Arthur HM

Dookun E

Owens A

Passos JF

Redgrave R

Richardson GD

Spyridopoulos I

Tual-Chalot A

Walaszczyk A

Cardiovascular disease is the leading cause of death in individuals over 60 years old. Ageing is associated with an increased prevalence of coronary artery disease and a poorer prognosis following acute myocardial infarction (MI). With age, senescent cells accumulate in tissues, including the heart, and contribute to age-related pathologies. However, the role of senescence in recovery following MI has not been investigated. In this study, we demonstrate that treatment of aged mice with the senolytic drug, navitoclax, eliminates senescent cardiomyocytes and attenuates pro-fibrotic protein expression in aged mice. Importantly, clearance of senescent cells improved myocardial remodelling and diastolic function as well as overall survival following MI. These data provide proof-of-concept evidence that senescent cells are major contributors to impaired function and increased mortality following MI and that senolytics are a potential new therapeutic avenue for MI

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Aging Cell. 2019;e12945.	 	 	 | 	1 of 4https://doi.org/10.1111/acel.12945wileyonlinelibrary.com/journal/acel1  | INTRODUCTION, RESULTS, DISCUSSIONCardiovascular disease (CVD) is a prevalent disease in the older pop‐ulation. The incidence of atherosclerotic coronary artery disease (CAD) increases with age and is present in over 50% of people over 60 years old (North & Sinclair, 2012). As such, older patients have a higher incidence of MI, which is accompanied by increased morbidity and mortality (Mehta et al., 2001).Cellular senescence is classically defined as the irreversible cell cycle arrest of somatic cells. While senescence can act as a potent an‐titumour mechanism, recent studies have shown that senescent cells accumulate in several tissues with age where they contribute to age‐dependent tissue dysfunction and several age‐related diseases (de Magalhaes & Passos, 2018; Kirkland & Tchkonia, 2017). Senescent cells are thought to contribute to aging via a pro‐oxidant phenotype (Passos et al., 2010) and the secretion of a senescence‐associated  Received:	16	January	2019  |  Revised:	13	February	2019  |  Accepted:	16	February	2019DOI: 10.1111/acel.12945  S H O R T  T A K EPharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarctionAnna Walaszczyk1 |   Emily Dookun1 |   Rachael Redgrave1 |   Simon Tual‐Chalot1 |   Stella Victorelli2,3 |   Ioakim Spyridopoulos1 |   Andrew Owens1 |   Helen M. Arthur1  |   João F. Passos2,3  |   Gavin D. Richardson1This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.1Cardiovascular Research Centre, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK2Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK3Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MinnesotaCorrespondenceGavin D. Richardson, Cardiovascular Research Centre, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.Email: Gavin.Richardson@ncl.ac.ukandJoão	F.	Passos,	Department	of	Physiology	and Biomedical Engineering, Mayo Clinic, Rochester, MN.Email Passos.Joao@mayo.eduFunding informationThis work was supported by British Heart	Foundation	PG/15/85/31744	and	PG/14/86/31177,	Wellcome	Trust	and	the	Newcastle Healthcare Charity.AbstractCardiovascular disease is the leading cause of death in individuals over 60 years old. Aging is associated with an increased prevalence of coronary artery disease and a poorer prognosis following acute myocardial infarction (MI). With age, senescent cells accumulate in tissues, including the heart, and contribute to age‐related pathol‐ogies. However, the role of senescence in recovery following MI has not been inves‐tigated. In this study, we demonstrate that treatment of aged mice with the senolytic drug, navitoclax, eliminates senescent cardiomyocytes and attenuates profibrotic protein expression in aged mice. Importantly, clearance of senescent cells improved myocardial remodelling and diastolic function as well as overall survival following MI. These data provide proof‐of‐concept evidence that senescent cells are major con‐tributors to impaired function and increased mortality following MI and that senolyt‐ics are a potential new therapeutic avenue for MI.K E Y W O R D Saging, cardiac, myocardial infarction, senescence, senolytics, survival2 of 4  |     WALASZCZYK et AL.     |  3 of 4WALASZCZYK et AL.secretory phenotype (SASP) (Coppe et al., 2008), which is pro‐in‐flammatory, profibrotic and can also promote senescence in sur‐rounding cells (Nelson et al., 2012).Senescence has been shown to occur in the heart during aging (Anderson et al., 2019) and contributes to the pathophysiology of a num‐ber of CVDs, as clearance of senescent cells in aged and atherosclerotic mice using both genetic and pharmacological approaches improves vascular and myocardial function and attenuates age‐dependent re‐modelling (Anderson et al., 2019; Roos et al., 2016; Zhu et al., 2015). However, the impact of senescent cells in MI has not been investigated thus far. In this study, we hypothesise that senescent cells contribute to the poor prognosis and survival of aged individuals following MI.F I G U R E  1  Aged	mice	display	increased	CM	senescence,	CM	hypertrophy,	increased	TGFβ2 expression and functional characteristics of myocardial aging, which are attenuated by navitoclax treatment. (a) Experimental design. (b) Percentage p16+ CMs by RNA in situ hybridization. Arrow indicates p16 expressing CMs (p16 red, troponin‐C green, DAPI blue), n = 8 per group. Scale bars = 50 µm. (c) WGA staining and quantification of CM cross‐sectional area, n	=	6–10	per	group.	(d)	TGFβ2 protein expression, n = 4–5 per group. (e) Examples of individual short axis cine‐MR images. Analysis of (f) left ventricular mass and (g) ejection fraction. (h) % change in wall thickness, n	=	14–34	per	group.	For	c	and	d,	scale	bars	=	100	µm.	Data	are	mean	±	SEM, ***p < 0.001; **p < 0.01; * p < 0.05 using Student's t test or one‐way ANOVAF I G U R E  2   Navitoclax treatment improves survival and functional outcome following MI. (a) Kaplan–Meier survival curve following MI, n = 20–45 per group. Dotted line indicates survival between the age of 104–109 weeks in a cohort of vehicle‐treated mice that received no MI (n = 9). (b) Examples of individual short axis cine‐MR images post‐MI. ED = end diastole, ES = end systole. (c) Ejection fraction at 1 and 4 weeks post‐MI, n = 9–15 per group. (d) Relative change in ejection fraction between 1 and 4 weeks post‐MI, n = 7–11 per group. Data are mean	±	SEM, ***p < 0.001; **p < 0.01; * p < 0.05 using one‐way ANOVA4 of 4  |     WALASZCZYK et AL.Previously we found that in addition to clearing senescent cells, navitoclax treatment reduced fibrosis and cardiomyocyte (CM) hy‐pertrophy in aged mice (Anderson et al., 2019) and considered that these beneficial effects may help to improve outcomes in aged mice following MI. We therefore performed a more detailed longitudinal study to examine this possibility and to explore potential mecha‐nisms (detailed methods are included in Appendix S1).Histological analysis was performed on a cohort of nonin‐farcted mice, to assess the baseline effects of navitoclax treat‐ment (50 mg kg−1 day−1	 in	 the	 regime	 shown	 in	 Figure	 1a).	 In	addition to decreasing CM hypertrophy, treatment reduced mark‐ers of CM senescence, indicating clearance of senescent cells from	the	hearts	of	treated	aged	mice	(Figure	1b–c).	Furthermore,	we found a significant reduction in expression of profibrotic TGFβ2	(Figure	1d),	which	we	previously	identified	as	a	key	com‐ponent	of	CM	SASP	(Anderson	et	al.,	2019).	Functionally,	navito‐clax treatment significantly reduced the age‐dependent increase in	 left	 ventricular	 (LV)	 mass	 (Figure	 1f)	 but	 did	 not	 impact	 on	ejection	fraction	(EF)	(Figure	1g).	Aged	mice	also	exhibited	a	de‐crease in the percentage change in diastole versus end systole LV wall thickness, indicating an increased LV rigidity compared with young animals, which was also partly rescued by navitoclax treatment	 (Figure	 1h).	 Clinically,	 increased	 ventricle	 stiffness	 is	related to fibrosis and hypertrophy during aging, is symptomatic of diastolic dysfunction and is observed in heart failure with pre‐served ejection fraction patients (Borlaug, 2014).To investigate whether treatment with navitoclax improved out‐come following MI, mice from each group were then subjected to ligation of the left anterior descending coronary artery (LAD), which mimics the pathophysiology of MI (Redgrave et al., 2017).We observed that aged mice had significantly higher mortal‐ity rates following MI (60% over 5 weeks) compared with young mice and that this outcome was rescued by prior navitoclax treat‐ment	 (Figure	2a).	This	high	mortality	also	contrasts	with	the	 low	mortality observed in a cohort of aged mice that received no MI (Figure	2a)	and	our	previously	published	data	that	demonstrated	a low mortality in male C57BL/6 mice between the same ages of 104–109 weeks (Jurk et al., 2014). Together these data suggest that reduced survival in old mice following LAD‐ligation was a re‐sult of the MI and was not due to aging alone. No difference in EF	was	observed	between	the	MI	groups	at	1	week.	However,	 in	contrast	to	young	mice,	old	mice	show	a	significant	reduction	in	EF	between 1 and 4 weeks post‐MI. Importantly, navitoclax was able to	rescue	this	functional	decline	(Figure	2b–d)	which	may	help	to	explain	the	improved	survival	of	this	group.	Furthermore,	expres‐sion of senescence markers p16 and p21 at 4 weeks following MI was reduced in the hearts of navitoclax‐treated mice, consistent with reduction of the senescence burden (supporting information Figure	S1A).Collectively, this study shows that pharmacological clearance of senescent cells in aging mice alleviates age‐dependent myocar‐dial remodelling and attenuates expression of profibrotic medi‐ators. Navitoclax improved the maintenance of cardiac function following MI, ultimately increasing survival. An important limita‐tion of this study is that our experimental strategy was not able to distinguish which senescent cell types are responsible for this effect, and it is possible that clearance of senescent cells in non‐cardiac organs impact on survival following MI. We have focussed our attention on CMs in this study as our earlier findings showed that, in the heart, markers of senescence accumulate primarily in CMs during aging (Anderson et al., 2019). However, further stud‐ies using animal models where senescent cells can be cleared in a cell‐type specific manner are required to formally show the con‐tribution of senescent CMs to cardiac recovery post‐MI. As seno‐lytics have now moved into clinical trials in other fibrotic diseases (Justice et al., 2019), we propose that senolytics may help to re‐duce the morbidity and mortality associated with MI in the older population.CONFLIC T OF INTERESTNone Declared.AUTHOR CONTRIBUTIONSAW, ED, RR, SV and ST‐C performed experiments. AO, HMA and IS contributed	to	supervision.	GDR	and	JFP	designed	and	supervised	the	study.	GDR,	JFP	and	HMA	wrote	the	manuscript	with	contribu‐tions from all authors.ORCIDHelen M. Arthur  https://orcid.org/0000-0001-6522-1363 João F. Passos  https://orcid.org/0000‐0001‐8765‐1890 Gavin D. Richardson  https://orcid.org/0000-0002-2310-9987 R E FE R E N C E SAnderson, R., Lagnado, A., Maggiorani, D., Walaszczyk, A., Dookun, E.,	 Chapman,	 J.,	 …	 Passos,	 J.	 F.	 (2019).	 Length-independent	telomere damage drives post‐mitotic cardiomyocyte senes‐cence. EMBO Journal, 38(5), e100492. https://doi.org/10.15252/embj.2018100492Borlaug, B. A. (2014). The pathophysiology of heart failure with pre‐served ejection fraction. Nature Reviews Cardiology, 11(9), 507–515. https://doi.org/10.1038/nrcardio.2014.83Coppé,	J.-P.,	Patil,	C.	K.,	Rodier,	F.,	Sun,	Y.	u.,	Muñoz,	D.	P.,	Goldstein,	J.,	… Campisi, J. (2008). Senescence‐associated secretory phenotypes reveal cell‐nonautonomous functions of oncogenic RAS and the p53	 tumor	suppressor.	PLoS Biology, 6(12),	2853–2868.	https://doi.org/10.1371/journal.pbio.0060301de	Magalhaes,	 J.	 P.,	&	Passos,	 J.	 F.	 (2018).	 Stress,	 cell	 senescence	and	organismal ageing. Mechanisms of Ageing and Development, 170, 2–9. https://doi.org/10.1016/j.mad.2017.07.001Jurk,	D.,	Wilson,	C.,	Passos,	J.	F.,	Oakley,	F.,	Correia-Melo,	C.,	Greaves,	L., … von Zglinicki, T. (2014). Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nature Communications, 2,	4172.	https://doi.org/10.1038/ncomms5172     |  5 of 6WALASZCZYK et AL.Justice, J. N., Nambiar, A. M., Tchkonia, T., LeBrasseur, N. K., Pascual, R., Hashmi, S. K., … Kirkland, J. L. (2019). Senolytics in idiopathic pulmo‐nary fibrosis: Results from a first‐in‐human, open‐label, pilot study. EBioMedicine. https://doi.org/10.1016/j.ebiom.2018.12.052Kirkland, J. L., & Tchkonia, T. (2017). Cellular senescence: A translational perspective. Ebiomedicine, 21, 21–28. https://doi.org/10.1016/j.ebiom.2017.04.013Redgrave, R. E., Tual‐Chalot, S., Davison, B. J., Singh, E., Hall, D., Amirrasouli, M., … Arthur, H. M. (2017). Cardiosphere‐Derived Cells Require Endoglin for Paracrine‐Mediated Angiogenesis. Stem cell re-ports, 8(5), 1287–1298.Mehta,	 R.	 H.,	 Rathore,	 S.	 S.,	 Radford,	 M.	 J.,	 Wang,	 Y.,	 Wang,	 Y.,	 &	Krumholz, H. M. (2001). Acute myocardial infarction in the elderly: Differences by age. Journal of the American College of Cardiology, 38(3),	736–741.	https://doi.org/10.1016/S0735-1097(01)01432-2Nelson, G., Wordsworth, J., Wang, C., Jurk, D., Lawless, C., Martin‐Ruiz, C., & von Zglinicki, T. (2012). A senescent cell bystander effect: Senescence‐induced senescence. Aging Cell, 11(2),	345–349.	https://doi.org/10.1111/j.1474‐9726.2012.00795.xNorth, B. J., & Sinclair, D. A. (2012). The intersection between aging and cardiovascular disease. Circulation Research, 110(8), 1097–1108. https://doi.org/10.1161/CIRCRESAHA.111.246876Passos,	J.	F.,	Nelson,	G.,	Wang,	C.,	Richter,	T.,	Simillion,	C.,	Proctor,	C.	J.,	…	von	Zglinicki,	T.	(2010).	Feedback	between	p21	and	reactive	oxy‐gen production is necessary for cell senescence. Molecular Systems Biology, 6,	347.	https://doi.org/10.1038/msb.2010.5Roos, C. M., Zhang, B., Palmer, A. K., Ogrodnik, M. B., Pirtskhalava, T., Thalji, N. M., … Miller, J. D. (2016). Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or athero‐sclerotic mice. Aging Cell, 15(5),	973–977.	https://doi.org/10.1111/acel.12458Zhu,	Y.	i.,	Tchkonia,	T.,	Pirtskhalava,	T.,	Gower,	A.	C.,	Ding,	H.,	Giorgadze,	N.,	…	Kirkland,	J.	L.	(2015).	The	Achilles'	heel	of	senescent	cells:	From	transcriptome to senolytic drugs. Aging Cell, 14(4), 644–658. https://doi.org/10.1111/acel.12344SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section at the end of the article.  How to cite this article: Walaszczyk A, Dookun E, Redgrave R, et al. Pharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarction. Aging Cell. 2019;e12945. https://doi.org/10.1111/acel.12945

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Pharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarction.

Pharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarction.

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