Caroline Gregg
Heat stress refers to an increase in an animal’s core body temperature in response to elevated environmental temperature. This can have detrimental effects on an animal’s productivity and survival. Heat-related mortality and production loss costs the American swine industry roughly a billion dollars every year. Currently, there is no fix to this problem other than environmental cooling methods. In order to develop a therapeutic to treat the negative consequences of heat stress, we must build an understanding of what occurs during heat stress at the cellular level by studying the activation of inflammatory pathways in skeletal muscle cells.
Samples of semitendinosus muscle tissue were taken from gilts raised in either thermoneutral (24ºC), heat stress (37ºC), or pair-fed thermoneutral (24ºC) conditions for 7 days. Pair-fed gilts were raised in standard temperature conditions but fed a restricted diet to control for the effects of lower feed intake that occurs during heat stress.
I isolated the nuclear fractions from those muscle samples to test for the nuclear content of transcription factors; HSF1, NF-kB, AP-1, and TFEB. These transcription factors code for genes that respond to cellular stress through inflammation, cytokine production, and autophagy. I then standardized protein concentration of every sample and used gel electrophoresis to separate out the proteins by molecular weight. After that, I transferred the protein to nitrocellulose membranes to run a western blot assay. Membranes were incubated with antibodies against the transcription factor of interest, so the antibodies bind to the membrane. Last, membranes were incubated with secondary antibody attached to a signal molecule that causes a color change reaction of the protein band on the membrane according to the amount of protein present.
We originally hypothesized that heat stress would cause an increase in inflammatory signaling, but we found no significant difference in transcription factor content between groups. The next step is to test muscle samples for mRNA content of genes controlled by these transcription factors to confirm that signaling did not change due to heat stress. Further research in the development of a therapeutic will involve understanding what signaling pathways are activated following chronic heat stress.
Duration: 04/29/2019
Principal Investigator(s): Joshua Selsby