Abstract 310: Nix in the Heart: Regulation of Developmental Apoptosis and Sarcoplasmic Reticular Calcium Stores Explains its Functional Diversity
The physiological role of many BH3-only Bcl-2 family proteins has not been characterized, and their importance outside of Bax- and Bak-dependent cell death is controversial. We used conditional gene targeting in the mouse to evaluate Nix, a hypertrophy-inducible cardiac BH-3 only family member. Germ-line Nix ablation (EIIa-Cre): 1. Diminished cardiac contractile performance without altering cardiomyocyte viability, and 2. Increased reticulocyte counts by altering the apoptotic balance during erythrocyte maturation. Erythroid cells from Nix null spleens provided an experimental cell system of Nix-mediated apoptosis: Nix-null splenocytes exhibited in vivo stage-specific resistance to apoptosis during erythroblast development, and diminished in vitro apoptosis stimulated by cytokine deprivation or calcium ionophore. Nix-GST fusion protein released cytochrome C without opening the permeability transition pores of isolated mitochondria, demonstrating a mechanism for apoptosis induction similar to Bax. Hearts of Nix null mice were structurally and histologically normal, but displayed diminished contractility (+dP/dt) at baseline and in response to step-wise increases in ventricular pacing rate or graded dobutamine infusion. Cardiac-specific Nix null mice (Nkx2.5-Cre) were similar. Isolated Nix null cardiomyocytes exhibited 30% decrease in peak shortening rate, 20% reduction in %fractional shortening and 30% decrease in calcium transient amplitude with field pacing and isoproterenol. Caffeine stimulated calcium release was depressed by 25%, indicating diminished SR calcium stores in Nix null myocytes. Likewise, Nix null embryonic fibroblasts had diminished calcium release by thapsigargin, implicating a primary abnormality of endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR). Thus, Nix is a functionally unique BH3-only family member that is essential for normal cardiac function by maintaining SR calcium homeostasis, and also serves a critical function in hematopoiesis by regulating apoptosis during erythroblast differentiation.