Sudden cardiac death (SCD) remains the leading cause of death worldwide and is responsible for up to 20% of all deaths in the USA (Benjamin et al., 2019; Deo and Albert, 2012). The incidence of SCD increases exponentially with age, which poses a growing problem as the portion of the US population 65 years or older is estimated to increase to 15% by 2030 (Benjamin et al., 2019; Partridge et al., 2018). The vast majority of SCDs are caused by ventricular tachycardia/ventricular fibrillation (VT/VF) subsequent to myocardial infarction (MI) (Kolettis, 2013). Immediate clinical reperfusion interventions can limit the extent of acute ischemic injury and acute arrhythmic mortality post-MI, however progressive tissue remodeling in the days-to-years post-MI can establish a substrate and trigger for potentially lethal arrhythmias (Mendonca Costa et al., 2018). This tissue remodeling process is well described in young animals (van den Borne et al., 2010), but less is known about what changes to this process occur with age and how these changes increase risk of arrhythmia initiation and propagation.

We have previously characterized the efficacy of the New Zealand White rabbit as a model of the aging human heart. We showed that the aging rabbit heart recapitulates cardiac dysfunctions observed in aging humans, including aortic stiffening, reduced ventricular compliance, increased interstitial fibrosis, abnormal conduction in the His-Purkinje system, and lower thresholds for induced ventricular arrhythmias (Cooper et al., 2012). We also demonstrated defects in autophagy and mitochondrial function in isolated aged rabbit myocytes resulting in increased mitochondrial reactive oxygen species which oxidized the ryanodine receptor and promoted spontaneous Ca²⁺ release (Cooper et al., 2013; Murphy et al., 2019). To better investigate the interaction between post-MI tissue remodeling and arrhythmogenesis with age, we have established a minimally invasive surgical procedure to reproducibly infarct the rabbit heart regardless of age or variable coronary anatomy, whereby an embolic coil occludes the left coronary artery (Morrissey et al., 2017). Infarction by this method results in electrophysiological remodeling characteristic of human MI, including functional reentry anchored at the infarct border zone (IBZ), reduction in ejection fraction, and lower thresholds for monomorphic VT induced by programmed stimulation (Morrissey et al., 2017; Ziv et al., 2012).

Cellular senescence is a stress response characterized by irreversible proliferation arrest, resistance to apoptosis, and secretion of a collection of inflammatory cytokines, growth factors, and proteases termed the senescence-associated secretory phenotype (SASP) (Coppé et al., 2010; Rodier et al., 2009; Campisi and Robert, 2014). Transient senescence plays beneficial roles in young animals in wound healing by limiting fibrosis, in tumor suppression, and in development (Wang et al., 2011; Storer et al., 2013; Demaria et al., 2014). However, senescent cells accumulate with age in many tissues and accelerate many aging-associated pathologies in a paracrine manner via SASP-mediated chronic inflammatory signaling and/or in a juxtacrine manner via transfer of intracellular proteins to neighboring cells (Prata et al., 2018; Vicente et al., 2016; Burton and Krizhanovsky, 2014; Shibamoto et al., 2019; Kirschner et al., 2020; Biran et al., 2015). Genetic or pharmacological elimination of senescent cells with age slows the onset and progression of many aging pathologies, including atherosclerosis, diabetes, idiopathic pulmonary fibrosis, osteoarthritis, and neurodegenerative diseases (Baker et al., 2016; Baker et al., 2011; Childs et al., 2015; Zhu et al., 2015; Chang et al., 2016; Kim and Kim, 2019; Kirkland et al., 2017; van Deursen, 2019; Kirkland and Tchkonia, 2017).

In the adult infarcted wild-type mouse heart, senescent cells arise in the scar and IBZ around day 3 post-MI and are largely cleared by day 7 (Shibamoto et al., 2019; Zhu et al., 2013). Most of these senescent cells are cardiac myofibroblasts, the main cell type responsible for secreting extracellular matrix components forming the scar. This observation is consistent with epidermal wound studies in mice, suggesting a role for transient myofibroblast senescence as a normal part of the wound healing process in limiting fibrosis (Demaria et al., 2014). Infarction of adult constitutive p53 KO mice, in which senescence induction is impeded, results in increased fibrosis and decreased inflammation in the scar 7 days post-MI (Zhu et al., 2013). In a cell culture model of isolated mouse cardiac fibroblasts, senescent cardiac fibroblasts limit the proliferation of non-senescent cardiac fibroblasts in a juxtacrine but not paracrine fashion (Shibamoto et al., 2019). These results suggest a beneficial role for transient myofibroblast senescence in limiting fibrosis post-MI. Conversely, insights toward a pathological role of chronic cardiac senescence come from aging mouse models of MI. Treatment of aged mice with the senolytic drug navitoclax (i.e. a drug that specifically eliminates senescent cells) before or after MI significantly reduces senescence burden, improves cardiac function, mitigates cardiac remodeling, and reduces scar size post-MI, although the effects on arrhythmias has not been studied to our knowledge (Dookun et al., 2020). These results suggest that acute induction of senescence followed by timely clearance limits excess scarring, but persistence of cardiac senescence as seen with age can promote cardiac fibrogenesis and worsen cardiac function and might lead to increased risk of arrhythmias. Although these findings in mice are important, the mechanisms underlying a relationship between age-associated senescence and arrhythmogenesis is not well understood, particularly in an animal model like the rabbit whose cardiac electrophysiology is more functionally relevant to humans than that of mice.

Potential pro-arrhythmic effects of senescent cells could occur through a paracrine fashion via SASP components. Indeed, treatment of isolated rat and mouse cardiomyocytes with exogenous SASP components including IL-6, IL-1β, and TNF-α have pro-arrhythmic effects in ion channel remodeling (Francis Stuart et al., 2016; Aromolaran et al., 2018). Senescent myofibroblasts might be able to interfere with cardiomyocyte electrophysiology from relatively long distances through chronic inflammatory signaling. Alternatively, the direct cell-cell coupling via gap junctions of senescent myofibroblasts to cardiomyocytes at the IBZ might alter their electrophysiology more than coupling with non-senescent myofibroblasts. Such a process would establish regional heterogeneities in ion channel activity, action potential duration (APD), and other electrophysiological factors anchored at the IBZ that would permit reentrant current and therefore VT/VF.

We hypothesized that aged infarcted rabbits experience a persistence of senescent myofibroblasts in the scar and IBZ compared to young, and that these senescent myofibroblasts act in a paracrine or juxtacrine fashion to induce pro-arrhythmic remodeling in IBZ cardiomyocytes. Here, we demonstrate that aged rabbits compared to young exhibit increased peri-procedural deaths mostly due to VT/VF, consistent with prolongation of APD and alternans at the IBZ associated with higher frequency VF. We observed no difference in the size of the scar or IBZ geometry over the first 12 weeks post-MI between young and aged rabbits. However, whereas senescent cells in the young rabbit scar were largely cleared by 3 weeks post-MI, senescence of mostly myofibroblasts remained high in the aged rabbits up to 12 weeks post-MI and correlated with increased local expression of inflammatory cytokines. In ventricular tissue samples from aged infarcted human patients, we observed elevated levels of senescence markers, and most senescent cells appeared to be myofibroblasts. Using primary adult rabbit cardiac fibroblasts, we induced senescence via treatment with the drug etoposide. With this method, we did not observe any effect of exogenous or co-cultured conditioned media from senescent cardiac fibroblasts on the I_Kr current or APD of treated rabbit myocytes compared to conditioned media from proliferating cardiac fibroblasts. However, in both aged rabbit and human cardiac tissue, we observed senescent cardiac myofibroblasts in close proximity to IBZ myocytes, with the gap junction protein Cx43 appearing to couple the cells. Previous studies in rabbits have demonstrated a role for Cx43 in the coupling of myocytes and fibroblasts as evidenced by dye transfer and optogenetic studies (Camelliti et al., 2004a; Camelliti et al., 2004b; Schultz et al., 2019). Based on our electrophysiological and volumetric measurements of senescent and non-senescent fibroblasts, we performed computational modeling which suggested that coupling of myocytes with senescent cardiac myofibroblasts would result in prolongation of APD and lower thresholds for conduction block. Altogether, our results indicate a pathological role for the persistence of senescent cardiac myofibroblasts in directly exacerbating arrhythmogenesis at least via a direct cell-cell interaction through gap junctions with age post-MI. These findings suggest that targeted elimination of senescent cells post-MI could be an effective therapeutic method to mitigate senescence burden and combat arrhythmias in aged infarcted individuals.

Although the age-associated accumulation of senescent cells and the related detriments to cardiac function and chronic inflammation have been previously described (Walaszczyk et al., 2019), the relationship between senescence arising during and after scar formation post-MI and arrhythmogenesis is not well understood. Our experiments show that accumulation of senescent myofibroblasts in the scar is associated with a tissue microenvironment more susceptible to cardiac arrhythmias and elevated inflammatory signaling. Additionally, our findings suggest that senescent myofibroblasts in the IBZ may be exerting pro-arrhythmic effects on surviving myocytes in a cell-cell interaction mediated by myocyte-fibroblast coupling via gap junctions. We propose that persistence of senescent myofibroblasts is a major driver of arrhythmogenic tissue remodeling with age post-MI, and that the timely pharmacological elimination of senescent cells might alleviate risk of arrhythmias particularly in elderly infarcted patients.

From our MI surgical procedures, we observed significantly increased mortality of aged rabbits mostly due to peri-procedural VT/VF (Figure 1). These results are consistent with the age-associated increase in out-of-hospital mortality from cardiac arrhythmias resulting from MI (Benjamin et al., 2019). The reason for this increase in peri-procedural mortality with age is likely related to our previous observations of increased interstitial fibrosis, disorganization of the His-Purkinje system, and altered myocyte Ca²⁺ handling with age in non-infarcted aged rabbits, in that these age-associated changes likely increase peri-procedural mortality by lowering the threshold for arrhythmias triggered by the acute ischemic insult (Cooper et al., 2012; Murphy et al., 2019; Terentyev et al., 2014). Based on our previous observations showing low levels of senescence in non-infarcted young and aged rabbits (not shown), we posit that senescence likely does not play a role in peri-procedural arrhythmias.

The anatomical characteristics of the scar, including scar size, functional IBZ area, and dense and interstitial fibrosis, could influence age-associated differences in arrhythmogenesis. However, the fact that we did not observe any significant difference in the timecourse of scar size, IBZ area, and overall or interstitial fibrosis suggests that age-associated changes in other cellular processes, including cellular senescence, are driving the increase arrhythmogenicity that we observe from optical mapping studies. Our findings of overall scar size are consistent with our previous study showing 30% infarcted area of the left ventricle from our MI surgical procedure, and the present data suggest no epicardial infarct expansion occurs over time in young or aged rabbits. (Morrissey et al., 2017).

Electrophysiological remodeling at the IBZ includes separation of myocyte bundles via interstitial and replacement fibrosis and significant disruptions of APD and reductions in conduction velocity, myocyte-myocyte coupling, and anisotropy ultimately permitting unidirectional conduction block, sustained reentry, and subsequent VT/VF (Kolettis, 2013; Rutherford et al., 2012; Di Diego and Antzelevitch, 2011). Although these changes are well characterized in young animal models, we sought to investigate age-associated changes in these processes and their underlying mechanisms. From our optical mapping studies, we observed aged rabbit hearts 3 weeks post-MI exhibited more extreme arrhythmogenic remodeling at the IBZ compared to young, including relative prolongation of APD as compared to the RZ, spatially discordant alternans near the IBZ at relatively slow heart rate, and higher frequency of sustained spontaneous VF following burst pacing. These pathological characteristics indicate that aged rabbits undergo remodeling at the IBZ differently than young animals, culminating in a stronger substrate for arrhythmias anchored at the IBZ. Specifically, prolonged APD near the IBZ has been shown to facilitate a reentrant circuit around the scar (Campos et al., 2019). Meanwhile, in young rabbits, the significantly shorter APD at the IBZ compared to the RZ and scar can also induce and maintain arrhythmia through increasing APD dispersion and AP conduction block. This finding of shortened APD in the border zone of young rabbits is consistent with previous data using relatively young Yucatan minipigs (Hegyi et al., 2018). However, young infarcted rabbits experience less arrhythmias than aged infarcted rabbits, as indicated by the lack of observed voltage alternans at slow heart rate and slower VF frequency. The observation of similarly slower conduction velocity at the IBZ relative to the RZ in both young and aged rabbits is consistent with our histological observations showing no difference in the number or length of IBZ protrusions (Figure 2B).

The accumulation of senescent cells in many tissues has been shown to exacerbate age-associated pathologies via paracrine or juxtacrine mechanisms. Studies in mice have demonstrated increased senescence in the scar post-MI with age, the pharmacological clearance of which limits scar size and improves cardiac function and outcome (Walaszczyk et al., 2019). However, little is known about whether these findings translate to large animal models with more electrophysiological relevance to the human heart, or the mechanisms through which senescent cells might promote arrhythmogenesis in the aged infarcted heart. Here, we show that cellular senescence as measured by SA-β-gal and γH2AX staining between young and aged rabbits is similar for the first 2 weeks post-MI but persists in aged rabbits at 3 and up to 12 weeks post-MI in the scar (Figure 4A–E) and is associated with higher local expression of inflammatory SASP factors (Figure 4H). Consistent with previous findings from our group and others (Zhu et al., 2013), we found very few SA-β-gal-positive cells in the RZ of young or aged rabbits. These data, along with our previous results assessing young and aged non-infarcted rabbits, suggest a low baseline of senescence in the normal young and aged rabbit heart which is reasonable considering the relatively low proliferation of myocardial cells under baseline conditions. The similarly low levels of SA-β-gal in young and aged rabbits 2 weeks post-sham infarction indicate that the age-associated significant difference in senescence post-MI was not an artifact of the MI procedure. Additionally, we did not observe any evidence of senescent cardiomyocytes in young or aged rabbits at any timepoint (Figure 4C, bottom) as well as from human ventricular tissues samples from SA-β-gal staining, which differs from previous reports of mouse tissue (Anderson et al., 2019). This important observation suggests that the rabbit heart could be a more appropriate animal model than the mouse to study the effects of cardiac aging relevant to humans. Interestingly, we observed no difference in either total fibrosis of the LV free wall or interstitial fibrosis over time between young and aged rabbits even though we observed persistence of senescence in the aged rabbit heart over time compared to young. It is well known that the content of the SASP changes over time and depending on cell type and the induction source, and so it is possible that pro-fibrotic and anti-fibrotic SASP factors are present in functionally equivalent amounts ultimately resulting in no difference in fibrosis. This hypothesis is supported by our RT-qPCR data, indicating upregulation in expression of the pro-fibrotic factors TGF-β, TIMP1, and TIMP2 as well as anti-fibrotic factor MMP9. Further investigation into this hypothesis is warranted.

From our immunofluorescence staining, the high degree of αSMA signal in young and aged rabbits at 1 and 2 weeks post-MI and the subsequent decrease in αSMA signal is consistent with genetic lineage tracing studies in mice which suggest that myofibroblasts remain present in the scar but lose αSMA expression (Fu et al., 2018). Our observation that the percent of γH2AX+ cells that are αSMA+ remains high at 2 weeks post-MI when αSMA signal begins to decline suggests that most of the senescent cells present in the scar are αSMA+ myofibroblasts that lose αSMA expression after 2 weeks post-MI. This observation is consistent with previous studies characterizing the identity of senescent cells in the scar post-MI in mice (Zhu et al., 2013; Meyer et al., 2016). The relatively lower percent of CD31+ cells at any timepoint between young and aged rabbits is consistent with previous reports of cellular composition in the scar (Pinto et al., 2016). The small percent of γH2AX+ foci that were CD31+ reinforces our conclusion that myofibroblasts are the majority of cells undergoing senescence in the young and aged heart post-MI.

Our RT-qPCR data of young and aged rabbits 3 weeks post-MI further demonstrates differences between the senescence profiles of the aged rabbit and the aged mouse. Consistent with our SA-β-gal and γH2AX data, we observed an increase in the expression of p16, p21, and p53 in aged rabbits compared to young. However, the only SASP factors for which we measured a significant change in expression in the scar zone between young and aged were CXCL2 and TIMP2, whereas the contents of the cardiac SASP were largely different between young and aged mice (albeit with a non-canonical SASP) (Anderson et al., 2019). Given these results, special attention should be placed on conclusions for the relevance of senescence studies performed in mice in regard to their clinical relevance for humans. Similar to our SA-β-gal data, we observed a similar but lower magnitude of changes in overall expression between the young and aged rabbit IBZ, and no differences in senescence and SASP gene expression between the young and aged RZ. The significant decrease in IL-6 expression in the aged IBZ compared to young IBZ is interesting, however senescence and SASP signatures are known to be variable depending on cell line and senescence induction method, and this decrease in IL-6 expression has also been observed in studies of young and aged non-infarcted murine myocytes (Anderson et al., 2019). Overall, our data suggest that senescence and the inflammatory SASP is largely confined to the scar.

To investigate the clinical relevance of our observations of persistent senescence in the aged infarcted rabbit heart, we measured senescence in aged infarcted human ventricular frozen sections. We observed a relatively high percent of cells in the scar exhibited nuclei with three or more γH2AX foci (Figure 5C), indicating the presence of senescent cells in the human tissue, which may contribute to arrhythmogenic tissue remodeling leading to increased incidence of potentially lethal arrhythmias in the chronic phase post-MI. These γH2AX senescence data are similar to what we observed in the aged rabbit 12 weeks post-MI. From our αSMA analysis of human tissue, we observed a high percent of cells were αSMA+ unlike the progressive decrease in αSMA+ cells over time that we observed in the young and aged rabbits. Because the specific timing of MI is difficult to diagnose especially out-of-hospital, we are unable to ascertain how long post-MI the samples were at time of death. If senescent cells indeed persist in the aged human scar as we hypothesize, it is possible that growth factors like TGF-β in the chronic SASP activate fibroblasts in the months-to-years post-MI. Alternatively, the content of the SASP could differ from what we observe in rabbits, contributing to differential activation of myofibroblasts. Similar to our observations in young and aged rabbits, our γH2AX and αSMA double immunofluorescence assays indicate that the majority of cells with three or more γH2AX nuclear foci were also αSMA+, further suggesting that myofibroblasts are the predominant cell type undergoing senescence. Relatively fewer cells with three or more γH2AX nuclear foci were either vWF+ or CD68+, consistent with previous reports in mice, suggesting relatively fewer senescent cells are endothelial cells or macrophages, respectively (Zhu et al., 2013).

To better study the mechanisms by which senescence might be promoting arrhythmias with age post-MI, we established an in vitro method to induce senescence in adult rabbit cardiac fibroblasts. Low doses of the drug etoposide induced senescence, measured by SA-β-gal and γH2AX staining as well as RT-qPCR in a time-depending manner, with the vast majority of cells positive for senescence markers after 12 days of treatment with 20 μM etoposide (Figure 6). These data establish a platform to future studies screening the efficacy of senolytic drugs to eliminate senescent cells post-MI.

To investigate whether senescent cells might have a paracrine effect on myocytes, we treated primary isolated myocytes from 3-week-old rabbits with conditioned media harvested from proliferating (proliferating-CM) or senescent (SASP-CM) fibroblasts (Figure 7). From our patch clamping studies, we observed no significant difference in I_Kr current or in APD between proliferating-CM or SASP-CM groups. Separately, we co-cultured isolated myocytes with proliferating or senescent cardiac fibroblasts for longer durations and also found no significant difference in I_Kr or APD. These findings suggest that senescent rabbit fibroblasts likely do not act in a paracrine manner to disrupt the electrophysiology of rabbit myocytes, but further study is needed to verify this. Because we are limited by the number of commercially available assays to determine the specific contents of the rabbit SASP, we are unable to exclude the possibility that the conditioned media lacked physiologically relevant levels of SASP factors to induce a significant effect. Besides, components in the media used or the absence thereof might have blunted a paracrine effect. One could also envisage that degradation of conditioned media occurred between harvest and treatment, as well as potential issues with incubation period and responsiveness of the 3-week-old myocytes to the SASP. Additionally, transfer of the cells from the conditioned media to the patch clamping media might have allowed for rapid recovery of the cardiomyocytes prior to patch clamp recording, which would not be detectable from the data presented.

We next investigated whether senescent fibroblasts might directly couple with myocytes at the IBZ to influence pathological electrophysiological remodeling. Homo- and hetero-typic coupling occurs in the healthy and diseased heart via connexins, including Cx43 (Kohl and Gourdie, 2014). We observed a significantly higher percentage of senescent cells neighboring IBZ myocytes in aged rabbits 3 weeks post-MI compared to young. Our immunofluorescence studies suggest that Cx43 facilitates homocellular coupling between senescent adult rabbit cardiac fibroblasts, as well as heterocellular coupling between senescent myofibroblasts and myocytes at the IBZ of aged rabbits and human frozen sections.

Our computational modeling of proliferating and senescent myofibroblasts as well as simulations of cardiomyocyte-myofibroblast interactions in young and aged tissue shed critical light for explaining mechanisms by which senescent myofibroblasts might promote arrhythmogenic tissue remodeling with age post-MI. Although electrical remodeling in the IBZ is a complex phenomenon with multiple contributing factors, our results indicate that myocyte-fibroblast interaction becomes a potential contributor to arrhythmogenesis in the aged MI condition. A large, flattened morphology is one of the hallmark characteristics of senescent cells in other models (Kuilman et al., 2010), and we observed this for cardiac myofibroblasts both in vitro and in vivo. The larger size of senescent myofibroblasts likely explains the greater extrapolated values for in vivo capacitance, and all differences in modeling between proliferating and senescent myofibroblasts are driven by the larger capacitance of senescent myofibroblasts. Importantly, the prolongation in APD from our modeling of aged condition relative to young is consistent with our optical mapping findings from young and aged rabbits 3 weeks post-MI and validates the relevance of our modeling. From our S1S2 modeling under young and aged conditions, we would predict that aged rabbits would have higher risks for conduction block than young rabbits. Although we did not observe a significant difference in the CL at which VF was induced, VF frequencies were higher and heterogeneous in the IBZ (Figure 3D), suggesting that the prolongation of APD in myocytes coupled to senescent myofibroblasts would result in lower threshold for conduction block and wavebreak, overall driving faster VF frequency. These observations are consistent with our hypothesis that myocyte coupling to senescent myofibroblasts in the IBZ of aged rabbits leads to pro-arrhythmic electrophysiological remodeling and increases risk of reentrant current anchored to the IBZ.

In summary, we have characterized an age-associated accumulation of senescent myofibroblasts in the aged infarcted rabbit heart, and our findings suggest that these senescent cells remodel the electrophysiology of IBZ myocytes at least via direct myocyte-fibroblast coupling mediated by Cx43, which would increase risk of cardiac arrhythmias. Previous studies have shown that pharmacological elimination of senescent cells with age via the senolytic drug navitoclax limits cardiac fibrosis in infarcted and non-infarcted mice and improves the survival and cardiac function of infarcted young and aged mice, but little is known about the effects of senescence on arrhythmogenesis with age (Walaszczyk et al., 2019; Anderson et al., 2019). Our findings demonstrate an important role of persistent senescence in arrhythmogenesis with age post-MI, suggesting the use of senolytic therapy to mitigate senescence burden with age might be promising strategy to mitigate chronic arrhythmias in aged infarcted individuals.

All data generated or analyzed during this study are included in the provided Source Data file.

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Decision letter after peer review:

Thank you for submitting your article "Myofibroblast Senescence Promotes Arrhythmogenic Remodeling in the Aged Infarcted Rabbit Heart" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Paul Delgado Olguin (Reviewer #1).

Both reviewers have responded positively to the overall science. Reviewer 1 considers that you have presented important results and convincing evidence linking myofibroblast senescence in the aged heart with a pro-arrhythmogenic phenotype and higher mortality after myocardial infarction in the aged rabbit heart. He/she does comment that the evidence supporting the contention that senescent myofibroblasts promote arrhythmia when coupled with cardiomyocytes is incomplete and would benefit from experimentally testing the in-silico model. He also comments that stronger evidence of coupling of myofibroblasts and cardiomyocytes linked to arrhythmia would raise interest from basic and clinician scientists studying cardiac function and disease. Reviewer 2's comments are of a more technical and presentational nature.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The reviewers make the following scientific comments which we would request you address:

Reviewer 1's comments concern the detailed interpretation of the results:

1) The cells in Figure 4F and G, do not appear to correspond to either myofibroblasts or endothelial cells. Given that γH2AX+ cells are significantly increased in the aged heart, could the results presented suggest that a different cell type might be more important for the aged heart's response to MI? Providing some insight into the identity of these cells would be helpful to better understand the results presented. For example, cardiomyocyte senescence could contribute to arrhythmic phenotypes.

2) The results presented show that treatment of cardiomyocytes with conditioned media from, and co-cultured with, senescent myofibroblasts did not change action potential duration in cardiomyocytes. This led to the conclusion that paracrine signalling is unlikely to contribute to a pro-arrhythmogenic phenotype. It is possible that cardiomyocytes do couple with myofibroblasts in the in vitro system used. In which case, the results presented would not favor the proposed model.

3) Another important possibility to be considered is that myofibroblasts might not have produced senescence-associated secretory phenotype-mediators at concentrations high enough to alter action potential duration in the conditions tested. Experimental evidence of the levels of selected mediators of the senescence-associated secretory phenotype in conditioned media would help assess a potential paracrine effect.

4) The evidence of coupling, i.e., the presence of connexin-43 in the interphase between αSMA+ and cardiomyocytes needs to be strengthened. Perhaps analyzing Z-stack 3D reconstructions would help to better define adjacent cells and more precisely reveal the localization of connexin-43.

Reviewer 2 has more technical issues to raise:

1) Some reflection on the media used to study paracrine effects is needed – more experiments here would be beneficial.

2) Patch clamp experiments – how does bath solution alter the effect of any limited paracrine effect – we are removing cells from the treatment media and putting them in physiological solutions – an opportunity to recover?

Essential revisions:

The reviewers make the following scientific comments which we would request you address:

Reviewer 1's comments concern the detailed interpretation of the results:

1) The cells in Figure 4F and G, do not appear to correspond to either myofibroblasts or endothelial cells. Given that γH2AX+ cells are significantly increased in the aged heart, could the results presented suggest that a different cell type might be more important for the aged heart's response to MI? Providing some insight into the identity of these cells would be helpful to better understand the results presented. For example, cardiomyocyte senescence could contribute to arrhythmic phenotypes.

Mouse lineage tracing studies have shown that myofibroblasts persist in the infarct border zone for long periods of time, however they lose aSMA expression up to at least 4 weeks post-MI (Kanisicak 2018 J Clin Invest, PMID 29664017). We suspect the gH2AX+ aSMA- cells are myofibroblasts that have lost aSMA expression. We attempted to verify this using stainings for other myofibroblast-specific markers (including vimentin, PDGFRα, DDR2, and Tcf21), but such stainings were not successful due to limited availability of verified primary antibodies against rabbit tissue for immunofluorescent microscopy. We did not observe evidence of cardiomyocyte senescence through SABgal staining or gH2AX staining.

2) The results presented show that treatment of cardiomyocytes with conditioned media from, and co-cultured with, senescent myofibroblasts did not change action potential duration in cardiomyocytes. This led to the conclusion that paracrine signalling is unlikely to contribute to a pro-arrhythmogenic phenotype. It is possible that cardiomyocytes do couple with myofibroblasts in the in vitro system used. In which case, the results presented would not favor the proposed model.

It is possible that cardiomyocytes do couple electrically with myofibroblasts in the in vitro system used. However, we have found no evidence of coupling in our co-culture assay. We recorded transient capacitive currents during a hyperpolarizing voltage step (-80 to -100 mV) in cardiomyocytes cultured on top of senescent myofibroblasts, as well as in single cardiomyocytes cultured alone. We fit the transient current decay with a two-exponential decay function. If a cardiomyocyte and myofibroblast were coupled, we would expect that the capacitive transient would have a significantly larger slow time constant and greater total capacitive charge (measured as an integral of the capacitive transient) than in a single myocyte in the absence of myofibroblasts. This is because in the coupled cells the total electric capacitance of cell membranes is larger, and the electrical access resistance of gap junctions to the myofibroblast is significantly higher than the pipette access resistance to the cardiomyocyte membrane. However, we found no significant difference in time constants between these two groups. A similar analysis was done to show coupling of descending vasa recta endothelial cells (Zhang 2006 Am J Physiol Regul Integr Comp Physiol, PMID 16840652). Furthermore, when we added 100 mM carbenoxolone to block gap junctions in the co-culture system, there was no significant difference in the slow time constants between the two groups of cells, indicating no significant electrical connections between the cardiomyocytes and the fibroblasts. A sentence mentioning this analysis has been added to the manuscript (lines 468-471).

3) Another important possibility to be considered is that myofibroblasts might not have produced senescence-associated secretory phenotype-mediators at concentrations high enough to alter action potential duration in the conditions tested. Experimental evidence of the levels of selected mediators of the senescence-associated secretory phenotype in conditioned media would help assess a potential paracrine effect.

This is possible, or alternatively the long-term effect of relatively low concentration paracrine signaling, as expected from aged infarcted animals, might have an effect on cardiomyocyte electrophysiology. Previous studies in mice and rats have indicated that exogenous administration of individual cytokines found in the SASP can alter the activity of ion channels and prolong cardiomyocyte APD (Stuart 2016 J Mol Cell Cardiol PMID 26739214). These are important questions to address to fully understand the spatiotemporal contribution of senescence towards inflammation and arrhythmias, however the results presented in this manuscript are not meant to rule out a paracrine effect. Further investigation is needed to characterize nuances in potential paracrine and juxtacrine effects but is outside the scope of this manuscript. Of note, other investigators reported that IL-6 may block I_Kr (Chowdhury 2021 Int J Mol Sci, PMID 34681909; Aromolaran 2018 PLoS One, PMID 30521586). Given that the conditioned media had no significant effects on I_Kr current density and APD, we feel that assessing the low levels of multiple cytokines in the media will not be productive.

4) The evidence of coupling, i.e., the presence of connexin-43 in the interphase between αSMA+ and cardiomyocytes needs to be strengthened. Perhaps analyzing Z-stack 3D reconstructions would help to better define adjacent cells and more precisely reveal the localization of connexin-43.

We appreciate the reviewer’s suggestion. Surely, the reviewer is aware of several elegant studies by Mahoney et al. (Mahoney 2016 Sci Rep, PMID 27244564), Quinn et al. (Quinn 2016 Proc Natl Acad Sci U S A, PMID 27930302), and Rubart et al. (Rubart 2018 Cardiovasc Res, PMID 29016731) that demonstrated electrical coupling between cardiomyocytes and myofibroblasts (nonmyocytes) in the infarcted mouse heart. The latter report also provided 3D reconstructions of fluorescence confocal images that were acquired from infarct border zones. Here (Figure 6B-D), the authors presented convincing data that showed the expression of connexins 43 and 45 between myocytes and myofibroblasts. As confocal imaging requires a compromise between resolution, scan time, and photobleaching of the samples, we decided to choose a high enough resolution (approximately 200um width, 200um height, 6um depth) to create informative 2D images (the juxtaposition of cardiomyocytes and senescent myofibroblasts). This information was added to the methods section, (lines 878-879). However, the resolution chosen is not high enough for any convincing 3D reconstruction as we felt at that time that an in-depth study of the aforementioned already established heterocellular coupling would not add any new information to the manuscript.

However, we would like to present the reviewer additional confocal images that show the presence of Cx43 between cardiomyocytes and myofibroblasts (indicated by yellow circles) confirming the aforementioned observations made by Rubert et al. in a larger animal. These additional images have been included in the manuscript as Figure 8—figure supplement 1 (line 541). Our data would also somewhat corroborate an earlier finding (Camelliti 2004 Circ Res, PMID 14976125) that reported coupling between neighboring fibroblasts and myocytes in rabbit sinoatrial node as evidenced by dye transfer.

Reviewer 2 has more technical issues to raise:

1) Some reflection on the media used to study paracrine effects is needed – more experiments here would be beneficial.

In our experience, rabbit cardiomyocytes deteriorate over course of hours in unusual or substandard media. Standard cardiomyocyte growth media used was DMEM + 7% FBS + 1X Pen/Strep, and standard cardiac fibroblasts growth media used was DMEM-F12 + 10% FBS + 1X Pen/Strep. Standard cardiomyocyte growth media as described in the methods section (lines 952-953) was used to co-culture the myocytes and fibroblasts 24-30 hours before the patch clamping. The discussion was revised to include reflection on how the media used might have masked a paracrine effect (lines 762-763), however a several-hour co-culture in substandard media might induce stress in cardiomyocytes that might also introduce artefacts.

2) Patch clamp experiments – how does bath solution alter the effect of any limited paracrine effect – we are removing cells from the treatment media and putting them in physiological solutions – an opportunity to recover?

Correct, the patch clamping media differed from the co-culture media, although the patch clamping media was introduced shortly before patch clamping. Any possible rapid recovery of cardiomyocytes immediately before patch clamping would therefore not be detected in our experiments. However, we believe that such a scenario is unlikely. This this addressed in the revised discussion (lines 765-767).

Author details

1. Brett C Baggett

   1. Brown University, Providence, United States
   2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Contributed equally with

   Kevin R Murphy and Elif Sengun

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2227-0004

2. Kevin R Murphy

   1. Brown University, Providence, United States
   2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Brett C Baggett and Elif Sengun

   Competing interests

   No competing interests declared

3. Elif Sengun

   1. Brown University, Providence, United States
   2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States
   3. Department of Pharmacology, Institute of Graduate Studies in Health Sciences, Istanbul University, Istanbul, Turkey

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing

   Contributed equally with

   Brett C Baggett and Kevin R Murphy

   Competing interests

   No competing interests declared

4. Eric Mi

   1. Brown University, Providence, United States
   2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. Yueming Cao

   1. Brown University, Providence, United States
   2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Data curation

   Competing interests

   No competing interests declared

6. Nilufer N Turan

   Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Resources, Data curation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Yichun Lu

   Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Resources, Data curation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

8. Lorraine Schofield

   Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Resources, Data curation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

9. Tae Yun Kim

   Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

10. Anatoli Y Kabakov

    1. Brown University, Providence, United States
    2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

11. Peter Bronk

    Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9067-2016

12. Zhilin Qu

    School of Medicine, University of California, Los Angeles, Los Angeles, United States

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

13. Patrizia Camelliti

    School of Biosciences and Medicine, University of Surrey, Guildford, United Kingdom

    Contribution

    Conceptualization, Resources, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

14. Patrycja Dubielecka

    1. Brown University, Providence, United States
    2. Department of Hematology, Rhode Island Hospital, Providence, United States

    Contribution

    Conceptualization, Resources, Supervision, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3987-0647

15. Dmitry Terentyev

    Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

    Contribution

    Conceptualization, Resources, Supervision, Writing – review and editing

    Competing interests

    No competing interests declared

16. Federica del Monte

    Medical University of South Carolina, Charleston, United States

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

17. Bum-Rak Choi

    Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Project administration, Writing – review and editing

    Competing interests

    No competing interests declared

18. John Sedivy

    Brown University, Providence, United States

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing, Provided critical input in in vitro experiments and overall experimental design

    Competing interests

    No competing interests declared

19. Gideon Koren

    1. Brown University, Providence, United States
    2. Cardiovascular Research Center, Rhode Island Hospital, Providence, United States

    Contribution

    Conceptualization, Resources, Software, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing, Supervised all experiments and writing of the article

    For correspondence

    gideon_koren@brown.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6211-5837

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