquired. A number of genes associated with inherited cardiomyopathy, such as the genes encoding for sarcomeric, cytoskeletal, nuclear membrane, lysosomal, desmosomal and mitochondrial proteins, have been identified (Wilde and Behr, 2013). However, the underlying mechanisms of acquired cardiomyopathy remain poorly understood. Therefore, understanding the pathogenesis of acquired cardiomyopathy is crucial to develop new therapies for its prevention and treatment. Tuberous sclerosis (TSC) is an autosomal dominant disorder affecting about 1 in 6000 individuals worldwide (Northrup and Krueger, 2013). The disease is characterized by the development of benign hamartomas in multiple organs. The cardiac manifestation of TSC is rhabdomyomas in fetuses and infants, and fat-containing lesions in adolescents and adults (Smythe et al., 1990; Shaaya et al., 2013). Inactivating mutations in either Tsc1 or Tsc2 gene have been detected in majority of patients suffering from TSC (Cheadle et al., 2000). Tsc1 null embryos die at mid-gestation (Kobayashi et al., 2001; Kwiatkowski et al., 2002). Mice devoid of Tsc1 in ventricular myocytes develop congenital dilated cardiomyopathy (Meikle et al., 2005). Tsc1 and Tsc2 are known to function as a complex which acts through the Rheb GTPase to inhibit the mechanistic target of rapamycin (mTOR). Loss of either Tsc1 or Tsc2 leads to constitutive activation of mTOR and hyperphosphorylation of its downstream targets S6 kinases and 4E-BP1 (Thoreen et al., 2012). Dysregulated mTOR signaling has been implicated in several major diseases, such as cardiovascular diseases, cancer, metabolic diseases, neurological diseases and inflammatory diseases (Dazert and Hall, 2011). Whether activation or inhibition of mTOR signaling happens during cardiomyopathy in human subjects remains to be determined. Nevertheless, mTOR is critical for the survival and proliferation of cardiomyocytes during mouse cardiac development (Zhu et al., 2013). Loss of mTOR in cardiomyocytes leads to a fatal, dilated cardiomyopathy due to apoptosis, autophagy and altered mitochondrial
structure (Zhang et al., 2010). Furthermore, hyperactivation of mTOR plays an important role in load-induced cardiac hypertrophy in mice (Mcmullen et al., 2004). To model acquired cardiomyopathy and further study the cardiac function of mTOR in the adult animal, we inducibly knocked out Tsc1 in the cardiomyocytes of 2-month-old mice. A gene-targeting strategy was used to generate aMHCMerCreMer/Tsc1flox/flox mice (aMHC-MCM/Tsc1flox/flox mice) (Fig. S1A). Two-month-old male aMHC-MCM/Tsc1flox/flox mice were consecutively administered tamoxifen for 6 days to induce cardiomyocyte-specific Tsc1 knockout (Tsc1-cKO mice) (Fig. S1B). As the control group, the age-matched male Tsc1flox/ flox mice also received tamoxifen-injection. After tamoxifen administration, the expression of Tsc1 protein in Tsc1-cKO mice decreased significantly. The level of phospho-S6, a hallmark of mTOR activity, increased markedly in the hearts of Tsc1-cKO mice (Fig. S1C), indicating that mTOR activity was upregulated (Materials and Methods in Supplementary Data). Tsc1-cKO mice began to die (one mouse) at the fifth day of tamoxifen administration, and 40% of Tsc1-cKO mice (eight mice) died at the last day (the sixth day) of tamoxifen administration. By the fourteenth day of the initiation of cardiac Tsc1 knockout, one more mouse died. Nevertheless, none of the remaining 10 mice died after that. Collectively, the mortality of Tsc1-cKO mice was as high as 50% within two weeks of induction of Tsc1 knockout. In contrast, none of the Tsc1flox/flox mice (control mice, n ¼ 17) died at the endpoint of our study (52 days) (Fig. 1A). At two weeks after the initiation of tamoxifen administration, the heart/body weight ratio (HW/BW) of the survived Tsc1-cKO mice was significantly higher than that of control mice (P < 0.05, Fig. 1B). In addition, the heart of the Tsc1cKO mice was perceptibly enlarged in comparison with that of the Tsc1flox/flox mice (Fig. 1C). Internal diameters of atrial and ventricular chambers were larger, and the left ventricular wall and interventricular septum were thinner than those of Tsc1flox/ flox mice (Fig. 1D). Echocardiography demonstrated left ventricular internal diameter (LVID) of Tsc1-cKO mice was larger than that of control mice: 4.30 0.21 mm vs. 3.80 0.19 mm (P < 0.05) in the end of diastole (LVIDd), and 3.53 0.24 mm vs.
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74
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77
Fig. 1. Cardiomyocyte-specific Tsc1 knockout induces a lethal cardiac dilation and impairs cardiac function. A: Tsc1-cKO led to a high mortality at the early stage. Mortality of Tsc1-cKO mice began five days after tamoxifen administration, and reached 50% by two weeks. Time was shown as days after tamoxifen administration. n ¼ 20 for Tsc1-cKO mice and n ¼ 17 for control mice (Ctrl) B: Heart/body weight ratio (HW/BW) from Tsc1-cKO mice and control mice. HW/BW ratio of the Tsc1-cKO mice was higher than that of control two weeks after tamoxifen administration. n ¼ 3 for each group. *P < 0.05 vs. the control group. C: Representative explanted hearts. Heart of the Tsc1-cKO mice was larger than that of control at one week after tamoxifen administration. D: Representative H&E-stained heart sections. Tsc1-cKO hearts had thinner ventricular walls and enlarged cardiac chambers at one week after tamoxifen administration. E: Movement of ventricular wall assessed using echocardiography. Tsc1-cKO mouse was associated with obviously weaker ventricular wall movement when compared with control at one week after tamoxifen administration. F: Re-expression of cardiac fetal genes, assayed by qRT-PCR and normalized for b-actin expression, was given as fold change. Tsc1-cKO hearts had a 2- to 16-fold increase in the re-expression of these genes at two weeks after tamoxifen administration. Data are presented from one experiment performed in three replicates and the experiment was repeated three times with similar results. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group. ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; b-MyHC, b-myosin heavy chain; SK.actin, a-skeletal actin.
2.29 0.34 mm (P < 0.01) in the end of systole (LVIDs) at one week since tamoxifen administration (Table 1). Left ventricular posterior wall in the end of systole (LVPWs) of Tsc1-cKO mice was thinner than that of Tsc1flox/flox mice, 1.01 0.12 mm vs. 1.25 0.09 mm (P < 0.05). In consistent with histological examination (Fig. 1D), left ventricular anterior wall in the end of systole (LVAWs) was also thinner
than that of control mice, even though statistical significance was not reached. In addition, left ventricular mass corrected for anterior wall (LV Mass (AW) Corrected) was heavier as well, 107.5 6.9 mg vs. 80.6 14.2 mg (P < 0.01). The enlarged ventricular chamber and the thinner left ventricular wall in addition to heavier heart weight observed in the first week remained at the second week since tamoxifen
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77
Table 1 Inducible Tsc1 knockout in cardiomyocytes caused cardiomyopathy in mice as assessed by echocardiography Parameter
Week after tamoxifen administration
Control
LVIDd (mm)
0
3.70 0.10
3.56 0.27
1
3.80 0.19
4.30 0.21*
LVIDs (mm)
LVPWd (mm)
LVPWs (mm)
LVAWd (mm)
LVAWs (mm)
EF (%)
FS (%)
LV Mass (AW) Corrected (mg)
Tsc1-cKO
2
3.40 0.29
3.68 0.21*
4
3.67 0.21
3.59 0.17
0
2.40 0.11
2.12 0.34
1
2.29 0.34
3.53 0.24**
2
1.90 0.41
2.46 0.24
4
2.19 0.42
2.29 0.25
0
0.71 0.04
0.81 0.10
1
0.76 0.04
0.81 0.13
2
0.89 0.11
0.81 0.04 1.01 0.11*
4
0.74 0.08
0
1.06 0.05
1.27 0.10
1
1.25 0.09
1.01 0.12*
2
1.37 0.17
1.12 0.06*
4
1.21 0.19
1.42 0.08
0
0.76 0.15
0.76 0.13
1
0.76 0.16
0.81 0.12
2
0.91 0.12
0.94 0.05
4
0.88 0.08
1.02 0.10*
0
1.28 0.10
1.33 0.16
1
1.36 0.26
1.08 0.11
2
1.38 0.18
1.43 0.07 1.59 0.11
4
1.50 0.09
0
65.0 4.2
71.8 6.5
1
70.6 8.6
37.8 4.3***
2
76.4 8.1
62.7 4.4*
4
71.8 10.5
66.6 6.0
0
35.0 3.1
40.6 5.4
1
39.9 7.2
18.1 2.3**
2
44.7 7.1
33.4 3.2*
4
41.0 8.4
36.2 4.4
0
73.8 9.5
75.3 7.0
1
80.6 14.2
107.5 6.9**
2
85.5 11.3
92.8 9.2**
4
83.8 12.5
110.6 15.3*
LVIDd/s, left ventricular internal diameter in the end of diastole/systole; LVPWd/s, left ventricular posterior wall thickness in the end of diastole/systole; LVAWd/s, left ventricular anterior wall thickness in the end of diastole/ systole; EF, ejection fraction; FS, fractional shortening; LV Mass (AW) Corrected, left ventricular mass (anterior wall) corrected. Five mice were assessed in each group. Measurements were given as mean SD. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group, ***P < 0.001 vs. the control group (t-test).
75
administration: LVIDd, 3.68 0.21 mm vs. 3.40 0.29 mm (P < 0.05); LVPWs, 1.12 0.06 mm vs. 1.37 0.17 mm (P < 0.05); and LV Mass (AW) Corrected, 92.8 9.2 mg vs. 85.5 11.3 mg (P < 0.01) (Table 1). Taken together, mice deficient of Tsc1 in adulthood developed dilated cardiomyopathy probably due to activation of mTOR. By the fourth week since tamoxifen administration, the size of heart and the thickness of left ventricular wall in Tsc1-cKO mice were progressively larger than those of Tsc1flox/flox mice: LV Mass (AW) Corrected, 110.6 15.3 mg vs. 83.8 12.5 mg (P < 0.05); LVPWd, 1.01 0.11 mm vs. 0.74 0.08 mm (P < 0.05); and LVAWd, 1.02 0.10 mm vs. 0.88 0.08 mm (P < 0.05) (Table 1). Therefore, the survived Tsc1-cKO mice experienced cardiac dilation to cardiac hypertrophy after Tsc1 knockout in the heart. Echocardiography revealed impaired cardiac function in Tsc1-cKO mice when compared to Tsc1flox/flox mice. At one week since tamoxifen administration, Tsc1-cKO mice had dramatically decreased ejection fraction (EF) and fractional shortening (FS), which represent cardiac systolic and diastolic function respectively: EF, 37.8 4.3% (Tsc1-cKO mice) vs. 70.6 8.6% (Tsc1flox/flox mice) (P < 0.001); FS, 18.1 2.3% vs. 39.9 7.2% (P < 0.01) (Table 1), and the movement of ventricular wall was obviously weaken (Fig. 1E). Nevertheless, the cardiac function of Tsc1-cKO mice restored over time. At two weeks since tamoxifen administration, EF was 62.7 4.4% (Tsc1-cKO mice) vs. 76.4 8.1% (Tsc1flox/flox mice) (P < 0.05); FS was 33.4 3.2% vs. 44.7 7.1% (P < 0.05). And at four weeks, there was no difference in the cardiac systolic and diastolic function between Tsc1-cKO mice and Tsc1flox/flox mice (Table 1). Fetal genes are normally expressed in embryonic and neonatal heart, and re-expressed in response to pathological stress (Crone et al., 2002). Tsc1-cKO myocardium had higher levels of mRNA for fetal genes, including atrial natriuretic factor (ANF ), brain natriuretic peptide (BNP), b-myosin heavy chain (b-MyHC ), and a1-skeletal actin (SK.actin) (Fig. 1F), indicative of cardiac dysfunction. The previous study has demonstrated that loss of mTOR in myocardium of adult mice causes cardiomyopathy. The present study indicates that cardiomyocyte-specific Tsc1 knockout in adult mice triggers the development of acquired cardiomyopathy with hyperactive mTOR signaling. Although cardiomyopathy often presents as a congenital disease, it may occur due to various non-inherited pathogeny, including ischemic heart disease (e.g., acute myocardial infarction), structural heart disease (e.g., valvular heart disease), myocarditis (e.g., giant cell myocarditis), and many other factors (Hershberger et al., 2010). Therefore, the inducible Tsc1-cKO in normal adult mice generated in our study recapitulates clinical acquired cardiomyopathy to some extent. Tsc1 null mouse embryos die between E9.5 and E13.5 possibly due to prominent defects in liver development and significant cardiac enlargement (Kwiatkowski et al., 2002). Endothelial Tsc1 knockout illustrates physiological Tsc1mTOR signaling in endothelial cells is crucial for cardiovascular development and embryogenesis (Ma et al., 2013). And mice with specific ventricular loss of Tsc1 develop dilated
76
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77
cardiomyopathy with a median survival of 6 months (Meikle et al., 2005). In our study, inducible Tsc1 knockout of cardiomyocytes in entire hearts led to cardiac dilation and impaired cardiac function with high mortality in adult mice. However, the remaining mice after two weeks of Tsc1 deficiency gradually restored their cardiac function in parallel with a switch of myocardium from dilation to hypertrophy. We speculate that these survived mice might have fewer Tsc1 knockout cardiomyocytes than their deceased counterparts. Cardiac hypertrophy is a compensated response to physiological or pathological stress, such as exercise and persistent pressure overload. In its early stage, hypertrophic cardiomyopathy is characterized by left ventricular hypertrophy, normal to smaller left ventricular cavity and may be hypercontractile (Hershberger et al., 2009). However, hypertrophic cardiomyopathy may occasionally show cardiac dilation and decreased systolic function, characteristics of dilated cardiomyopathy (Maron et al., 2003). Therefore, whether hypertrophy is beneficial for cardiac function remains controversial (Frey et al., 2004). Here, Tsc1-cKO mice developed larger atrial and ventricular chambers, thinner left ventricular wall and interventricular septum than control mice at one week after tamoxifen administration. However, the ventricular wall became thicker than that of control mice over time in association with improved survival. Our study suggests cardiac hypertrophy may be beneficial. mTOR integrates both intracellular and extracellular signals through multiple pathways to regulate cell growth, proliferation, metabolism, function and survival (Dazert and Hall, 2011). It is essential for cardiac development and growth during embryogenesis (Zhu et al., 2013). Malfunction of mTOR pathway leads to hypertrophic or dilated cardiomyopathy (Meikle et al., 2005; Zhang et al., 2010). mTOR inhibitor rapamycin can rapidly normalize cardiomyocyte size, attenuate or reverse cardiac hypertrophy. It can improve cardiac function in a setting of left ventricular hypertrophy and cardiac dysfunction as well (Mcmullen et al., 2004; Marin et al., 2011). And it can also reverse vascular smooth muscle Tsc1 knockout-induced ventricular hypertrophy (Malhowski et al., 2011). After cardiomyocyte-specific knockout of Tsc1, the level of phospho-S6 increased in myocardium (Fig. S1C), suggestive of mTOR activation in this acquired cardiomyopathy mouse model. Therefore, the benefit of rapamycin ought to be investigated in the treatment of cardiomyocyte-specific Tsc1 knockout-induced cardiomyopathy in adult mice. Our study has implicated cardiomyocyte Tsc1 deficiencymediated mTOR activation in the induction of acquired cardiomyopathy. This acquired cardiomyopathy mouse model should be very valuable in the study of pathogenesis of cardiomyopathy. And targeting mTOR should be a promising strategy in the treatment of acquired cardiomyopathy. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 81270288) and the Chinese Ministry of Science and Technology (No. 2011CB965002).
SUPPLEMENTARY DATA Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2013.11.005. Yi Chena,1, Fang Wangb,1, Chunjia Lib, Lianmei Wangb, Hongbing Zhangb, Hongbing Yanc,* a
Department of Cardiology, Beijing An Zhen Hospital, Capital Medical University, Beijing 100029, China
b
Department of Physiology, Institute of Basic Medical Sciences and School of Basic Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100005, China c Center for Coronary Heart Disease, Cardiovascular Institute & Fu Wai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100037, China
*Corresponding author. Tel: þ86 10 8839 7408, fax: þ86 10 6831 1790. E-mail address: [email protected] (H. Yan) 1 These authors contribute equally to this work. Received 10 October 2013 Revised 4 November 2013 Accepted 14 November 2013 Available online 3 December 2013
REFERENCES Cheadle, J.P., Reeve, M.P., Sampson, J.R., Kwiatkowski, D.J., 2000. Molecular genetic advances in tuberous sclerosis. Hum. Genet. 107, 97e114. Crone, S.A., Zhao, Y.Y., Fan, L., Gu, Y., Minamisawa, S., Liu, Y., Peterson, K.L., Chen, J., Kahn, R., Condorelli, G., Ross Jr., J., Chien, K.R., Lee, K.F., 2002. ErbB2 is essential in the prevention of dilated cardiomyopathy. Nat. Med. 8, 459e465. Dazert, E., Hall, M.N., 2011. mTOR signaling in disease. Curr. Opin. Cell Biol. 23, 744e755. Frey, N., Katus, H.A., Olson, E.N., Hill, J.A., 2004. Hypertrophy of the heart: a new therapeutic target? Circulation 109, 1580e1589. Hershberger, R.E., Cowan, J., Morales, A., Siegfried, J.D., 2009. Progress with genetic cardiomyopathies: screening, counseling, and testing in dilated, hypertrophic, and arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ. Heart Fail. 2, 253e261. Hershberger, R.E., Morales, A., Siegfried, J.D., 2010. Clinical and genetic issues in dilated cardiomyopathy: a review for genetics professionals. Genet. Med. 12, 655e667. Kobayashi, T., Minowa, O., Sugitani, Y., Takai, S., Mitani, H., Kobayashi, E., Noda, T., Hino, O., 2001. A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice. Proc. Natl. Acad. Sci. USA 98, 8762e8767. Kwiatkowski, D.J., Zhang, H., Bandura, J.L., Heiberger, K.M., Glogauer, M., El-Hashemite, N., Onda, H., 2002. A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and upregulation of p70S6 kinase activity in Tsc1 null cells. Hum. Mol. Genet. 11, 525e534. Malhowski, A.J., Hira, H., Bashiruddin, S., Warburton, R., Goto, J., Robert, B., Kwiatkowski, D.J., Finlay, G.A., 2011. Smooth muscle protein-22-mediated deletion of Tsc1 results in cardiac hypertrophy that is mTORC1-mediated and reversed by rapamycin. Hum. Mol. Genet. 20, 1290e1305. Ma, A., Wang, L., Gao, Y., Chang, Z., Peng, H., Zeng, N., Gui, Y.S., Tian, X., Li, X., Cai, B., Zhang, H., Xu, K.F., 2013. Tsc1 deficiency-mediated mTOR hyperactivation in vascular endothelial cells causes angiogenesis defects and embryonic lethality. Hum. Mol. Genet. 23, 693e705. Marin, T.M., Keith, K., Davies, B., Conner, D.A., Guha, P., Kalaitzidis, D., Wu, X., Lauriol, J., Wang, B., Bauer, M., Bronson, R., Franchini, K.G.,
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77 Neel, B.G., Kontaridis, M.I., 2011. Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation. J. Clin. Invest. 121, 1026e1043. Maron, B.J., McKenna, W.J., Danielson, G.K., Kappenberger, L.J., Kuhn, H.J., Seidman, C.E., Shah, P.M., Spencer 3rd, W.H., Spirito, P., Ten Cate, F.J., Wigle, E.D., Task Force on Clinical Expert Consensus Documents. American College of Cardiology, Committee for Practice Guidelines, European Society of Cardiology, 2003. American College of Cardiology/ European Society of Cardiology clinical expert consensus document on hypertrophic cardiomyopathy. A report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus Documents and the European Society of Cardiology Committee for practice guidelines. J. Am. Coll. Cardiol. 42, 1687e1713. Mcmullen, J.R., Sherwood, M.C., Tarnavski, O., Zhang, L., Dorfman, A.L., Shioi, T., Izumo, S., 2004. Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload. Circulation 109, 3050e3055. Meikle, L., McMullen, J.R., Sherwood, M.C., Lader, A.S., Walker, V., Chan, J.A., Kwiatkowski, D.J., 2005. A mouse model of cardiac rhabdomyoma generated by loss of Tsc1 in ventricular myocytes. Hum. Mol. Genet. 14, 429e435. Northrup, H., Krueger, D.A., International Tuberous Sclerosis Complex Consensus Group, 2013. Tuberous sclerosis complex diagnostic criteria
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update: recommendations of the 2012 international tuberous sclerosis complex consensus conference. Pediatr. Neurol. 49, 243e254. Shaaya, E.A., Hirshberg, J.S., Rabe, O.T., Thibert, R.L., Inglessis, I., Sharma, A., Thiele, E.A., 2013. Cardiac fat-containing lesions are common in tuberous sclerosis complex. Am. J. Med. Genet. A 161, 1662e1665. Smythe, J.F., Dyck, J.D., Smallhorn, J.F., Freedom, R.M., 1990. Natural history of cardiac rhabdomyoma in infancy and childhood. Am. J. Cardiol. 66, 1247e1249. Thoreen, C.C., Chantranupong, L., Keys, H.R., Wang, T., Gray, N.S., Sabatini, D.M., 2012. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109e113. Wilde, A.A., Behr, E.R., 2013. Genetic testing for inherited cardiac disease. Nat. Rev. Cardiol. 10, 571e583. Zhang, D., Contu, R., Latronico, M.V., Zhang, J., Rizzi, R., Catalucci, D., Miyamoto, S., Huang, K., Ceci, M., Gu, Y., Dalton, N.D., Peterson, K.L., Guan, K.L., Brown, J.H., Chen, J., Sonenberg, N., Condorelli, G., 2010. MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice. J. Clin. Invest. 120, 2805e2816. Zhu, Y., Pires, K.M., Whitehead, K.J., Olsen, C.D., Wayment, B., Zhang, Y.C., Bugger, H., Ilkun, O., Litwin, S.E., Thomas, G., Kozma, S.C., Abel, E.D., 2013. Mechanistic target of rapamycin (mTOR) is essential for murine embryonic heart development and growth. PLoS ONE 8, e54221.
ScienceDirect Journal of Genetics and Genomics 41 (2014) 73e77
JGG LETTER TO THE EDITOR
Acquired Cardiomyopathy Caused by Cardiac Tsc1 Deficiency Cardiomyopathies are myocardial disorders with enlarged, thick, or rigid cardiac muscles. Because no effective treatment is available to prevent the deterioration of the myocardium function, the prognosis of cardiomyopathy is usually poor. Cardiomyopathies can be inherited or acquired. A number of genes associated with inherited cardiomyopathy, such as the genes encoding for sarcomeric, cytoskeletal, nuclear membrane, lysosomal, desmosomal and mitochondrial proteins, have been identified (Wilde and Behr, 2013). However, the underlying mechanisms of acquired cardiomyopathy remain poorly understood. Therefore, understanding the pathogenesis of acquired cardiomyopathy is crucial to develop new therapies for its prevention and treatment. Tuberous sclerosis (TSC) is an autosomal dominant disorder affecting about 1 in 6000 individuals worldwide (Northrup and Krueger, 2013). The disease is characterized by the development of benign hamartomas in multiple organs. The cardiac manifestation of TSC is rhabdomyomas in fetuses and infants, and fat-containing lesions in adolescents and adults (Smythe et al., 1990; Shaaya et al., 2013). Inactivating mutations in either Tsc1 or Tsc2 gene have been detected in majority of patients suffering from TSC (Cheadle et al., 2000). Tsc1 null embryos die at mid-gestation (Kobayashi et al., 2001; Kwiatkowski et al., 2002). Mice devoid of Tsc1 in ventricular myocytes develop congenital dilated cardiomyopathy (Meikle et al., 2005). Tsc1 and Tsc2 are known to function as a complex which acts through the Rheb GTPase to inhibit the mechanistic target of rapamycin (mTOR). Loss of either Tsc1 or Tsc2 leads to constitutive activation of mTOR and hyperphosphorylation of its downstream targets S6 kinases and 4E-BP1 (Thoreen et al., 2012). Dysregulated mTOR signaling has been implicated in several major diseases, such as cardiovascular diseases, cancer, metabolic diseases, neurological diseases and inflammatory diseases (Dazert and Hall, 2011). Whether activation or inhibition of mTOR signaling happens during cardiomyopathy in human subjects remains to be determined. Nevertheless, mTOR is critical for the survival and proliferation of cardiomyocytes during mouse cardiac development (Zhu et al., 2013). Loss of mTOR in cardiomyocytes leads to a fatal, dilated cardiomyopathy due to apoptosis, autophagy and altered mitochondrial
structure (Zhang et al., 2010). Furthermore, hyperactivation of mTOR plays an important role in load-induced cardiac hypertrophy in mice (Mcmullen et al., 2004). To model acquired cardiomyopathy and further study the cardiac function of mTOR in the adult animal, we inducibly knocked out Tsc1 in the cardiomyocytes of 2-month-old mice. A gene-targeting strategy was used to generate aMHCMerCreMer/Tsc1flox/flox mice (aMHC-MCM/Tsc1flox/flox mice) (Fig. S1A). Two-month-old male aMHC-MCM/Tsc1flox/flox mice were consecutively administered tamoxifen for 6 days to induce cardiomyocyte-specific Tsc1 knockout (Tsc1-cKO mice) (Fig. S1B). As the control group, the age-matched male Tsc1flox/ flox mice also received tamoxifen-injection. After tamoxifen administration, the expression of Tsc1 protein in Tsc1-cKO mice decreased significantly. The level of phospho-S6, a hallmark of mTOR activity, increased markedly in the hearts of Tsc1-cKO mice (Fig. S1C), indicating that mTOR activity was upregulated (Materials and Methods in Supplementary Data). Tsc1-cKO mice began to die (one mouse) at the fifth day of tamoxifen administration, and 40% of Tsc1-cKO mice (eight mice) died at the last day (the sixth day) of tamoxifen administration. By the fourteenth day of the initiation of cardiac Tsc1 knockout, one more mouse died. Nevertheless, none of the remaining 10 mice died after that. Collectively, the mortality of Tsc1-cKO mice was as high as 50% within two weeks of induction of Tsc1 knockout. In contrast, none of the Tsc1flox/flox mice (control mice, n ¼ 17) died at the endpoint of our study (52 days) (Fig. 1A). At two weeks after the initiation of tamoxifen administration, the heart/body weight ratio (HW/BW) of the survived Tsc1-cKO mice was significantly higher than that of control mice (P < 0.05, Fig. 1B). In addition, the heart of the Tsc1cKO mice was perceptibly enlarged in comparison with that of the Tsc1flox/flox mice (Fig. 1C). Internal diameters of atrial and ventricular chambers were larger, and the left ventricular wall and interventricular septum were thinner than those of Tsc1flox/ flox mice (Fig. 1D). Echocardiography demonstrated left ventricular internal diameter (LVID) of Tsc1-cKO mice was larger than that of control mice: 4.30 0.21 mm vs. 3.80 0.19 mm (P < 0.05) in the end of diastole (LVIDd), and 3.53 0.24 mm vs.
1673-8527/$ - see front matter Copyright Ó 2013, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.jgg.2013.11.005
74
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77
Fig. 1. Cardiomyocyte-specific Tsc1 knockout induces a lethal cardiac dilation and impairs cardiac function. A: Tsc1-cKO led to a high mortality at the early stage. Mortality of Tsc1-cKO mice began five days after tamoxifen administration, and reached 50% by two weeks. Time was shown as days after tamoxifen administration. n ¼ 20 for Tsc1-cKO mice and n ¼ 17 for control mice (Ctrl) B: Heart/body weight ratio (HW/BW) from Tsc1-cKO mice and control mice. HW/BW ratio of the Tsc1-cKO mice was higher than that of control two weeks after tamoxifen administration. n ¼ 3 for each group. *P < 0.05 vs. the control group. C: Representative explanted hearts. Heart of the Tsc1-cKO mice was larger than that of control at one week after tamoxifen administration. D: Representative H&E-stained heart sections. Tsc1-cKO hearts had thinner ventricular walls and enlarged cardiac chambers at one week after tamoxifen administration. E: Movement of ventricular wall assessed using echocardiography. Tsc1-cKO mouse was associated with obviously weaker ventricular wall movement when compared with control at one week after tamoxifen administration. F: Re-expression of cardiac fetal genes, assayed by qRT-PCR and normalized for b-actin expression, was given as fold change. Tsc1-cKO hearts had a 2- to 16-fold increase in the re-expression of these genes at two weeks after tamoxifen administration. Data are presented from one experiment performed in three replicates and the experiment was repeated three times with similar results. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group. ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; b-MyHC, b-myosin heavy chain; SK.actin, a-skeletal actin.
2.29 0.34 mm (P < 0.01) in the end of systole (LVIDs) at one week since tamoxifen administration (Table 1). Left ventricular posterior wall in the end of systole (LVPWs) of Tsc1-cKO mice was thinner than that of Tsc1flox/flox mice, 1.01 0.12 mm vs. 1.25 0.09 mm (P < 0.05). In consistent with histological examination (Fig. 1D), left ventricular anterior wall in the end of systole (LVAWs) was also thinner
than that of control mice, even though statistical significance was not reached. In addition, left ventricular mass corrected for anterior wall (LV Mass (AW) Corrected) was heavier as well, 107.5 6.9 mg vs. 80.6 14.2 mg (P < 0.01). The enlarged ventricular chamber and the thinner left ventricular wall in addition to heavier heart weight observed in the first week remained at the second week since tamoxifen
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77
Table 1 Inducible Tsc1 knockout in cardiomyocytes caused cardiomyopathy in mice as assessed by echocardiography Parameter
Week after tamoxifen administration
Control
LVIDd (mm)
0
3.70 0.10
3.56 0.27
1
3.80 0.19
4.30 0.21*
LVIDs (mm)
LVPWd (mm)
LVPWs (mm)
LVAWd (mm)
LVAWs (mm)
EF (%)
FS (%)
LV Mass (AW) Corrected (mg)
Tsc1-cKO
2
3.40 0.29
3.68 0.21*
4
3.67 0.21
3.59 0.17
0
2.40 0.11
2.12 0.34
1
2.29 0.34
3.53 0.24**
2
1.90 0.41
2.46 0.24
4
2.19 0.42
2.29 0.25
0
0.71 0.04
0.81 0.10
1
0.76 0.04
0.81 0.13
2
0.89 0.11
0.81 0.04 1.01 0.11*
4
0.74 0.08
0
1.06 0.05
1.27 0.10
1
1.25 0.09
1.01 0.12*
2
1.37 0.17
1.12 0.06*
4
1.21 0.19
1.42 0.08
0
0.76 0.15
0.76 0.13
1
0.76 0.16
0.81 0.12
2
0.91 0.12
0.94 0.05
4
0.88 0.08
1.02 0.10*
0
1.28 0.10
1.33 0.16
1
1.36 0.26
1.08 0.11
2
1.38 0.18
1.43 0.07 1.59 0.11
4
1.50 0.09
0
65.0 4.2
71.8 6.5
1
70.6 8.6
37.8 4.3***
2
76.4 8.1
62.7 4.4*
4
71.8 10.5
66.6 6.0
0
35.0 3.1
40.6 5.4
1
39.9 7.2
18.1 2.3**
2
44.7 7.1
33.4 3.2*
4
41.0 8.4
36.2 4.4
0
73.8 9.5
75.3 7.0
1
80.6 14.2
107.5 6.9**
2
85.5 11.3
92.8 9.2**
4
83.8 12.5
110.6 15.3*
LVIDd/s, left ventricular internal diameter in the end of diastole/systole; LVPWd/s, left ventricular posterior wall thickness in the end of diastole/systole; LVAWd/s, left ventricular anterior wall thickness in the end of diastole/ systole; EF, ejection fraction; FS, fractional shortening; LV Mass (AW) Corrected, left ventricular mass (anterior wall) corrected. Five mice were assessed in each group. Measurements were given as mean SD. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group, ***P < 0.001 vs. the control group (t-test).
75
administration: LVIDd, 3.68 0.21 mm vs. 3.40 0.29 mm (P < 0.05); LVPWs, 1.12 0.06 mm vs. 1.37 0.17 mm (P < 0.05); and LV Mass (AW) Corrected, 92.8 9.2 mg vs. 85.5 11.3 mg (P < 0.01) (Table 1). Taken together, mice deficient of Tsc1 in adulthood developed dilated cardiomyopathy probably due to activation of mTOR. By the fourth week since tamoxifen administration, the size of heart and the thickness of left ventricular wall in Tsc1-cKO mice were progressively larger than those of Tsc1flox/flox mice: LV Mass (AW) Corrected, 110.6 15.3 mg vs. 83.8 12.5 mg (P < 0.05); LVPWd, 1.01 0.11 mm vs. 0.74 0.08 mm (P < 0.05); and LVAWd, 1.02 0.10 mm vs. 0.88 0.08 mm (P < 0.05) (Table 1). Therefore, the survived Tsc1-cKO mice experienced cardiac dilation to cardiac hypertrophy after Tsc1 knockout in the heart. Echocardiography revealed impaired cardiac function in Tsc1-cKO mice when compared to Tsc1flox/flox mice. At one week since tamoxifen administration, Tsc1-cKO mice had dramatically decreased ejection fraction (EF) and fractional shortening (FS), which represent cardiac systolic and diastolic function respectively: EF, 37.8 4.3% (Tsc1-cKO mice) vs. 70.6 8.6% (Tsc1flox/flox mice) (P < 0.001); FS, 18.1 2.3% vs. 39.9 7.2% (P < 0.01) (Table 1), and the movement of ventricular wall was obviously weaken (Fig. 1E). Nevertheless, the cardiac function of Tsc1-cKO mice restored over time. At two weeks since tamoxifen administration, EF was 62.7 4.4% (Tsc1-cKO mice) vs. 76.4 8.1% (Tsc1flox/flox mice) (P < 0.05); FS was 33.4 3.2% vs. 44.7 7.1% (P < 0.05). And at four weeks, there was no difference in the cardiac systolic and diastolic function between Tsc1-cKO mice and Tsc1flox/flox mice (Table 1). Fetal genes are normally expressed in embryonic and neonatal heart, and re-expressed in response to pathological stress (Crone et al., 2002). Tsc1-cKO myocardium had higher levels of mRNA for fetal genes, including atrial natriuretic factor (ANF ), brain natriuretic peptide (BNP), b-myosin heavy chain (b-MyHC ), and a1-skeletal actin (SK.actin) (Fig. 1F), indicative of cardiac dysfunction. The previous study has demonstrated that loss of mTOR in myocardium of adult mice causes cardiomyopathy. The present study indicates that cardiomyocyte-specific Tsc1 knockout in adult mice triggers the development of acquired cardiomyopathy with hyperactive mTOR signaling. Although cardiomyopathy often presents as a congenital disease, it may occur due to various non-inherited pathogeny, including ischemic heart disease (e.g., acute myocardial infarction), structural heart disease (e.g., valvular heart disease), myocarditis (e.g., giant cell myocarditis), and many other factors (Hershberger et al., 2010). Therefore, the inducible Tsc1-cKO in normal adult mice generated in our study recapitulates clinical acquired cardiomyopathy to some extent. Tsc1 null mouse embryos die between E9.5 and E13.5 possibly due to prominent defects in liver development and significant cardiac enlargement (Kwiatkowski et al., 2002). Endothelial Tsc1 knockout illustrates physiological Tsc1mTOR signaling in endothelial cells is crucial for cardiovascular development and embryogenesis (Ma et al., 2013). And mice with specific ventricular loss of Tsc1 develop dilated
76
Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 73e77
cardiomyopathy with a median survival of 6 months (Meikle et al., 2005). In our study, inducible Tsc1 knockout of cardiomyocytes in entire hearts led to cardiac dilation and impaired cardiac function with high mortality in adult mice. However, the remaining mice after two weeks of Tsc1 deficiency gradually restored their cardiac function in parallel with a switch of myocardium from dilation to hypertrophy. We speculate that these survived mice might have fewer Tsc1 knockout cardiomyocytes than their deceased counterparts. Cardiac hypertrophy is a compensated response to physiological or pathological stress, such as exercise and persistent pressure overload. In its early stage, hypertrophic cardiomyopathy is characterized by left ventricular hypertrophy, normal to smaller left ventricular cavity and may be hypercontractile (Hershberger et al., 2009). However, hypertrophic cardiomyopathy may occasionally show cardiac dilation and decreased systolic function, characteristics of dilated cardiomyopathy (Maron et al., 2003). Therefore, whether hypertrophy is beneficial for cardiac function remains controversial (Frey et al., 2004). Here, Tsc1-cKO mice developed larger atrial and ventricular chambers, thinner left ventricular wall and interventricular septum than control mice at one week after tamoxifen administration. However, the ventricular wall became thicker than that of control mice over time in association with improved survival. Our study suggests cardiac hypertrophy may be beneficial. mTOR integrates both intracellular and extracellular signals through multiple pathways to regulate cell growth, proliferation, metabolism, function and survival (Dazert and Hall, 2011). It is essential for cardiac development and growth during embryogenesis (Zhu et al., 2013). Malfunction of mTOR pathway leads to hypertrophic or dilated cardiomyopathy (Meikle et al., 2005; Zhang et al., 2010). mTOR inhibitor rapamycin can rapidly normalize cardiomyocyte size, attenuate or reverse cardiac hypertrophy. It can improve cardiac function in a setting of left ventricular hypertrophy and cardiac dysfunction as well (Mcmullen et al., 2004; Marin et al., 2011). And it can also reverse vascular smooth muscle Tsc1 knockout-induced ventricular hypertrophy (Malhowski et al., 2011). After cardiomyocyte-specific knockout of Tsc1, the level of phospho-S6 increased in myocardium (Fig. S1C), suggestive of mTOR activation in this acquired cardiomyopathy mouse model. Therefore, the benefit of rapamycin ought to be investigated in the treatment of cardiomyocyte-specific Tsc1 knockout-induced cardiomyopathy in adult mice. Our study has implicated cardiomyocyte Tsc1 deficiencymediated mTOR activation in the induction of acquired cardiomyopathy. This acquired cardiomyopathy mouse model should be very valuable in the study of pathogenesis of cardiomyopathy. And targeting mTOR should be a promising strategy in the treatment of acquired cardiomyopathy. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 81270288) and the Chinese Ministry of Science and Technology (No. 2011CB965002).
SUPPLEMENTARY DATA Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2013.11.005. Yi Chena,1, Fang Wangb,1, Chunjia Lib, Lianmei Wangb, Hongbing Zhangb, Hongbing Yanc,* a
Department of Cardiology, Beijing An Zhen Hospital, Capital Medical University, Beijing 100029, China
b
Department of Physiology, Institute of Basic Medical Sciences and School of Basic Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100005, China c Center for Coronary Heart Disease, Cardiovascular Institute & Fu Wai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100037, China
*Corresponding author. Tel: þ86 10 8839 7408, fax: þ86 10 6831 1790. E-mail address: [email protected] (H. Yan) 1 These authors contribute equally to this work. Received 10 October 2013 Revised 4 November 2013 Accepted 14 November 2013 Available online 3 December 2013
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