The anti-apoptotic protein HAX-1 is a regulator of cardiac function

Abstract

The HS-1 associated protein X-1 (HAX-1) is a ubiquitously expressed protein that protects cardiomyocytes from programmed cell death. Here we identify HAX-1 as a regulator of contractility and calcium cycling in the heart. HAX-1 overexpression reduced sarcoplasmic reticulum Ca-ATPase (SERCA2) pump activity in isolated cardiomyocytes and in vivo, leading to depressed myocyte calcium kinetics and mechanics. Conversely, downregulation of HAX-1 enhanced calcium cycling and contractility. The inhibitory effects of HAX-1 were abolished upon phosphorylation of phospholamban, which plays a fundamental role in controlling basal contractility and constitutes a key downstream effector of the beta-adrenergic signaling cascade. Mechanistically, HAX-1 promoted formation of phospholamban monomers, the active/inhibitory units of the calcium pump. Indeed, ablation of PLN rescued HAX-1 inhibition of contractility in vivo. Thus, HAX-1 represents a regulatory mechanism in cardiac calcium cycling and its responses to sympathetic stimulation, implicating its importance in calcium homeostasis and cell survival.

Fig. 1.

Effects of acute HAX-1 overexpression or downregulation on rat cardiomyocyte contractile function. Adenoviruses with sense and anti-sense mouse HAX-1 gene insertion were generated according to standard procedures. An adenovirus expressing GFP was used as a control. Rat adult cardiomyocytes were isolated and infected with Ad.GFP (GFP), Ad.HAX-1 (HAX-1), and Ad.HAX-1-antisense (HAX-1-AS). (A) Representative images of infected cardiomyocytes under light microscopy (LM, upper panel) and fluorescence microscopy (FM, lower panel). (B) Immunofluorescence analysis of HAX-1 localization in GFP and HAX-1 infected cardiomyocytes, using PLN as a SR marker. To investigate the contractile function, myocytes were infected with GFP, HAX-1, and HAX-1-AS for 24 h and contractility was subsequently recorded. (C) Representative curves of myocyte contraction under basal conditions. (D) Percentage of fractional shortening (FS%), (E) rate of contraction (PdL/dt, μm/s), and (F) rate of relaxation (NdL/dt, μm/s) under basal conditions. For the calcium transients, cells were loaded with fura-2 for 30 min, and then calcium transients were recorded and analyzed. (G) Representative curves of calcium transients under basal conditions. (H) Calcium amplitude, indicated as the fura-2 ratio of 340/380 nm; (I) Tau under basal conditions. For SR calcium load indicating by caffeine-induced calcium release from SR, 10 mM caffeine was added to the buffer. (J) Peak of SR calcium load, indicated by the fura-2 ratio of 340/380 nm. n = 4–5 hearts (6–8 cells/heart) for each group. Data are mean ± SEM. *, P < 0.05, compared to Ad.GFP group.

Fig. 2.

Generation and characterization of cardiac-specific HAX-1 overexpressing mice. HAX-1 transgenic mice were generated, using mouse HAX-1 cDNA under the control of an α-MHC promoter and human growth hormone polyA (HGH polyA). (A) Quantitative immunoblotting of heart homogenates from HAX-1 overexpressors (OE) and wild-type (WT). (B) Immunofluorescence analysis of HAX-1 localization in cardiomyocytes from WT and OE mice, using PLN as a SR marker. To analyze the contractile function, cardiomyocytes from OE or age-matched WT mice were isolated; contractility and calcium transients were recorded and analyzed under basal conditions (Basal) and upon isoproterenol stimulation (Iso, 100 nM). (C, E, and G) Percentage of fractional shortening (FS%), rate of contraction (PdL/dt, μm/s) and rate of relaxation (NdL/dt, μm/s). (D) Calcium amplitude, indicated by fura-2 ratio of 340/380 nm. (F) Tau. (H) Peak of SR calcium load, indicated by the fura-2 ratio of 340/380 nm. (I) Initial rates of oxalate-supported SR Ca uptake in cardiac homogenates from WT (○) and OE (●) mice. (Inset) The EC₅₀ of SERCA for calcium in OE and WT hearts. Values were normalized to V_max values. (J) Representative immunoblots (upper panel) and relative protein levels (lower panel) of PLN monomers and pentamers in WT and OE heart homogenates. Data are mean ± SEM. n = 6–8 hearts for each group (for C–H, 8–10 cells/heart). *, P < 0.05, compared to wild-type hearts.

Fig. 3.

Effect of HAX-1 deficiency on cardiomyocyte contractile function. Cardiomyocytes from HAX-1 heterozygous mice (HE) or age-matched wild-type (WT) were isolated; contractility and calcium transients, as well as caffeine induced calcium release from SR were recorded and analyzed. (A, C. and E) Percentage of fractional shortening (FS%), rate of contraction (PdL/dt, μm/s) and rate of relaxation (NdL/dt, μm/s). (B) Calcium amplitude, indicated by the fura-2 ratio of 340/380 nm. (D) Time to 50% decay of calcium (T₅₀, s). (F) Tau. (G) Peak of SR calcium load, indicated by the fura-2 ratio of 340/380 nm. A–G are all under basal conditions (Basal) and upon isoproterenol stimulation (Iso). (H) Initial rates of oxalate-supported SR calcium uptake in hearts from WT (□) and HE (■) mice. (H, inset) The EC₅₀ of SERCA for calcium in HE and WT hearts. Data are mean ± SEM. n = 4–6 hearts for each group (for A–G, 8–10 cells/heart). *, P < 0.05, compared to wild-type hearts.

Fig. 4.

Effect of HAX-1 on PLN-PLN and PLN-SERCA fluorescence resonance energy transfer (FRET). (A) Fluorescence microscopy showed that acceptor-selective photobleaching of YFP-PLN fluorescence resulted in an increase in CFP-SERCA fluorescence, indicating FRET. (B) Image quantification showed CFP-SERCA fluorescence (blue circles) increased exponentially as YFP-PLN (green triangles) was photobleached. The magnitude of CFP-SERCA fluorescence enhancement was greater in cells cotransfected with HAX-1 (black squares), suggesting increased FRET compared to control. (C) CFP-PLN fluorescence (blue circles) increased as YFP-PLN (green triangles) was bleached, indicating intrapentameric FRET. The magnitude of the CFP-PLN fluorescence enhancement was smaller in cells cotransfected with HAX-1 (black squares), suggesting reduced FRET. (D) Summary of FRET microscopy results. Coexpression of HAX-1 caused a 32% decrease in overall FRET between CFP-PLN and YFP-PLN, and a 35% increase in overall FRET between CFP-SERCA and YFP-PLN. Data are mean ± SEM, *, P < 0.01, compared with control. (E) A model for HAX-1 regulation of cardiac calcium handling. HAX-1 shifts the PLN pentamer/monomer equilibrium toward the active, monomeric species, and promotes formation of the PLN-SERCA regulatory complex.

Fig. 5.

Effect of HAX-1 overexpression on cardiomyocyte contractile performance in the absence of PLN. Cardiomyocytes were isolated from HAX-1 overexpression in the PLN knockout background (OE/KO), PLN knockout (PLN KO), and HAX-1 overexpression (HAX-1 OE) mice as well as age-matched wild-type (WT). Contractility and calcium transients as well as caffeine-induced SR calcium release from SR were recorded and analyzed. (A) Percentage of fractional shortening (FS%); (C) Rate of contraction (PdL/dt, μm/s); and (E) Rate of relaxation (NdL/dt, μm/s). (B) Calcium amplitude, indicated by fura-2 ratio of 340/380 nm. (D) Peak of SR calcium load, indicated by the fura-2 ratio of 340/380 nm. A–E are all under basal conditions. Data are mean ± SEM, n = 4–5 hearts (8–10 cells/heart) for each group. (F) Initial rates of oxalate-supported SR calcium uptake, and (G) EC₅₀ of calcium uptake in heart homogenates from WT, HAX-1 OE, PLN KO and OE/KO mice. Data are mean ± SEM. n = 6–8 hearts for each group. *, P < 0.05, compared to WTs. ^, P < 0.05, compared to HAX-1 OEs.