Interleukin-1α dependent survival of cardiac fibroblasts is associated with StAR/STARD1 expression and improved cardiac remodeling and function after myocardial infarction

Highlights

• StAR is a novel myocardial infarction (MI) dependent cardiac fibroblasts (CFs) protein.

• StAR expression is exclusively upregulated by IL-1α, the proximal trigger of the inflammatory response to MI.

• IL-1α primed CFs in culture acquire StAR dependent antiapoptotic resistance.

• In IL-1α deficient mice, MI results in apoptosis of StAR-less CFs, adverse cardiac remodeling and worsened heart function.

• The cardioprotective impact of IL-1α is probably mediated by a novel anti-apoptotic StAR activity.

Abstract

Aims

One unaddressed aspect of healing after myocardial infarction (MI) is how non-myocyte cells that survived the ischemic injury, keep withstanding additional cellular damage by stress forms typically arising during the post-infarction inflammation. Here we aimed to determine if cell survival is conferred by expression of a mitochondrial protein novel to the cardiac proteome, known as steroidogenic acute regulatory protein, (StAR/STARD1). Further studies aimed to unravel the regulation and role of the non-steroidogenic cardiac StAR after MI.

Methods and results

Following permanent ligation of the left anterior descending coronary artery in mouse heart, timeline western blot analyses showed that StAR expression corresponds to the inflammatory response to MI. Following the identification of StAR in mitochondria of cardiac fibroblasts in culture, confocal microscopy immunohistochemistry (IHC) identified StAR expression in left ventricular (LV) activated interstitial fibroblasts, adventitial fibroblasts and endothelial cells. Further work with the primary fibroblasts model revealed that interleukin-1α (IL-1α) signaling via NF-κB and p38 MAPK pathways efficiently upregulates the expression of the Star gene products. At the functional level, IL-1α primed fibroblasts were protected against apoptosis when exposed to cisplatin mimicry of in vivo apoptotic stress; yet, the protective impact of IL-1α was lost upon siRNA mediated StAR downregulation. At the physiological level, StAR expression was nullified during post-MI inflammation in a mouse model with global IL-1α deficiency, concomitantly resulting in a 4-fold elevation of apoptotic fibroblasts. Serial echocardiography and IHC studies of mice examined 24 days after MI revealed aggravation of LV dysfunction, LV dilatation, anterior wall thinning and adverse tissue remodeling when compared with loxP control hearts.

Conclusions

This study calls attention to overlooked aspects of cellular responses evolved under the stress conditions associated with the default inflammatory response to MI. Our observations suggest that LV IL-1α is cardioprotective, and at least one mechanism of this action is mediated by induction of StAR expression in border zone fibroblasts, which renders them apoptosis resistant. This acquired survival feature also has long-term ramifications on the heart recovery by diminishing adverse remodeling and improving the heart function after MI.

Introduction

The first step toward healing after myocardial infarction (MI) is activation of the innate/sterile inflammatory response [1]. The predominant, yet not exclusive, trigger of the inflammatory response is the proinflammatory interleukin-1α (IL-1α) [2,3]. A result of the infarction ischemia is a passive release of the cytoplasmic and nuclear IL-1α content [4] from cardiomyocytes and possibly other cell types undergoing necrotic lysis in the injured tissue [2,3]. Also, IL-1α is only proximal to the MI-related release of additional intracellular substances and extracellular matrix (ECM) degradation products that can trigger the cardiac immune response, collectively called danger associated molecular patterns (DAMPs), or alarmins [1,5]. Immediate cell targets of IL-1α and DAMPs are resident interstitial fibroblasts in the infarct border zone that were spared during the ischemic demise; IL-1α binding to these cells results in a massive synthesis and secretion of more proinflammatory cytokines and chemokines, such as IL-6 and CCL2/MCP-1, which ignite the post-MI inflammation by recruiting immune cells to the site of the injury [1,[6], [7], [8]]. Concomitantly, resident fibroblasts of the infarct border zone are subjected to an additional mode of activation via the mechanical tension generated by loss of tissue volume due the lytic collapse of the necrotic cardiomyocytes. This stress dependent activation of the fibroblasts leads to proliferation, periostin (POSTN) expression, and early differentiation to smooth muscle α-actin (SMA) bearing myofibroblasts, which rapidly repopulate the infarct vacant space [[9], [10], [11]]. Finally, upon resolution of the inflammatory phase, fully differentiated myofibroblasts shape the nature of cardiac remodeling by deposition of collagen type I and other ECM components, so that the resulting scar tissue prevents left ventricular wall rupture [[12], [13], [14]].

An unaddressed aspect of the heart response to MI is the question how the border zone fibroblasts endure through the inflammatory response, when the injured tissue is infiltrated by neutrophils and proinflammatory monocytes/macrophages that release reactive oxygen species (ROS) required for their clearing activities [5,13,15]. We recently revealed that the post-MI injured left ventricular (LV) tissue expresses a protein new to the cardiac protein landscape, the steroidogenic acute regulatory protein, StAR [16]. The working hypothesis underlying the present study aimed to examine if StAR supports cell survival after MI. Originally, StAR discovery [17,18] unveiled a mechanism by which StAR activity provides cholesterol transfer to the inner membrane (IMM) of steroidogenic mitochondria, where the sterol serves as substrate for the synthesis of the first steroid by the IMM mitochondrial P450 enzyme complex of CYP11A1 [19]. In steroidogenic cells of the adrenal cortex and the gonads, the nuclear encoded Star gene is upregulated by trophic hormones and the cAMP signaling pathway [20,21]. In the heart, however, StAR expression cannot support steroidogenesis since CYP11A1 and 3βHSD [19,22], the two obligatory enzymes required for de novo steroid hormone synthesis from cholesterol, are missing [16,23]. We therefore hypothesized that cardiac StAR should have a new alternative activity to be harnessed for a non-steroidogenic function in the heart.

To identify the cell types that express StAR, we applied antibodies to cardiomyocytes-specific troponin I, to fibroblast-specific markers such as platelet derived growth factor receptor-α (PDGFRα) and periostin (POSTN), and endothelial cell marker, CD31/PECAM1 [24]. PDGFRα is required for the generation of the interstitial fibroblast population during development, and remains essential for cardiac fibroblast survival in adult mice [11,25,26]. Periostin is a 90 kDa hetero-functional matricellular protein [27,28] previously found instrumental for cardiac healing after MI [29,30].

Our in vitro experiments with cardiac fibroblasts revealed that IL-1α signaling is pivotal for StAR expression and function. Interleukin-1α, −1β, −33 and IL-18 comprise the leading members of the IL-1 gene family [31]. Interestingly, the modes of activation and release of IL-1α and IL-1β are profoundly different; in contrast to the passive release of IL-1α, secretion of IL-1β is highly regulated in the context of the innate and adaptive immune responses [[32], [33], [34], [35]]. What unifies the two cytokines is their independent binding to a mutual receptor type I (IL-1R1) that in turn recruits a receptor accessory protein (IL-1R3) in order to activate downstream intracellular signaling. Relevant to this study are the two arms of the IL-1R1 signaling patterns, culminating in gene activation by NF-κB/AP-1 pathways, and activation of MAP kinase pathways (p38, JNK, ERK) [8,34,36,37].

Finally, we also examined the physiological impact of IL-1α absence on the recovery outcomes after MI in a global IL-1α deficient mouse model, which turned out to agree with the predicted centrality of IL-1α, and possibly StAR, in cardiac repair and remodeling after MI.