Abstract
Sudden blockage of arteries supplying the heart muscle contributes to millions of heart attacks (myocardial infarction, MI) around the world. Although re-opening these arteries (reperfusion) saves MI patients from immediate death, approximately 50% of these patients go on to develop chronic heart failure (CHF) and die within a 5-year period; however, why some patients accelerate towards CHF while others do not remains unclear. Here we show, using large animal models of reperfused MI, that intramyocardial hemorrhage – the most damaging form of reperfusion injury (evident in nearly 40% of reperfused ST-elevation MI patients) – drives delayed infarct healing and is centrally responsible for continuous fatty degeneration of the infarcted myocardium contributing to adverse remodeling of the heart. Specifically, we show that the fatty degeneration of the hemorrhagic MI zone stems from iron-induced macrophage activation, lipid peroxidation, foam cell formation, ceroid production, foam cell apoptosis and iron recycling. We also demonstrate that timely reduction of iron within the hemorrhagic MI zone reduces fatty infiltration and directs the heart towards favorable remodeling. Collectively, our findings elucidate why some, but not all, MIs are destined to CHF and help define a potential therapeutic strategy to mitigate post-MI CHF independent of MI size.
Introduction
Myocardial infarction (MI) from sudden obstruction of a coronary artery afflicts ~1 million people in the US yearly1. Prompt restoration of blood flow through the epicardial arteries (reperfusion) during the acute phase of MI (lasting hours to days: Fig. 1a) has been a major advance and has reduced immediate death from acute MI with partial recovery of left ventricle function during the sub-acute phase of MI (lasting days to weeks: Fig. 1a). However, adverse remodeling of the left ventricle (LV) of the heart in the chronic phase of MI (lasting months to years: Fig. 1a) often results in chronic heart failure (CHF), which increases mortality. The incidence of post-MI CHF has increased in recent decades1,2. In the US, more than 2 million patients are affected, with >250,000 new cases reported every year and >300,000 deaths/yr due to CHF1. Although many studies have investigated the mechanisms underlying chronic heart failure post-MI, why some MI patients accelerate toward heart failure while others do not remain unclear.
A growing appreciation underlying the development of CHF in the post-MI settings is that not all acute MIs are the same. One of the most common, and perhaps the most damaging forms of tissue injury in the setting of revascularized acute MI is intramyocardial hemorrhage—a condition leading to bleeding within the heart muscle (myocardium). It is estimated that hemorrhage within acute MI is evident in nearly half of the successfully revascularized patients3,4. Studies have shown that hemorrhagic acute MIs are associated with adverse LV remodeling and poor prognosis in the ensuing chronic phase of MI compared to acute MIs without hemorrhage3. However, a causal connection between hemorrhagic MI and adverse LV remodeling has not been established.
Previously, the presence of iron following acute MI was believed to be short-lived and cleared by macrophages within weeks after the MI5. The perceived role of iron in MI was focused on the ability of iron to promote free-radical formation via the Fenton Reaction with the ensuing death of cardiomyocytes, thereby contributing to MI expansion during the acute phase of MI6. It was further believed that physiological ramifications of MI size enhanced alterations in neurohormones and pro-inflammatory cytokine release following MI and that this was responsible for anatomic and functional remodeling during the chronic phase of MI resulting in CHF7,8,9,10. In this model, infarct size is believed to be the persistent driver of LV remodeling and poor prognosis: the role of iron is only transient.
A growing body of evidence now indicates that hemorrhagic MIs lead to chronic iron deposition and that such deposits facilitate the perpetual recruitment of macrophages for months and years during the chronic phase of MI11,12. Current evidence also suggests that the macrophages recruited to the site of iron-rich MI take up the iron and are polarized into a pro-inflammatory phenotype. Studies outside the myocardium have demonstrated that iron-laden macrophages can transform into foam cells when they oxidize lipids encountered in their vicinity to form ceroids (lipopigments) within the cells13,14. Further, in the chronic phase of MI, fat deposition within the infarction zone is a common finding15,16 and is known to significantly impair cardiac energetics, function17,18 and adverse outcomes including CHF in post-MI patients19. To date, however, factors driving fat deposition within the MI scar are not understood, which greatly impedes the discovery and development of effective therapeutics to combat CHF.
Based on these collective observations, we hypothesized that the iron from hemorrhagic MI is centrally responsible for the fatty transformation of infarcted tissue leading to loss of cardiac function (Fig. 1a), which can be mitigated through timely reduction of iron from the MI zone; We also hypothesized that this fatty degeneration of infarct scar is a consequence of continuous iron-induced macrophage activation, lipid oxidation, foam cell formation, ceroid production, foam cell apoptosis, and iron recycling (Fig. 1b).
We tested our hypothesis using clinically relevant large animal models of reperfused MI with and without intramyocardial hemorrhage. Specifically, we used serial in vivo cardiac magnetic resonance imaging (cardiac MRI), histological evaluations, and Western blot analyses to study the spatial and temporal relationships between iron and fat by following the animals over a 6-month period after MI. We also tested whether a clinically available, intracellular iron chelator delivered starting 3 days post-MI for 8 weeks can decrease the iron and fat content within the hemorrhagic MI zone and alter the course of the functional state of the heart in the chronic MI period. We studied this in two groups of animals matched for MI size and iron content, with one group receiving the iron chelator and another not. Our findings support the hypothesis that iron is causally involved in adverse LV remodeling in the post-MI period through fatty degeneration of the infarcted myocardium. Notably, our observations here help define a new potential therapeutic dimension for CHF, with the possibility to mitigate the development of post-MI CHF independent of MI size, days after the acute event.
Results
Extent of fat deposition within infarcts depends on acute iron concentration of hemorrhagic MI
We investigated the temporal evolution of fat deposition and its relation to iron within MI using serial cardiac MRI in a validated canine model of reperfused MIs with (IMH+) and without (IMH−) hemorrhage, noting that cardiac MRI was performed on day 3 (D3), week 8 (Wk8), and month 6 (M6), post-MI. Confounder-corrected R2* (or 1/T2*, validated measure of iron concentration ([Fe]) in MI)20 and proton density fat-fraction (PDFF) maps were constructed and analyzed for [Fe] and fat-fraction within MI relative to the remote myocardium21. Acute MI size (%LV) at D3 in IMH+ animals was 35.2 ± 2.1%, compared to 12.9 ± 2.1% in IMH− animals (p < 0.01). Size of IMH (%LV) in IMH+ animals was 9.5 ± 1.9%. Representative findings on R2* and PDFF maps from an animal with hemorrhagic MI followed over a 6-month period post-MI are shown (Fig. 2a). In hemorrhagic cases, R2* was not different between D3, Wk8, and M6 (43.4 ± 2.2 at D3, 38.7 ± 1.25 at Wk8, and 41.7 ± 1.50 at M6, p = 0.33) suggesting that [Fe] was approximately constant, with only small elevation, between D3 and M6, post-MI. However, PDFF increased significantly from D3, Wk8 to M6, from 1.86 ± 0.11 (D3) to 2.07 ± 0.14 (Wk8), to 4.09 ± 0.33 (M6), p = 2.8 × 10−8; that is an increase of ~10% by Wk8 and ~220% by M6 relative to D3. In non-hemorrhagic cases, no significant difference was found between the various time points with respect to R2* (32.1 ± 2.3 (D3), 29.9 ± 1.2 (Wk8), and 30.0 ± 2.3 (M6), p = 0.69) and PDFF (1.97 ± 0.16 (D3), 2.16 ± 0.27 (Wk8), and 2.44 ± 0.20 (M6), p = 0.36). Compared to IMH+ groups IMH− groups showed greater R2* at D3 (p = 0.0048), Wk8 (p = 0.00035), and M6 (p = 0.0015); however, PDFF was not different at D3 (p = 0.55) and at Wk8 (p = 0.36) but significantly different at M6 (p = 0.028) (Fig. 2b)…
