Energetic and Cellular Adaptations

Question 1:
What energetic differences exist between marine and terrestrial cells?

Decreased glycolysis activation and increased spare respiratory capacity in marine mammal cells

Question 2:
What cellular adaptations could lead to these differences?

Increased mitochondrial volume in Ziphius cavirostris cells

Question 3: What are the underlying gene mechanisms for these cellular adaptations?

Increased gene copy of BPGM (Oxygen affinity), increased gene expression of PGC1a (mitochondrial biogenesis) in hypoxia in marine cells

Question 4: How can these Structural and Gene Mechanisms be modified to prove their direct phenotypic effect?

MitoCeption and CRISPRko experimentation to recreate human cellular phenotypes in marine cells and vice versa!

Hypothesis formation:

Species Guide

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Question 1: Cellular Energetics

We found a significant increase in marine mammal spare respiratory capacity when compared to terrestrial mammals

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(A) Example profile of the Seahorse XFe96 Analyzer Mito Stress Test when injected with 0.5uM, 1uM, and 2uM FCCP and 0.5 AA.

(B) Real time Oxygen Consumption Rate of various species.

(C) Spare respiratory capacity calculated as the difference between maximal respiration and basal respiration with each species compared to NHDF using a three-way ANOVA. GMA p=.0017, TTR p=.0106. n=5.

(D) Maximal mitochondrial respiration calculated as the difference between maximal respiration and non-mitochondrial respiration with each species compared to NHDF using a three-way ANOVA. GMA p=.001, KB p=.002. n=5

Question 2: Cellular Adaptations

We found a significant increase in marine mammal mitochondrial volume when compared to terrestrial mammals

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(A) Immunofluorescent imaging of NHDF and ZC cells with nuclei stained with DAPI (blue), F-actin with 488 Phalloidin (green), and mitochondria with TOM20-Cy5 (red).

(B) Comparison of maximum fluorescence intensity of red mitochondria analyzed with ImageJ and compared with a two-tailed t test with p<0.0001, NHDF n=11 ZC n=11

Question 3: Gene Mechanisms

We started our gene deep-dive with PGC1a, a gene responsible for mitochondrial biogenesis

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(A) PGC1a gene expression increased in marine mammal cells exposed to hypoxia when compared to normoxia. Decreased PGC1a expression in human cells when exposed to hypoxia.

(B) Mechanism of interaction between PGC1a, HIF1a, and mitochondrial metabolism (Luo et al. 2016)

(C) PGC1 proteins present across species. Total protein concentration measured with control GAPDH. No significant difference in protein levels when normalized to GAPDH total protein across species.

Question 4: Manipulating Cells to Observe Phenotypic Responses

We manipulated cell structural adaptations through MitoCeption procedures, still undergoing experimentation

(A) Procedure example of MitoCeption. GMA cell mitochondria labeled with red MitoTracker were transferred to BDF cells labeled with green CellTracker.

(B) Immunofluorescent imaging following MitoCeption procedure showing presence of red mitochondria in donor images but not in control images.

(C) Future experimentation to confirm the presence of GMA mitochondria in the BDF cells via qPCR. Once the presence of GMA mitochondria is confirmed, the cells will be exposed to hypoxia and normoxia and analyzed via flow cytometry to determine if BDF cells with GMA mitochondria tolerate hypoxia better than BDF cells with BDF mitochondria.

We manipulated genes with CRISPRko procedures, still undergoing experimentation

Oligonucleotide gRNA primers designed and ligated to lentiCRISPRv2 plasmid. Future experimentation to transfect whale cells with CRISPRko plasmid and evaluate spare respiratory capacity and mitochondrial function in these cells.