Myocardial stress and autophagy: mechanisms and potential therapies

Abstract

Autophagy is a ubiquitous cellular catabolic process responsive to energy stress. Research over the past decade has revealed that cardiomyocyte autophagy is a prominent homeostatic pathway, important in adaptation to altered myocardial metabolic demand. The cellular machinery of autophagy involves targeted direction of macromolecules and organelles for lysosomal degradation. Activation of autophagy has been identified as cardioprotective in some settings (that is, ischaemia and ischaemic preconditioning). In other situations, sustained autophagy has been linked with cardiopathology (for example, sustained pressure overload and heart failure). Perturbation of autophagy in diabetic cardiomyopathy has also been observed and is associated with both adaptive and maladaptive responses to stress. Emerging research findings indicate that various forms of selective autophagy operate in parallel to manage various types of catabolic cellular cargo including mitochondria, large proteins, glycogen, and stored lipids. In this Review, induction of autophagy associated with cardiac benefit or detriment is considered. The various static and dynamic approaches used to measure autophagy are critiqued, and current inconsistencies in the understanding of autophagy regulation in the heart are highlighted. The prospects for pharmacological intervention to achieve therapeutic manipulation of autophagic processes are also discussed.

Figure 1.. Autophagy machinery.

The degradation of cellular components by autophagy is coordinated by a number of protein complexes and vesicle fusion events. The pre-autophagosome membrane can originate from the endoplasmic reticulum (ER) and requires the ULK complex (containing ULK1/2, Atg13, FIP200, Atg101) and movement of Beclin1 away from Bcl2 and into the PI3K complex (containing PI3K(III), Beclin1, Atg14). The PI3K complex produces PI(3)P lipid which attracts WIPI, Atg9 and Atg2 to the pre-autophagosome to position these key proteins ready to expand the membrane into an autophagosome. The Atg16L1 complex (containing Atg16L1, Atg12, Atg5) is recruited to the forming autophagosome membrane by Rab33. Atg8 family proteins are incorporated into the autophagosome membrane via Atg7-and Atg3-mediated lipidation (PE), anchoring them into the autophagosome double-membrane. Cellular cargo to be degraded are recruited into the autophagosome by adaptor proteins and bind to an Atg8 family member. The mechanism of autophagosome closure around the cargo is not well established, but may involve Atg2, Atg8 and Atg9. Autophagosomes can traffick along the cytoskeleton and various proteins from the Rab (Rab5, Rab7), UVRAG and SNARE (Stx7, Stx8, Stx17, VTI1b, and Vamp7) families are implicated in lysosome fusion. LAMP1 and LAMP2 have been implicated in various lysosomal functions including maintaining lysosomal membrane integrity, lysosome-autophagosome fusion, and lysosome motility. The vacuolar-type H+ ATPase (V-ATPase) pumps protons across the lysosomal membrane to acidify the lumen. Hydrolases in the lysosome (blue circles) degrade the autophagosome contents. Chloroquine (CQ) and bafilomycin are pharmacological agents used experimentally to block autophagosome/lysosome fusion (by neutralizing or preventing acidification) to assess autophagosome clearance.

Figure 2.. Cargo-selective autophagy.

A new understanding of cargo-selective autophagy pathways is emerging. Macrophagy involves the breakdown of protein macromolecules with the p62 adaptor protein recruiting the protein aggregates into the autophagosome to bind to LC3B. Negative feedback regulation of macrophagy by proteolysis of the cargo, yielding amino acids - especially branched chain amino acids, is mediated through re-activation of mTOR. Mitophagy involves the recruitment of mitochondria to the autophagosome by a number of different adaptor proteins, including Bnip3, Nix and p62, which bind to LC3B in the autophagosome membrane. Mitophagy is stimulated by reactive oxygen species (ROS), collapse of the mitochondrial membrane potential (mito Ψ) and low ATP levels. Glycophagy involves the tagging of glycogen with the adaptor protein, starch-binding protein domain 1 (STBD1) which recruits glycogen into the autophagosome by binding to GABARAPL1. Glycophagy may be regulated by its breakdown product, glucose.

Figure 3.. Physiological regulation of autophagy in the heart.

Cardiomyocyte energy status regulates autophagy via metabolic signaling. A substrate deficit (e.g. during nutrient deprivation or hypoxia) leads to low ATP levels which stimulate AMPK activity. AMPK activates autophagy via stimulating ULK1 and PI3K(III) and inhibiting autophagy suppressors such as mTORC1 and JNK which promotes the interaction of Bcl2 and Beclin1, thus preventing Beclin1 from moving to the PI3K(III) complex for autophagy. A growth or cell survival stimulus activates insulin/insulin-like growth factor (IGF) signaling in cardiomyocytes leading to activation of the IRS1/PI3K(I)/Akt pathway. Rheb activation results in inhibition of autophagy via activation of autophagy suppressors (primarily mTORC1) and inhibition of transcription factors FOXO1/3 and TFEB. AMPK: AMP-activated kinase; Atg: autophagy; ULK1: unc-51 like autophagy activating kinase 1; PI3K(III): phosphoinositide-3-kinase class 3, mTORC1: mammalian target of rapamycin complex 1; JNK: c-Jun N-terminal kinase; Bcl2: B-cell lymphoma 2; IGF: insulin growth factor; IRS1: insulin receptor substrate 1; PI3K(I): phosphoinositide-3-kinase class 1; Rheb: Ras homolog enriched in brain; FOXO: Forkhead box; TFEB: transcription factor EB.

Figure 4.. Autophagy and ischemia-reperfusion.

Cartoon depicting the transverse view of the heart showing ischemic ‘at risk’ zone (grey shaded) and post-reperfusion necrotic area (black). During ischemia (blue zone), activation of autophagy is linked with ATP depletion, activation of AMPK and GSK3β, and inhibition of mTORC1. During reperfusion (pink zone), a surge in the production of reactive oxygen species (ROS) is associated with inhibited GSK3β activity and ATP levels are at least partially restored. Question marks highlight areas of uncertainty requiring further investigation. Some indication that sustained mTORC1 inhibition and AMPK activation is linked with elevated autophagy during reperfusion is evident. AMPK: AMP-activated kinase; mTORC1: mammalian target of rapamycin complex 1; GSK3β: glycogen synthase kinase 3β.