Senescent cardiomyocytes contribute to cardiac dysfunction following myocardial infarction

Myocardial infarction is a leading cause of morbidity and mortality. While reperfusion is now standard therapy, pathological remodeling leading to heart failure remains a clinical problem. Cellular senescence has been shown to contribute to disease pathophysiology and treatment with the senolytic navitoclax attenuates inflammation, reduces adverse myocardial remodeling and results in improved functional recovery. However, it remains unclear which senescent cell populations contribute to these processes. To identify whether senescent cardiomyocytes contribute to disease pathophysiology post-myocardial infarction, we established a transgenic model in which p16 (CDKN2A) expression was specifically knocked-out in the cardiomyocyte population. Following myocardial infarction, mice lacking cardiomyocyte p16 expression demonstrated no difference in cardiomyocyte hypertrophy but exhibited improved cardiac function and significantly reduced scar size in comparison to control animals. This data demonstrates that senescent cardiomyocytes participate in pathological myocardial remodeling. Importantly, inhibition of cardiomyocyte senescence led to reduced senescence-associated inflammation and decreased senescence-associated markers within other myocardial lineages, consistent with the hypothesis that cardiomyocytes promote pathological remodeling by spreading senescence to other cell-types. Collectively this study presents a novel demonstration that senescent cardiomyocytes are major contributors to myocardial remodeling and dysfunction following a myocardial infarction. Therefore, to maximize the potential for clinical translation, it is important to further understand the mechanisms underlying cardiomyocyte senescence and how to optimize senolytic strategies to target this cell lineage.

Importantly, inhibition of cardiomyocyte senescence led to reduced senescence-associated in ammation and decreased senescence-associated markers within other myocardial lineages, consistent with the hypothesis that cardiomyocytes promote pathological remodeling by spreading senescence to other celltypes. Collectively this study presents a novel demonstration that senescent cardiomyocytes are major contributors to myocardial remodeling and dysfunction following a myocardial infarction. Therefore, to maximize the potential for clinical translation, it is important to further understand the mechanisms underlying cardiomyocyte senescence and how to optimize senolytic strategies to target this cell lineage.

Introduction, Results, Discussion
Myocardial infarction (MI), is the leading cause of death and disability in developed countries. 1 Even, with reperfusion therapy, pathological myocardial remodelling can impact patient health by progressively impairing cardiac function, leading to heart failure. 2 Several independent studies have shown that MI causes senescence in numerous myocardial cell types, including cardiomyocytes (CMs), broblasts and endothelial cells 3,4 (reviewed; 5,6 ). Post-MI treatment with mechanistically diverse senolytic compounds, reduces senescent cell number, decreases in ammation, and improves heart function, suggesting that senescence contributes to disease pathophysiology. 4,7 Although the role of broblast senescence has been studied in this disease setting 8 it remains unclear if senescent CMs actively participate in disease pathophysiology. Indeed, it has been proposed that as CMs lack a meaningful regenerative capacity, 9,10 their elimination could in fact be detrimental and senotherapies improve outcomes despite, rather than as a result of CMs apoptosis. 6,7 Moreover, while senolytic treatment improves outcome following MI, studies have failed to address the possibility that the observed bene ts are due to non-myocardial senescent cell elimination or peripheral off-target effects. 6,7 For example, the molecular pathways in uenced by senolytics are not uniquely expressed by senescent cells 11 and non-resident myocardial cell populations which contribute to remodelling, including T-lymphocytes 12,13 and platelets, 14 are in uenced by senolytic treatment. 15,16 To continue the development of effective senotherapies, it's imperative that the contribution of individual senescent cell populations to disease progression is understood.
The cyclin dependant kinase inhibitor p16 plays a key role in regulating CM senescence. p16 is increased in CMs with age and in response to myocardial infarction. 4,17 Furthermore, in aged INK-ATTAC mice, pharmacogenetic clearance of p16, reduces the percentage of CMs expressing alternative markers of senescence, reducing CM hypertrophy, and improving heart function. 17 Therefore, to investigate the speci c role of CM senescence in disease pathophysiology following MI with reperfusion we employed a transgenic model that allows the CM speci c inactivation of CDKN2A (which encodes p16). 4 CM-p16 KO and CM-p16 WT mice were subjected to cardiac ischaemia reperfusion (IR) and assessed 5 weeks postsurgery ( Fig. 1a and b; supplementary experimental procedures). No animals died post-surgically in either group, and no differences in weight were observed between groups both pre-and post-IR ( Supplementary   Fig. 1). At 5 weeks the peri-infarct region of CM-p16 KO mice contained signi cantly fewer p16-expressing CMs compared to CM-p16 WT controls, indicating that p16 was effectively knocked out in CMs ( Fig. 1c and d). CMs expressing p21 (a senescence marker) were also reduced in the peri-infarct region of CM-p16 KO mice compared to CM-p16 WT , but to a lesser extent than p16 ( Fig. 1e and f). Additionally, a decrease in senescence associated secretory phenotype (SASP) proteins with described roles in myocardial remodelling 18 was also observed in the myocardium of p16-CM KO mice ( Fig. 1g and h). IR induces telomere foci of DNA damage (TAF) within CMs and TAF can trigger activation of p16 and p21 associated senescence pathways. 17 As would be expected given that TAF induction is upstream of p16 activation there was no difference in mean TAF number or the percentage of CMs with TAF between the two groups. This is also suggestive that the degree of insult and stress as a result of IR was similar between the two groups ( Fig. 1i and j). These data indicate that, while p16 independent senescence may still occur in the CMs following inactivation of p16 INK4 , CM senescence was dampened in the CM-p16 KO model.
To determine if senescent CMs contribute to maladaptive myocardial remodelling, and therefore impaired cardiac function after IR, the mice were analysed using cardiac magnetic resonance imaging (MRI) ( Fig.   2a and b). At 5 weeks post-IR CM-p16 KO mice had a signi cantly higher ejection fraction compared to CM-16 WT littermates (Fig. 2b). This was attributed to improved preservation of left ventricular (LV) systolic volume, as no signi cant difference in LV end diastolic volume was observed between the groups. A trend in improved stroke volume was also evident in CM-p16 KO mice ( Supplementary Fig. 2).
Senescence has been previously linked to pathological CM hypertrophy. 17,19 We therefore aimed to identify if improved maintenance of cardiac function was a result of the attenuation of pathological hypertrophy in the CM-16 KO hearts. No differences were observed in hypertrophy measured at an organ or cellular level as LV mass indexed to tibia length and mean CM area were consistent between experimental groups (Fig. 2b, c and d). This data suggests that, in the acute setting, senescence is not a leading driver of hypertrophy and alternative pathways, for example the angiotensin II and the reninangiotensin-aldosterone system pathways may be more important. 20 Interestingly, scar size was signi cantly reduced in CM-p16 KO mice, suggesting that CM senescence contributes to this aspect of pathological remodelling after IR (Figure e and f). This data together with the observed reduction in SASP expression in the myocardium of p16-CM KO mice led us to hypothesise that CM senescence and SASP has a paracrine in uence on surrounding non-CM cell populations. Supporting this hypothesis, CM-p16 KO mice demonstrated a signi cant reduction in p16 expressing interstitial cells and reduced senescence associated-β-galactosidase staining in the myo broblast rich area of infarct at 5 weeks post IR (Fig. 2g, h  and i). Furthermore, studies have demonstrated that senescence induction promotes myo broblast differentiation and enhances collagen deposition. 8, 21 This, together with our current study provides a mechanism by which senescent CMs promote scar formation and cardiac dysfunction following IR.
Collectively, this study demonstrates for the rst time, that following IR, CM senescence is detrimental to cardiac function and promotes myocardial remodelling, adding to the growing body of evidence which suggests that post-mitotic cell senescence plays a crucial role in the pathophysiology of various diseases. The data also indicates that this is a result of SASP expression, which induces paracrine senescence and promotes brosis and scar production. This suggests a more in-depth study of CM SASP could uncover unique biomarkers that are more accurate in predicting myocardial dysfunction compared to SASP from other cell types found across multiple organ systems. In conclusion, it is crucial that future therapeutic approaches consider the signi cant contribution of senescent CMs to myocardial remodelling and dysfunction.