Introduction

Myocardial infarction (MI) is the most common cause of cardiac injury in humans and results in acute loss of heart tissue, which becomes replaced with a fibrous scar that later might lead to post-MI heart failure. Unlike humans, some fish can regenerate their hearts after injury. Astyanax mexicanus is a single fish species comprising two different forms: a blind cave-dwelling and an eyed surface-dwelling form. Over several millions of years, geological events that occurred in Northern Mexico caused surface fish living in rivers to enter caves. Subsequently, with retreating river waters, river fish got trapped in numerous caves and evolved into different cavefish populations. Our laboratory has discovered that surface fish are able to fully regenerate their hearts, while cavefish from the PachÏŒn cave form a permanent fibrotic scar like humans and also identified key differences in their immune responses (Stockdale et al., 2018). 

Objectives

Here, we characterized the immune response to cardiac injury in A. mexicanus and hypothesized that the differences in immune response underlie the differential regenerative response between surface fish and cavefish. 

Methods 

To induce cardiac injury, cryoinjury was performed on the surface and cavefish. Hearts were isolated at timepoints from 0 to 60 days post-cryoinjury (dpci, n=10-18 per timepoint) and processed for scRNA-seq (0-14dpci), in situ hybridisation (0-60dpci) and (immuno-) histochemistry (0-60dpci). Pharmacological inhibition was performed by intraperitoneal injection between 7 to 11dpci (n=10 per group) or water immersion from 7 to 14dpci daily (n=15 per group). 

Results

Using scRNA-seq, we found that immediately after injury, PachÏŒn showed a strong innate immune response peaking at 1 and 3dpci whereas surface fish had a prolonged response throughout at least 14 days after injury. Additionally, surface fish showed a stronger adaptive immune response after 7dpci which was absent in PachÏŒn. Both the adaptive and innate surface fish response was transcriptionally different from PachÏŒn. In situ hybridization experiments validated the scRNA-seq findings, PachÏŒn showed an immediate substantial influx of innate cells into the wound, but this response was decreased and resolved by 7dpci (P<0.05). Surface fish innate immune response was prolonged throughout 60 days after injury whereas PachÏŒn innate cell numbers remained lower from 3 to 60dpci (P<0.005, P<0.0001). Moreover, surface fish showed a significant increase in adaptive cells starting at 7dpci and peaking at 30dpci (P<0.0001), whereas PachÏŒn adaptive response was at negligible levels at all time points from 0 to 60dpci. Suppressing the late immune response pharmacologically using either clodronate liposomes or dexamethasone significantly inhibited the leukocyte response at 30dpci and resulted in larger wound sizes (P<0.05) at both 30 and 60dpci suggesting that the prolonged leukocyte response might be essential for surface fish regeneration.

Conclusions

Prolonged innate and adaptive responses might be one of the key regulators of successful heart regeneration. Understanding the signals and mechanisms that govern regeneration and scarring in A. mexicanus could be useful to create new therapeutic targets to stimulate the regeneration of the human heart.