Sensitivity and activation of endoplasmic reticulum stress response and apoptosis in the perinatal sheep heart

Abstract

Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA (P = 0.0096) and remained low at 1 dPN (P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth (P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth (P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart.NEW & NOTEWORTHY Cardiomyocyte apoptosis occurs even in the healthy, normally developing perinatal myocardium. As cardiomyocyte number is a critical contributor to heart health, the sensitivity of cardiomyocytes to endoplasmic reticulum stress leading to apoptosis is an important consideration. This study suggests that the heart has less robust protective mechanisms in response to endoplasmic reticulum stress immediately before and after birth, and that more cardiomyocyte death can be induced by stress in this period.

Keywords: cardiac; caspases; fetal development; myocardium; unfolded protein response.

Graphical abstract

Figure 1.

Schematic of the endoplasmic reticulum (ER) stress response pathway. ATF, activating transcription factor; BAX, Bcl-2-associated X-protein; DDIT3, DNA damage-inducible transcript; EIF2A, eukaryotic initiation factor 2; ERO1A, ER oxidoreductase 1; GRP78, 78-kDa glucose-regulated protein; UPR, unfolded protein response.

Figure 2.

Endoplasmic reticulum (ER) stress-induced ER stress response- and apoptosis-related protein expression in cultured cardiomyocytes. Thapsigargin was used to create ER stress in primary cardiomyocyte cultures. Measurements were made of 78-kDa glucose-regulated protein (GRP78) protein (78 kDa) (A), eukaryotic initiation factor 2 (EIF2A) protein phosphorylation (38 kDa) (B), cleaved caspase 3 protein (17 kDa) (C), cleaved caspase 7 protein (18 kDa) (D), cleaved caspase 12 protein (42 kDa) (E), and representative Western blot images (white balance and saturation adjusted for visual assessment) (F). n = 3 females (open symbols) and 3 male (closed symbols) animals per group. Groups were compared by two-way analysis of variance; if justified, multiple comparisons were performed by the Šidák test (ages with different letters are statistically different). dGA, days of gestational age; dPN, day postnatal. Raw values are shown with means ± SD.

Figure 3.

Endoplasmic reticulum (ER) stress-induced caspase 12 activity and cell death in cultured cardiomyocytes. Thapsigargin was used to create ER stress in primary cultures of cardiomyocytes and measurements were made of caspase 12 activity levels were assessed (A), cell survival as indicated by metabolic activity (B), and cell apoptosis as measured by TdT-mediated dUTP nick-end labeling (TUNEL) staining (C). n = 3 female (F; open symbols) and 3 male (M; closed symbols) animals per group. Cell culture and treatment, and assessment of caspase 12 activity, were performed in triplicate. Cell culture and treatment, and assessment of cell metabolic activity, were performed in triplicate and was repeated twice. Cell culture and treatment, and assessment of cell apoptosis, were repeated three times. Groups were compared by two-way analysis of variance; if justified, multiple comparisons were performed by the Šidák test (ages with different letters are statistically different). dGA, days of gestational age; dPN, day postnatal. Raw values are shown with means ± SD.

Figure 4.

Lipid peroxidation in sheep serum and left ventricle. Oxidative stress as measured by levels of lipid peroxidation by-product malondialdehyde (MDA) in serum (A) and myocardium (B). n = 3 female (F; open symbols) and 3 male (M; closed symbols) animals per group. Groups were compared by two-way analysis of variance; if justified, multiple comparisons were performed by the Šidák test (ages with different letters are statistically different). Raw values are shown with means ± SD. dGA, days of gestational age; dPN, day postnatal.

Figure 5.

Endoplasmic reticulum (ER) stress response-related mRNA and protein expression levels in sheep left ventricle. Measurements were made from left ventricular myocardium of 78-kDa glucose-regulated protein (GRP78) (A), also known as BiP or HSPA5, protein (78 kDa); activating transcription factor 6 (ATF6) mRNA (B); C/EBP homologous protein (CHOP) (C), also known as DNA damage-inducible transcript 3 (DDIT3) mRNA; endoplasmic reticulum oxidoreductase 1 (ERO1A) mRNA (D); eukaryotic initiation factor 2 (EIF2A) mRNA (E); EIF2A protein phosphorylation (38 kDa) (F); activating transcription factor 4 (ATF4) mRNA (G); and representative Western blot images (white balance and saturation adjusted for visual assessment) (H). n = 3 female (open symbols) and 3 male (closed symbols) animals per group. Groups were compared by two-way analysis of variance; if justified multiple comparisons were performed by the Šidák test (ages with different letters are statistically different). dGA, days of gestational age; dPN, day postnatal. Raw values are shown with means ± SD.

Figure 6.

Apoptosis-related mRNA and protein expression levels in sheep left ventricle. Measurements were made from the left ventricular myocardium of Bcl-2-associated agonist of cell death (BAD) mRNA (A), Bcl-2-associated X-protein (BAX) mRNA (B), caspase 3 (CASP3) mRNA (C), cleaved caspase 3 protein (17 kDa) (D), cleaved caspase 7 protein (18 kDa) (E), caspase 8 (CASP8) mRNA (F), caspase 9 (CASP9) mRNA (G), cleaved caspase 12 protein (42 kDa) (H), cytochrome-c (CYCS) mRNA (I), and representative Western blot images (white balance and saturation adjusted for visual assessment) (J). n = 3 female (open symbols) and 3 male (closed symbols) animals per group. Groups were compared by two-way analysis of variance; if justified multiple comparisons were performed by the Šidák test (ages with different letters are statistically different). dGA, days of gestational age; dPN, day postnatal. Raw values are shown with means ± SD.

Figure 7.

Total nucleic acids in sheep left ventricle. Measurements were made from the left ventricular myocardium of total RNA levels (A) and total messenger RNA (mRNA) levels (B). n = 4 female (open symbols) and 3 male (closed symbols) animals per group. Groups were compared by two-way analysis of variance; if justified multiple comparisons were performed by the Šidák test (ages with different letters are statistically different). dGA, days of gestational age; dPN, day postnatal. Raw values are shown with means ± SD.