Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation

Abstract

Background: Derailment of proteostasis, the homeostasis of production, function, and breakdown of proteins, contributes importantly to the self-perpetuating nature of atrial fibrillation (AF), the most common heart rhythm disorder in humans. Autophagy plays an important role in proteostasis by degrading aberrant proteins and organelles. Herein, we investigated the role of autophagy and its activation pathway in experimental and clinical AF.

Methods and results: Tachypacing of HL-1 atrial cardiomyocytes causes a gradual and significant activation of autophagy, as evidenced by enhanced LC3B-II expression, autophagic flux and autophagosome formation, and degradation of p62, resulting in reduction of Ca²⁺ amplitude. Autophagy is activated downstream of endoplasmic reticulum (ER) stress: blocking ER stress by the chemical chaperone 4-phenyl butyrate, overexpression of the ER chaperone-protein heat shock protein A5, or overexpression of a phosphorylation-blocked mutant of eukaryotic initiation factor 2α (eIF2α) prevents autophagy activation and Ca²⁺-transient loss in tachypaced HL-1 cardiomyocytes. Moreover, pharmacological inhibition of ER stress in tachypaced Drosophila confirms its role in derailing cardiomyocyte function. In vivo treatment with sodium salt of phenyl butyrate protected atrial-tachypaced dog cardiomyocytes from electrical remodeling (action potential duration shortening, L-type Ca²⁺-current reduction), cellular Ca²⁺-handling/contractile dysfunction, and ER stress and autophagy; it also attenuated AF progression. Finally, atrial tissue from patients with persistent AF reveals activation of autophagy and induction of ER stress, which correlates with markers of cardiomyocyte damage.

Conclusions: These results identify ER stress-associated autophagy as an important pathway in AF progression and demonstrate the potential therapeutic action of the ER-stress inhibitor 4-phenyl butyrate.

Keywords: 4PBA; Drosophila; Endoplasmic Reticulum stress; HSPA5; atrial fibrillation; autophagy; drug research; molecular biology; structural biology; tachypacing.

Figure 1

Tachypacing (TP) induces autophagosome formation and enhanced activation of autophagy. A, Representative Western blot of TP‐induced autophagy markers p62 (molecular weight [MW], 62), LC3B‐I and LC3B‐II (MWs, 14 and 16, respectively), and loading control GAPDH (MW, 37). HL‐1 cardiomyocytes were normal paced (NP) or TP for the duration indicated. B, Quantified data showing a significant reduction in p62 levels after 6 or more hours of TP (N=4). C, Quantified data showing a significant increase in LC3B‐II levels, beginning after 2 hours of TP (N=5). D, Confocal images of TP HL‐1 cardiomyocytes, for the period as indicated, transfected with LC3B–green fluorescent protein (GFP) plasmid. E, Confocal images of TP HL‐1 cardiomyocytes for the period as indicated. Endogenous LC3B was visualized by immunostaining. Green puncta indicate autophagosomes. F, Quantified data showing accumulation of LC3B‐GFP punctae/cardiomyocytes during TP (n/N=35/3). G, Representative Western blot of HL‐1 cardiomyocytes NP vs TP for the duration, as indicated, in the presence or absence of bafilomycin A1 (BAF). H, Quantification of the autophagic flux by determining the difference in LC3B‐II levels in the presence vs absence of BAF (N=4). Note that all NP data are shown after 8 hours of observation. *P≤0.05, **P≤0.01, ***P≤0.001 vs NP.

Figure 2

Tachypacing (TP)–induced autophagy does not involve mammalian target of rapamycin complex (mTORC) signaling. Top panels: Western blots of proteins within mTORC signaling. Bottom panels: Quantified data of the ratio of phosphorylated proteins normalized for basal protein levels. Phosphorylated mTOR S2448 (mTORC1; molecular weight [MW], 289; N=3; A), phosphorylated mTOR S2481 (mTORC2; MW, 289; N=3; B), phosphorylated ribosomal protein S6 (S6RP) S235/236 (downstream of mTORC1; MW, 32; N=3; C), and phosphorylated protein kinase B (Akt) S473 (downstream of mTORC2 and endoplasmic reticulum stress; MW, 60; N=3; D) in response to TP for the duration, as indicated, compared with normal pacing (NP). Note that all NP data shown are after 8 hours of observation. **P≤0.01, ***P≤0.001 vs NP.

Figure 3

Tachypacing (TP) augments levels of endoplasmic reticulum (ER) stress markers and the autophagy gene ATG12. A, Representative Western blot of phosphorylated eIF2α S51 (molecular weight [MW], 38), an ER stress marker, basal eIF2α (MW, 36), and GAPDH levels during normal pacing (NP) or in response to TP for the indicated duration. B, Quantified data of the ratio of phosphorylated eIF2α S51 normalized for basal eIF2α protein levels (N=3). C, Quantitative real‐time polymerase chain reaction of ER stress markers ATF4, ATF6, CHOP, and heat shock protein (HSP) A5 and the autophagy‐related gene ATG12 in response to TP for the indicated duration relative to NP (N=3). Note that all NP data shown are after 8 hours of observation. **P≤0.01, ***P≤0.001 vs NP.

Figure 4

Patients with persistent atrial fibrillation (PeAF) show markers of endoplasmic reticulum (ER) stress and autophagy. A, Electron microscopic image of left atrial appendage (LAA) of a patient in sinus rhythm (SR), showing normal sarcomere structures and absence of autophagosomes and autolysosomes. B, Image of LAA of a patient in SR, showing normal sarcomere structures and absence of perinuclear autophagosomes and autolysosomes at higher magnification. C, Electron microscopic image of LAA of a patient with PeAF, which shows the presence of autophagosomes and autolysosomes with an electron‐dense core with a perinuclear (N) localization. D, Image of LAA of a patient with PeAF at a higher magnification, showing the presence of autophagosomes and autolysosomes. E, Top panel: Representative Western blot of the autophagy markers LC3B‐II and p62 and the ER stress chaperone‐protein heat shock protein (HSP) A5 in atrial appendages of patients with PeAF vs those in SR. Bottom panel: Quantified data of the autophagy markers LC3B‐II and p62 and the ER stress chaperone‐protein HSPA5 in atrial appendages of patients with PeAF vs those in SR. F through J, Significant correlations between levels of the autophagy marker p62 and markers of cardiomyocyte structural remodeling in patients with PeAF and patients in SR. F, Cardiac troponin T (cTnT). G, Cardiac troponin I (cTnI). H, α‐Tubulin (Tub). I Myolysis. J, HSPA5. RAA indicates right atrial appendage. **P≤0.01, ***P≤0.001 vs SR.

Figure 5

Inhibition of endoplasmic reticulum (ER) stress and autophagy protects against tachypacing (TP)–induced contractile dysfunction in HL‐1 cardiomyocytes and Drosophila melanogaster. A, Representative Western blot of ER stress marker (eIF2α‐PS51) and autophagy markers (LC3B‐II and p62) in HL‐1 cardiomyocytes pretreated with dimethyl sulfoxide (DMSO; control [C]), the autophagy modulator pepstatin A (PepA) or bafilomycin A1 (BAF), or the molecular chaperone 4‐phenyl butyrate (4PBA). B, Quantified data showing that HL‐1 cardiomyocytes treated with 4PBA reveal attenuation of TP‐induced increase in eIF2α‐PS51, LC3B‐II induction, and reduction in p62. PepA and BAF inhibit lysosomal cathepsin D/E and lysosomal fusion, respectively, and therefore result in an induction of LC3B‐II levels and attenuation of p62 reduction without affecting upstream eIF2α‐PS51 levels. Open bars represent normal‐paced (NP) cardiomyocytes, whereas closed bars represent TP cardiomyocytes after 8 hours of observation. N=3. C, Confocal images of NP and TP HL‐1 cardiomyocytes after 8 hours of observation, stained for myosin heavy chain with DMSO (C), 4PBA, BAF, or PepA pretreatment. D, Quantified data showing the number of puncta for the conditions as indicated, all obtained after 8 hours of observation. 4PBA pretreatment protects against the formation of perinuclear puncta (n/N=60/3). E, Representative Ca²⁺ transients (CaT; 5 seconds) of HL‐1 cardiomyocytes after NP or TP. HL‐1 cardiomyocytes were pretreated with the autophagy modulators PepA or BAF, or the chemical chaperone 4PBA, followed by NP or TP and measurement of CaT. F, Quantified CaT amplitude of HL‐1 cardiomyocytes after NP or TP (n/N=60/4). HL‐1 cardiomyocytes were pretreated with the autophagy modulators PepA or BAF or the ER chaperone 4PBA. G, Representative CaT (5 seconds) of HL‐1 cardiomyocytes transfected with empty plasmid (C) or ER chaperone heat shock protein (HSP) A5, followed by NP or TP. H, Quantified CaT amplitude of NP and TP HL‐1 cardiomyocytes transiently transfected with empty plasmid or HSPA5 (n/N=30/3). I, Representative CaT (5 seconds) of HL‐1 cardiomyocytes transfected with empty plasmid (C), eIF2α wild‐type, nonphosphorylated (S52A), or phosphorylated mimetic (S52D) mutant and followed by NP or TP. J, Quantified CaT amplitude of NP and TP cardiomyocytes transiently transfected with empty plasmid (C), eIF2α wild‐type, constitutively nonphosphorylated (S52A), or constitutively phosphorylated (S52D) mutant (n/N=30/3). K, Representative heart wall contractions of Drosophila monitored before TP (sinus rhythm [SR]) and after TP with DMSO (C) or PepA, BAF, or 4PBA pretreatment. L, Quantified data showing heart wall contraction rates of Drosophila before and after TP with DMSO (C) or PepA, BAF, or 4PBA treatment. Open bars represent NP (in HL‐1 cardiomyocytes) or spontaneous heart rate (SR; in Drosophila), and closed bars represent TP HL‐1 cardiomyocytes or Drosophila. N=9 to 15 prepupae for each group. Note that all NP data shown are after 8 hours of observation. *P≤0.05, **P≤0.01, ***P≤0.001 vs control NP or before TP; ^# P≤0.05, ^## P≤0.01, ^### P≤0.001 vs control (after) TP.

Figure 6

Sodium salt of phenyl butyrate (Na‐PBA) protects against atrial remodeling in a dog model for atrial fibrillation (AF). Atrial tachypacing (ATP) induces atrial remodeling, measured as shortening of action potential duration (ADP90; A), reduced L‐type Ca²⁺ current (ICaL; B), and increased diastolic calcium levels in cardiomyocytes (n=15–40 cardiomyocytes; C). D, Representative calcium transient (CaT) and cell shortening (CS) tracers for the conditions, as indicated. Furthermore, ATP results in loss of CaT amplitude (E), loss of contractility (F), reduced adaptation of the effective refractory period (ERP) at different basic cycle lengths (BCLs; G), and increased duration of induced AF (H). All ATP‐induced atrial remodeling end points were significantly attenuated by Na‐PBA treatment. C indicates control. *P≤0.05, **P≤0.01, ***P≤0.001 vs C; ^# P≤0.05, ^## P≤0.01, ^### P≤0.001 vs ATP.

Figure 7

Sodium salt of phenyl butyrate (Na‐PBA) protects against endoplasmic reticulum stress and autophagy in a dog model for atrial fibrillation. A, Top panel: Representative Western blot. Bottom panel: Quantified data revealing a significant reduction in p62 levels in atrial tachypacing (ATP), which was not significantly reduced by Na‐PBA treatment compared with control (C) dogs. B, Representative Western blot of LC3B‐I/II and loading control β‐actin (molecular weight [MW], 43) in groups, as indicated. ATP causes significant induction in LC3B‐II levels, which was significantly reduced in case of Na‐PBA treatment. C, Representative Western blot of heat shock protein (HSP) A5, showing a trend (P=0.058) in induction of HSPA5, which was not altered in Na‐PBA–treated group. D, Representative Western blot of myosin heavy chain (MHC; MW, 230) in groups, as indicated. ATP causes a significant reduction in MHC, which was not changed in case of Na‐PBA treatment. N=7 dogs for each group. *P≤0.05 vs C, ^# P≤0.05 vs ATP.

Figure 8

Proposed model for the role of autophagy and disease progression in atrial fibrillation (AF). AF triggers endoplasmic reticulum (ER) stress in cardiomyocytes, followed by the ER stress response, which results in activation of ATF6 and upregulation of heat shock protein (HSP) A5 expression in an attempt to restore ER homeostasis. ER stress then activates downstream phosphorylation of eIF2α.43 In turn, this results in the activation of the transcription factor ATF4, which regulates the expression of autophagy genes and LC3B, causing activation of autophagy by stimulating induction and elongation of autophagosomes. Initially, AF‐induced activation of autophagy may preserve cardiomyocyte proteostasis; however, excessive stress‐induced autophagy contributes to loss of contractile function and cardiac remodeling. ER stress–induced autophagy appears maladaptive, because inhibition of autophagy via 4‐phenyl butyrate (4PBA), HSPA5, or nonphosphorylatable mutant eIF2α (S51A) overexpression, pepstatin A (PepA), and bafilomycin A1 (BAF) prevented AF‐associated remodeling and progression in our studies. MHC indicates β‐myosin heavy chain 7.