背景介紹:
心衰竭，一種心肌功能退化與心臟纖維化的過程，影響老人比年輕人多。單純老化的過程就會引起心臟纖維化嗎? 理論上而言，因為老化的細胞會分泌發炎與促進纖維化的因子進而誘發心肌纖維化。成大醫院田寮的研究發現，健康的老人家，比起年輕人，他們的心臟比較大比較硬。然而這個研究也同時發現老人家有較多的共病例如高血壓與糖尿病。而高血壓與糖尿病就可能造成比較大或比較硬的心臟。所以年紀大不見得是導致心臟變硬變大的唯一原因，而老化本身是否足以造成心臟纖維化仍然未知。
從另一個觀點來看，並非所有的人年老後都會發生心臟衰竭，有些人或許具有演化上的優勢避免年老時發生心臟衰竭。然而是哪一種遺傳上的優勢可以避免老化時發生心臟纖維化，目前仍未有確切的定論。ROCK基因表現與心臟纖維化有關。降低ROCK基因表現可以減緩心臟纖維化。因此我們期望可以看到老化本身就會引起心臟纖維化，而透過降低ROCK基因表現可以減緩老化造成的心臟纖維化。
假設:
我們假設隨者老化，心肌纖維化持續，心臟功能下降。降低ROCK基因表現可以減緩老化造成的心臟纖維化。
方法:
以心臟超音波評估年輕，老化，野生與 ROCK2 gene knock down (基因剔除)老鼠左心室收縮功能。但是因為老鼠的心臟功能可能到老也不會下降，因此我們注射一個月angiotensin II來引發高血壓，接者以超音波評估心臟功能。所有老鼠在實驗結束時被安樂死，之後我們以心肌病理切片評估心室纖維化。
結果:
不論是年輕的，老的野生型老鼠左心室形狀與收縮功能都沒變化。老的ROCK2 gene knock down (基因剔除)老鼠比起年輕的ROCK2基因剔除老鼠，心臟較大但心室收縮功能不變。
用angiotensin II誘發高血壓後，病理切片上可見，老的野生型老鼠比起其他類型的老鼠(年輕的野生型，年輕的，老的ROCK2基因剔除)有明顯的心臟纖維化。而這個病理上的變化無法從心臟超音波早期偵測。
結論:
不論是野生型或ROCK2基因剔除的老鼠，年紀本身可能無法造成心臟纖維化。然而同時合併其他的共病(或壓力)，例如高血壓就能造成心臟纖維化。ROCK2基因剔除的老鼠可以緩和高血壓與年紀導致的心肌纖維化。

Introduction:
Heart failure, a process of myocytes dysfunction and accumulation of fibrosis, effected more old than young people. Can aging alone induce cardiac fibrosis? Theoretically, it is possible because age-related cell senescence associated secretory phenotype increase inflammatory and profibrogenic factors and promotes cardiac fibrosis. Our group had found relative healthy old people had larger and stiffer heart in “TaninLio Old People” study. However, the result was confounded by other comorbid disease such as hypertension or diabetes. It remained unknown if aging alone can cause cardiac fibrosis.
From another point of view, not all old people developed heart failure. Someone may have evolutional advantage to avoid cardiac fibrosis. But we remained unknow which kind of genetic variant can prevent cardiac fibrosis. ROCK expression is associated with cardiac fibrosis and reduction it’s expression can attenuate cardiac fibrosis. So, we hoped we can see age-related cardiac fibrosis and this fibrosis can be attenuated by reduction in ROCK expression.
Hypothesis:
We hypothesize myocardial fibrosis is in progression and heart function is in deterioration during aging. “Reduction in ROCK expression” can attenuate aging induced cardiac fibrosis
Because mice seldom have heart failure, we introduced hypertensive stress and test the cardiac fibrosis in different genetic groups.
Experimental design:
We have four group of mice, young, old wild type and ROCK2 knock down mice (ROCK2+/-). We evaluate their left ventricular contractile function, cardiac fibrosis, hypertrophy by echocardiography. After angiotensin II infusion, four group of mice and had hypertension. One month later, we evaluate their heart function by echocardiography. All mice are euthanatized, and we analyzed the cardiac fibrosis pathologically.
Results:
There is no difference in the geometry or myocardial contractile function between the old and young wild type mice. The old ROCK2+/- mice have enlarged heart size than young ROCK2+/- mice. After angiotensin II treatment, the old wild type mice have more prominent cardiac fibrosis than the other mice (young wild type, young ROCK2+/- and old ROCK2+/-). The cardiac fibrosis cannot be detected by echocardiography.
Conclusion:
Aging alone cannot cause cardiac fibrosis in wild type or ROCK2 knock down mice. It must be combined with other stress, like hypertension to cause cardiac fibrosis. Reduction in ROCK2 expression can attenuate hypertension induced cardiac fibrosis.

Reference
1. Mozaffarian D, Benjamin EJ, Go AS. Heart Disease and Stroke Statistics-2016 Update: A Report from the American Heart Association. Circulation 2016;133(4):e38-360.
2. Chang WT, Chen JS, Hung YK, Tsai WC, Juang JN, Liu PY. Characterization of aging-associated cardiac diastolic dysfunction. PLoS One. 2014 May 28;9(5):e97455.
3. Aurigemma GP, Zile MR, Gaasch WH. Contractile behavior of the left ventricle in diastolic heart failure: with emphasis on regional systolic function. Circulation. 2006 Jan 17;113(2):296-304.
4. Zile MR, Baicu CF, Ikonomidis JS, Stroud RE, Nietert PJ, et al. Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin. circulation 2015;131:1247-59.
5. Heymans S, González A, Pizard A, Papageorgiou AP, López-Andrés N, et al. Searching for new mechanisms of myocardial fibrosis with diagnostic and/or therapeutic potential. Eur J Heart Fail. 2015;17(8):764-71.
6. Travers JG, Kamal FA, Robbins J, Yutzey KE, Blaxall BC. Cardiac Fibrosis: The Fibroblast Awakens. Circ Res. 2016;118(6):1021-40
7. Moore-Morris T, Guimarães-Camboa N, Banerjee I, Zambon AC, Kisseleva T, et al. Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest. 2014 Jul;124(7):2921-34.
8. Duffield JS, Lupher M, Thannickal VJ, Wynn TA. Host responses in tissue repair and fibrosis. Annu Rev Pathol 2013; 8: 241-76.
9. Wynn TA1, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med. 2012;18(7):1028-40.
10. Martinod K, Witsch T, Erpenbeck L, Savchenko A, Hayashi H, et al. Peptidylarginine deiminase 4 promotes age-related organ fibrosis. J Exp Med. 2017;214(2):439-458.
11. Dimmeler S, Zeiher AM. Netting Insights into Fibrosis. N Engl J Med. 2017 Apr 13;376(15):1475-1477.
12. Wilson, M.S. et al. Bleomycin and IL-1β–mediated pulmonary fibrosis is IL-17A dependent. J. Exp. Med. 207, 535–552 (2010)
13. Lee, C.G. et al. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor-β1. J. Exp. Med. 194, 809–822 (2001).
14. Lee, J.H. et al. Interleukin-13 induces dramatically different transcriptional programs in three human airway cell types. Am. J. Respir. Cell Mol. Biol. 25, 474–485 (2001).
15. Shimizu T1, Liao JK. Rho Kinases and Cardiac Remodeling. Circ J. 2016 Jun 24;80(7):1491-8.
16. Shimizu T, Narang N, Chen P, Yu B, Knapp M, Janardanan J, Blair J, Liao JK. Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction. JCI Insight. 2017 Jul 6;2(13). pii: 93187.
17. Hata T, Soga J, Hidaka T, Idei N, Fujii Y, Fujimura N, et al. Calcium channel blocker and Rho-associated kinase activity in patients with hypertension. J Hypertens 2011; 29: 373 – 379.
18. Do.e Z, Fukumoto Y, Takaki A, Tawara S, Ohashi J, Nakano M, et al. Evidence for Rho-kinase activation in patients with pulmonary arterial hypertension. Circ J 2009; 73: 1731 – 1739.
19. Liu PY, Chen JH, Lin LJ, Liao JK. Increased Rho kinase activity in a Taiwanese population with metabolic syndrome. J Am Coll Cardiol 2007; 49: 1619 – 1624.
20. Liu PY, Liu YW, Lin LJ, Chen JH, Liao JK. Evidence for statin pleiotropy in humans: Differential effects of statins and ezetimibe on rho-associated coiled-coil containing protein kinase activity, endothelial function, and inflammation. Circulation 2009; 119: 131 – 138.
21. Nohria A, Grunert ME, Rikitake Y, Noma K, Prsic A, Ganz P, et al. Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease. Circ Res 2006; 99: 1426 – 1432.
22. Masumoto A1, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, et al. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension. 2001 Dec 1;38(6):1307-10.
23. Fukumoto Y1, Yamada N, Matsubara H, Mizoguchi M, Uchino K et al. Double-blind, placebo-controlled clinical trial with a rho-kinase inhibitor in pulmonary arterial hypertension. Circ J. 2013;77(10):2619-25.
24. Xiao JW1, Zhu XY, Wang QG, Zhang DZ, Cui CS, et al. Acute effects of Rho-kinase inhibitor fasudil on pulmonary arterial hypertension in patients with congenital heart defects. Circ J. 2015;79(6):1342-8.
25. Masumoto A1, Mohri M, Shimokawa H, Urakami L, Usui M, et al. Suppression of coronary artery spasm by the Rho-kinase inhibitor fasudil in patients with vasospastic angina. Circulation. 2002 Apr 2;105(13):1545-7.
26. Shimokawa H1, Hiramori K, Iinuma H, Hosoda S, Kishida H, et al. Anti-anginal effect of fasudil, a Rho-kinase inhibitor, in patients with stable effort angina: a multicenter study. J Cardiovasc Pharmacol. 2002 Nov;40(5):751-61.
27. Shibuya M1, Hirai S, Seto M, Satoh S, Ohtomo E, et al. Effects of fasudil in acute ischemic stroke: results of a prospective placebo-controlled double-blind trial. J Neurol Sci. 2005 Nov 15;238(1-2):31-9.
28. W. Waters, Janette K. Burgess, Doan TM. Ngo, Aaron L. Sverdlov, Darryl A. Knight, et al. The Role of Pathological Aging in Cardiac and Pulmonary Fibrosis. Aging Dis. 2019 Apr; 10(2): 419–428
29. Amzulescu MS, De Craene M, Langet H, Pasquet A, Vancraeynest D, et al. Myocardial strain imaging: review of general principles, validation, and sources of discrepancies. Eur Heart J Cardiovasc Imaging. 2019 Jun 1;20(6):605-619.
30. Liu YW, Lee WH, Lin CC, Huang YY, Lee WT et al. Left ventricular diastolic wall strain and myocardial fibrosis in treated hypertension. Int J Cardiol. 2014 Mar 15;172(2):e304-6.