Endoplasmic reticulum stress response favors cardiomyocyte apoptosis near birth
Authors: Bose Karthikeyan , Louey Samantha , Jonker Sonnet 
Published: May 2024

INTRODUCTION: One-third of cardiomyocyte are lost just before birth in the normally developing heart. The underlying reasons for this cell loss are unknown. We studied the role of the endoplasmic reticulum (ER) stress response in regulation of cardiomyocyte apoptosis in perinatal sheep hearts. We hypothesized that the signaling balance from survival to apoptosis changes just before the cardiomyocyte numbers decline, and that the ER stress response contributes. METHODS: Snap-frozen left ventricular (LV) samples and cultured cardiomyocytes from normally-growing lambs at 135 d of gestational age (dGA; term=147 dGA), 143 dGA and 1 d postnatal age (dPN) were used for this study (n=6 each age).Gene and protein regulation was studied by RT-PCR and western blot analysis. Parameters were compared by age by 1-way ANOVA followed, if justified, by Šídák's multiple comparisons test. RESULTS: In the LV, the ER stress master regulatory protein GRP78 was similar within fetal ages, but declined 32% between 143 dGA and 1 dPN (P= 0.0005), indicating less capacity for responding to misfolded proteins. eIF2α, downstream of GRP78, is critical for deciding the survival/apoptosis outcome following ER stress. Phosphorylation of eIF2α doubled between 135 dGA and 143 dGA (P=0.0015), implying increased ER stress in this period, and declined again at 1 dPN (p<0.0001 vs. 143 dGA). Cleaved apoptosis effector caspase 3 protein peaked after birth; levels at 1 dPN were 1- to 2-fold higher than at other ages (p<0.0001 vs. 135 dGA, P=0.002 vs. 143 dGA). Together, these data suggest that survival signaling in the ER stress pathway may be blunted between 135 and 143 dGA, as evidenced by diminished GRP78 expression and elevated eIF2α phosphorylation, resulting in activation of the apoptosis pathway. To better understand the mechanisms regulating dynamic perinatal changes in the ER stress response pathway, we studied sensitivity to ER stress (thapsigargin: 5 μM, 12 h) in cultured LV cardiomyocytes. In response to thapsigargin, GRP78 protein levels rose ~3-fold higher in cells from 135 dGA compared to 143 dGA or 1 dPN hearts (p<0.0001), supporting diminishing survival signaling closer to the time of birth. Phosphorylation of eIF2α followed a similar pattern and was highest in 135 dGA cells (p<0.0001 vs. 143 dGA and 1 dPN). This contrasts to the greater levels of phospho-eIF2α at 143 dGA in the tissue analysis. Thapsigargin stimulated 2-fold more cleavage of caspase 3 at 1 dPN than 135 dGA (P=0.0243), a similar timing as found in LV tissue. CONCLUSION: Findings suggest that at two weeks before birth, heart and cardiomyocytes are resilient to ER stress and stimulation of apoptosis, whereas nearer the time of birth there is a blunted ER stress response and increased activation of apoptotic pathways. R01HL142483 (Jonker), Collins Foundation (Bose). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.