Hypoxic chambers may cause myocardial repair after a heart attack

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Abstract​

Background: Oxygen supplementation in myocardial infarction (MI) remains controversial. Inflammation is widely believed to play a central role in myocardial repair. A better understanding of these processes may lead to the design of novel strategies complementary to MI treatment.
Methods: To investigate the role of hypoxia in inflammation and myocardial repair after acute MI, we placed MI mice in a tolerable mild hypoxia (10% O2) chamber for 7 days and then transferred the mice to ambient air for another 3 weeks.
Results: We found that the cumulative survival rate of the MI mice under hypoxia was significantly higher than that under oxygen supplementation. Hypoxia promoted postinfarction myocardial repair. Importantly, we found that hypoxia modulated the phenotypic transition of blood monocytes from pro-inflammatory to pro-reparative in a timely manner, leading to the subsequent discontinuation of inflammation in myocardial tissues and promotion of myocardial repair post-MI. Specifically, cultured bone marrow-derived macrophages (BMDMs) primed by hypoxia in vitro exhibited improved reparative capacities and differed from M1 and M2 macrophages through the AMPKα2 signaling pathway. The deletion of AMPKα2 in monocytes/macrophages prevented the phenotypic transition induced by hypoxia and could not promote myocardial repair after MI when transplanted into the myocardium.
Conclusions: Taken together, our work demonstrates that hypoxia promotes postinfarction myocardial repair by modulating the blood monocyte/macrophage phenotypic transition from pro-inflammatory to pro-reparative in a timely manner through the AMPKα2 signaling pathway. Hypoxia priming might be an attractive translational strategy for MI treatment by amplifying immune cells during early inflammation and subsequent resolution and repair.

Introduction​

A large population of inflammatory cells, including neutrophils and monocytes, are mobilized to the myocardium during acute myocardial infarction (AMI) 1. This process occurs in a time-dependent manner, and the rapid infiltration of neutrophils is followed by an influx of monocytes 2. Monocytes/macrophages play different roles during early inflammatory and late repair processes 3, 4. However, the specific environmental cues that induce monocyte/macrophage polarization and promote cardiac repair after AMI remain unclear 5.

Oxygen supplementation has been a cornerstone of supportive care in patients suspected of having AMI for more than a century and is widely endorsed by international guidelines 6, 7. However, clinical studies have increasingly confirmed that oxygen supplementation has no beneficial effects when administered to ST-elevation myocardial infarction (STEMI) patients with no hypoxemia 8, 9. Moreover, some studies proposed that the administration of oxygen supplementation therapy to acutely ill patients could be toxic and increase mortality and morbidity 10.

Recently, studies have confirmed that hypoxia is a major regulatory factor responsible for maintaining the proliferative capacity of cardiomyocytes in neonatal mice and inducing the regeneration of cardiomyocytes in adult mice 11-13. Hypoxia can promote macrophage phenotypic transition to a specific type or the M2 phenotype in solid tumors 14. Therefore, the role of hypoxia in heart recovery after MI seems to be protective, and further studies are needed to confirm this phenomenon.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is a highly conserved, key regulator of intracellular energy homeostasis. The catalytic α subunit has the following two isoforms: α1 is localized to the cytosol, while α2 can translocate to the nucleus 15. AMPKα2 has been shown to regulate the stability of hypoxia-inducible factor-1α (HIF-1α) and neutrophil survival, thereby determining further myeloid cell recruitment and repair potential after hindlimb ischemia 16. AMPK is upregulated under hypoxic conditions and promotes cardiomyocyte adaptation to chronic hypoxia 17. However, the role of AMPK in modulating the functions of monocytes/macrophages under AMI is still largely unknown.

In the present study, we investigated the role of hypoxia in cardiac repair after MI and the mechanism involved.
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Discussion​

Since Steele et al first demonstrated that oxygen can alleviate angina pectoris a century ago, oxygen supplementation has become the standard treatment for patients with AMI. It appears quite logical and biologically plausible to give oxygen to patients with AMI to improve oxygenation in ischemic myocardial tissue and decrease ischemic pain. However, several clinical trials have confirmed that oxygen supplementation might be harmful or at least not beneficial for short-term or long-term mortality and increase the rehospitalization rate 8-10. Therefore, supplemental oxygen is not recommended for routine use in AMI patients without hypoxemia. However, without considering the possible systemic harmful effect of hypoxemia, whether hypoxemia is beneficial for heart recovery after AMI is unclear. In the present study, we found that systemic hypoxia could reduce the scar size, promote cardiomyocyte proliferation, and most importantly, promote heart function recovery and increase survival. Importantly, mild systemic hypoxia was tolerable in the mice after MI. To the best of our knowledge, this report is the first to show that mild hypoxia during the early period after AMI could promote myocardial infarct repair.

Studies have confirmed the close relationship between hypoxia and cardiomyocyte proliferation. The transition to an oxygen-rich postnatal environment is a key factor resulting in the cell cycle arrest of cardiomyocytes 11, which is consistent with the fact that neonatal mice gradually lose cardiomyocyte proliferation capacity in the first postnatal week 12. In adult mice, when treated with a extremely low concentration of oxygen (7% O2) for approximately 2 weeks, cardiomyocyte regains proliferative abilities and promotes myocardial repair through cardiomyocyte proliferation 13. These findings provide an interesting therapeutic strategy for AMI by promoting cardiomyocyte proliferation under hypoxia. In the current study, we found evidence of cardiomyocyte proliferation, such as H3 phosphorylation and positive staining for Aurora B kinase, in cardiomyocytes under hypoxia.

Myocyte metabolism is a critical factor related to the oxygen supply and is intimately linked to the lack of oxygen supply promoting preferential glucose utilization over fatty acids as a more efficient supply of ATP. Adaptive metabolism promotes fetal gene expression, including GLUT1, to prioritize glucose utilization. A previous study showed that an increase in glucose metabolism promotes cardiac regeneration in the neonatal mouse heart 23. Neonatal mice have been shown to regenerate their hearts during a transient window of time of approximately 1 week after birth 24. A previous study showed that the hypoxic nature of the zebrafish external and circulatory environments prevents the activation of the DNA damage response and cell cycle arrest of myocytes. Mitochondrial ROS-mediated activation of the DNA damage response is an important upstream event that mediates cell cycle arrest in postnatal cardiomyocytes 12. The maintenance of the regenerative capacity may be related to hypoxic conditions. We previously found that hypoxia could prolong the cardiomyocyte regeneration window in neonatal mice 25. Meanwhile, cardiomyocyte proliferation in the cyanotic infant group was also observed to be significantly increased compared with that in the acyanotic infant group 13. Furthermore, decreased mitochondrial content with corresponding changes in metabolites, decreased radical oxygen species (ROS), and decreased DNA damage were observed in hypoxic hearts 26. Hypoxia can induce a switch in energy metabolism from mitochondrial oxidative phosphorylation to glycolysis 27. These results suggest that cardiomyocyte metabolism is hindered under hypoxia, which might be another contributor to the attenuation of remodeling after MI.

The inflammatory responses play crucial role in myocardial ischemic injury and repair. Early after MI, tissue necrosis initiates inflammation and dynamically recruits monocytes/macrophages. Infarct myocardial healing begins approximately 4 days after MI in murine models 28. During the processes of myocardial injury and repair, monocytes/macrophages are vital in clearing necrotic tissue and tissue healing 29. Any treatment that impairs or deletes macrophages will damage the process of myocardial repair 30. Modulating the function of monocytes/macrophages in different ways has obtained complex results 31,32. In nonreperfusion MI, the early period of inflammation is critical for the clearance of necrotic cells. In the present study, we found that systemic tolerable hypoxia enhanced the monocyte/macrophage transition from the pro-inflammatory to the pro-reparative subtype on approximately the 5th day after MI. Importantly, this tolerable hypoxia promoted the monocyte phenotypic transition without affecting the pro-inflammatory effect in the first 3 days after MI. Therefore, this tolerable hypoxia might be an ideal strategy to promote myocardial infarct repair.

A previous study showed that hypobaric hypoxia caused by exposure to altitude results in a significant reduction in the number of circulating peripheral dendritic cells 33. Another study reported that circulating inflammatory monocytes were increased in hypoxic mice. In hypoxic mice, the percentage and number of circulating monocytes, including pro-inflammatory Ly6Chigh monocytes, increased at different time points after hypoxic exposure 34. In contrast, we found that under the specific context of MI, hypoxia could halt inflammation in a timely manner and promote cardiac repair. Moreover, hypoxia influences the proliferation and lineage differentiation of bone marrow hematopoietic stem cells. Whether hypoxia might have an impact on myelopoiesis in the spleen or bone marrow is worthy of attention. Myeloid cells were reported to be derived from the bone marrow, and the spleen drove a substantial increase in the number of monocytes in the blood following MI 35. Monocytes are generated through two distinct cellular pathways. The earliest monocytes arise from granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs). GMPs and MDPs are proposed to arise from the hierarchical model of a common myeloid progenitor (CMP) 36. In our additional experiments, we found no impact on GMPs and MDPs in either proliferation assays or absolute cell numbers. This finding might be related to the short time (7 days) of hypoxia treatment after MI, proving that relatively short period of hypoxia has few side effects on MI mice.

The numbers and phenotypes of macrophages dynamically vary across different heart diseases. According to their functions and locations, different names have been used to distinguish macrophages. The terms “M1 macrophage” and “M2 macrophage” were recommended two decades ago to classify the macrophage subtypes. These terms were based on analyses of macrophages from C57BL/6 mice, and M1 macrophages were induced by LPS and IFN-r via the STAT-1 pathway, while M2 macrophages were induced by IL-4 via the STAT-6 signaling pathway. Our observations of macrophages revealed that MH macrophages were distinct from M1 and M2 macrophages. We found that M macrophages possessed improved tissue repair abilities and weak inflammatory responses. Recent research shows that oxygen is immediately reduced after injury and that the phenotypic transition of macrophages occurs simultaneously . We propose that hypoxia regulates the inflammatory response in the border zone of MI by altering the release of inflammatory factors, shifting from a pro-inflammatory to an anti-inflammatory environment. We also observed that hypoxia could upregulate genes related to macrophage skewing in myocardial tissue, strongly suggesting that hypoxia-regulated monocytes/macrophages might be critical for cardiac repair after MI. Importantly, we confirmed that this modulation was mainly based on blood monocytes and was not regulated by the tissue microenvironment. This finding offers a great opportunity for clinical translation as we could isolate monocytes/macrophages, stimulate them with hypoxia in vitro.

The detailed molecular mechanisms of monocyte/macrophage responses to hypoxia are still largely unknown. We found that MH macrophages exhibited a “pooled” change in gene expression, mainly in genes associated with metabolic modulation and hypoxia. This finding is consistent with a previous report suggesting that metabolic modulation is critical for macrophage phenotypic transition 38. Inhibiting the expression of AMPKα impaired the expression of HIF-1α in the nucleus under hypoxia or low glucose conditions. Studies have shown that AMPKα1 is a key molecule through which macrophages promote angiogenesis and tissue repair under normoxia and that the depletion of AMPKα1 impairs macrophage promotion of arteriogenesis 40. However, there have only been a few reports on the role of AMPKα2 in macrophages. In contrast to the AMPKα1 subunit, AMPKα2 can modulate gene and protein expression by translocating to the nucleus through many signaling pathways. In neutrophils, AMPKα2 regulates α-ketoglutarate generation, HIF-1α stability, and neutrophil survival, which, in turn, further facilitates myeloid cell recruitment and repair potential. In the present study, we found that AMPKα2, but not AMPKα1, was upregulated in hypoxia-primed monocytes/macrophages and that AMPKα2 was a key regulator of monocyte/macrophage phenotypic transition.

The latest results demonstrated that the production of interleukin (IL)-10 by macrophages promotes anti-inflammatory functions and prevents fibrosis after tissue damage, which could lead to new therapeutic perspectives for inflammatory diseases 42. In our study, MH macrophages expressed higher levels of IL-10 than M1 and M2 macrophages. IL-10 is considered to have a cardioprotective effect on improving the LV function and diminishing pathological remodeling 43. Recombinant IL-10 could preserve cardiac function, attenuate maladaptive remodeling and improve long-term survival in mice 44. IL-10 has been reported to increase the expression of intracellular galectin-3 through the activation of STAT3 in macrophages, which is essential for osteopontin-producing reparative macrophage polarization after myocardial infarction 21. We believe that inflammatory factors, such as IL-10, may be the key mediators secreted by hypoxia-primed macrophages that exert cardioprotective effects.

Currently, promising cardioprotective strategies for MI include the combination of ischemic postconditioning and remote ischemic preconditioning 45, which aims to reduce the infarct size and attenuate adverse cardiac remodeling and progression to heart failure 45. Meanwhile, clinical trials have reported that macrophage transplantation could be safe and feasible 46, 47. These results provide the basis for future prospective randomized clinical trials and hinder the potential clinical applications and future prospects of our results.

Conclusion​

Our data suggest that hypoxia promotes the monocyte/macrophage phenotypic transition from pro-inflammatory to pro-reparative in a timely manner and promotes postinfarction myocardial repair. Hypoxia priming might be an attractive translational strategy for MI treatment by amplifying immune cells during early inflammation and subsequent resolution and repair.

Hypoxia-primed monocytes/macrophages enhance postinfarction myocardial repair

Background: Oxygen supplementation in myocardial infarction (MI) remains controversial. Inflammation is widely believed to play a central role in myocardial repair. A better understanding of these processes may lead to the design of novel strategies complementary ...

www.ncbi.nlm.nih.gov

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How much oxygen is enough?​

Only a few years ago, back when I was in medical school, I was taught that the goal of oxygen therapy should be to push saturation above 95%, and that anyone who comes in to hospital with a saturation below that should receive oxygen therapy (with the exception of people with chronic obstructive pulmonary disease, whose bodies have adjusted to lower oxygen levels). For those who aren’t used to the terminology, the definition of oxygen saturation is the proportion of haemoglobin molecules in the arteries that are “saturated” with oxygen. A normal level for a healthy person is usually 97% or higher.

In the last few years, however, there’s been a bit of a shift in thinking. It started with the realization that people with heart attacks who were treated oxygen didn’t do any better than those who weren’t. This is a good example of a medical reversal – it seemed logical to give oxygen to people with heart attacks, because a heart attack is a blockage in one of the arteries that supply the heart, which means that the heart muscle isn’t getting enough oxygen. By increasing the oxygen level in the blood stream, even if only a small amount of that oxygen is able to get past the blockage, it should do some good. At least that was the thinking.

Unfortunately, logic often turns out be wrong, at least in medicine. As it turns out, oxygen isn’t the utterly benign substance it’s often made out to be. First, oxygen causes blood vessels to constrict, so by pushing up the oxygen saturation you could actually be worsening the blockage. Second, oxygen has an unfortunate tendency to form reactive oxygen species (ROS), which can wreak all kinds of havoc in our cells. We’ve evolved mechanisms to deal with these, but when a part of the body is depleted of oxygen for a while, many of our defences to deal with ROS are diminshed. If you then push up the oxygen arriving in an area enormously, say an area of heart muscle that’s been deprived of oxygen for a while but that now once again has good flow thanks to an intervention to remove the blockage, you might actually increase the damage to that area.

Long story short, it turned out that oxygen wasn’t good to give to people with heart attacks. It might even be bad. Thankfully, that practice has now stopped in most places. The realization that oxygen might actually be harmful in certain situations has led to a change in thinking about oxygen therapy. No longer is it viewed uncritically as a universal panacea that can be doled out generously to everyone who comes in through the doors of the hospital. At least that’s the case in theory. In practice, lots of patients still get oxygen who don’t need it.

When hospital staff see a patient who is out of breath, they will often will shove a mask on their face and crank up the oxygen, regardless of what the patient’s saturation level is. On a superficial level, I guess this makes some sense. If someone’s breathless, they must need oxygen, right?

Wrong. There are many things that can cause a sensation of breathlessness, and a lack of oxygen is just one of them. Our bodies are actually not very good at detecting changes to the oxygen level in the blood stream. If a patient has a saturation of 90% and is out of breath, then the breathlessness is not caused by the somewhat low oxygen. I’ll repeat that sentence, just to be sure you didn’t miss it. If a patient has a saturation of 90% and is out of breath, then the breathlessness is not caused by the somewhat low oxygen. Our bodies are in fact amazingly poor at noticing the oxygen level in the blood stream, and don’t really start to pay attention until the oxygen saturation drops well below 80%.

For the most part, our breathing rate is determined by the level of carbon dioxide in the blood stream (or to be more technically correct, the pH), not by the level of oxygen. It is much more central to our continued life on this planet to keep carbon dioxide within strict limits than it is to make sure that oxygen is always kept at a very high level. That’s why people who engage in free diving will often intentionally hyperventilate before going under the water. The purpose isn’t to increase the oxygen content in the bloodstream (which is anyway already at 100% or thereabouts). It’s to decrease the carbon dioxide content, which will allow them to hold their breath longer, since it will take longer for the carbon dioxide levels to reach the point where their bodies force them to take a breath.

Ok, that was a rather long preamble, but I think I’ve set the stage sufficiently now. A study was published recently in the New England Journal of Medicine that sought to answer the question of what a reasonable level of oxygen supplementation is in patients who are having trouble oxygenating themselves.

This was a randomized trial carried out at 35 intensive care units (ICU’s) in seven different European countries. In order to be included in the trial, participants had to be over the age of 18 and have respiratory failure for which they were receiving at least ten liters of oxygen. Unusual causes of respiratory failure which require special treatment, such as carbon monoxide poisoning and cyanide poisoning, were excluded from the study.

Participants were randomized to an oxygen saturation target of either 90% or 96% (technically they were randomized to a PaO2 of 60 mmHg or 90 mmHg, but it’s roughly the same thing). The study wasn’t blinded, since treating a patient to a specific target requires knowing what their saturation is.

2,928 patients were included in the trial, a nice big number that should show a difference in mortality if there is one, especially considering that the mortality rate in patients treated in ICU’s is high.

The median actual saturation level in the low oxygen group ended up being 93%, while the median in the high oxygen group ended up being 96%. In other words, not a huge difference. Maybe the nurses felt squeamish about letting the oxygen sit at 90%, as my experience tells me they are wont to be. Obviously, this will make the study a little less useful than it could have been.

Let’s get to the results.

At the 90-day point after recruitment in to the study, 42,9% of patients in the low oxygen group had died, as compared with 42,4% in the high oxygen group. This absolute difference of 0,5% was nowhere close to being statistically significant (p-value 0,64).

When it comes to serious adverse events, 36,1% of participants in the low oxygen group suffered a serious adverse event, as compared with 38,1% in the high oxygen group. Again the difference was nowhere close to being statistically significant (p-value 0,24).

In other words, there was no meaningful difference between the groups in terms of either mortality or serious adverse events. This was a big, high quality study, and it is telling us that we don’t need to push patients’ oxygen saturation up over 95%. If the saturation happens to be at 93% then that’s just fine, no extra oxygen required.

If every nurse, nursing assistant, and doctor became aware of this, hospitals could probably cut down on oxygen use enormously, and since the perceived need for oxygen therapy is one of the main reasons for keeping patients in hospital rather than sending them home, it would frequently also allow for earlier discharge. Considering that hospitals in many western countries are constantly operating at 100% capacity (which was the case long before the advent of covid-19), this could free up a lot of hospital beds.

How much oxygen is enough?

This article discusses oxygen therapy, and what a reasonable target should be for oxygen saturation, in light of a new study published in the New England Journal of Medicine.

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