Introduction:

Cardiovascular diseases remain one of the leading causes of mortality in the world. With limited capacity for regeneration, an infarcted human heart does not replace its damaged tissues, but forms a noncontractile fibrous scar instead. This detrimentally impacts the functionality of the myocardium, and is a leading cause of chronic heart failure across the globe. Similarly, while humans rely on satellite cells for skeletal muscle regeneration, their capacity for regeneration is often insufficient to fully repair extensive injuries or chronic muscle degeneration, limiting successful recovery and inducing impaired muscle function. As a result, searching for strategies that enhance endogenous repair effectiveness or promote de novo tissue regeneration is crucial. In this research project, I will be exploring the role of calcium signalling involved in both cardiac and skeletal muscle regeneration. This investigation will be conducted through in-vivo testing within zebrafish (Danio rerio) and Mexican tetra surface and cavefish morphs (Astyanax mexicanus), while combining pharmacological testing, immunohistochemistry, staining techniques, statistical analysis and Fiji image processing.

Objectives:

1. Investigating wild-type cavefish with intraperitoneal injection of Nifedipine (L-type calcium channel inhibitor) and Alfacalcidol (Vitamin D analog) 21 days after cryoinjury, through AFOG staining and Von Kossa staining

2. Evaluating cardiomyocyte proliferation and re-differentiation in wild-type cavefish and surface fish morphs 7 days & 14 days after cryoinjury, through immunohistochemistry (PCNA and embcmhc) and Von Kossa staining (calcium deposit)

3. Investigating the regenerative capacities of wild-type zebrafish with intraperitoneal injection of Nifedipine (L-type calcium channel inhibitor) 14 days after cryoinjury in relation to effects on mitochondria, through calcium assay, Von Kossa staining and immunohistochemistry (MitoTrack).

4. Immunohistochemistry (PCNA slush, proliferation vs apoptosis - cleaved caspase3 / tunel staining) in muscles of cavefish after cryoinjury (whole course) - characterisation of response (using antibody/RNA scope)

5. Live imaging – pacing (heart + muscle) with calcium dye (Cal520) to see changes in calcium levels within the model.

6. Utilise FIJI image processing and quantification with proper normalisation, while integrating statistical analysis.

Anticipated Outcomes:

As a pilot experiment, this research project will be evaluated based on the development and optimisation of methodologies and the extent to which our findings can help advance our understanding of tissue regeneration through modelling in Astyanax mexicanus and Danio rerio species. Since the cavefish and surface morphs of Astyanax mexicanus are not widely studied previously, the project's objectives and methodologies operate under constant amendments. This project explores how calcium signalling may correlate with the differences in regenerative capabilities within these two morphs, while comparing it to zebrafish models to understand how their contrasting regenerative efficacies can be explained despite a similar genetic background.