Mesaconine alleviates doxorubicin-triggered cardiotoxicity and heart failure by activating PINK1-dependent cardiac mitophagy.

Aberrant mitophagy has been identified as a driver for energy metabolism disorder in most cardiac pathological processes. However, finding effective targeted agents and uncovering their precise modulatory mechanisms remain unconquered. Fuzi, the lateral roots of Aconitum carmichaelii, shows unique efficacy in reviving Yang for resuscitation, which has been widely used in clinics. As a main cardiotonic component of Fuzi, mesaconine has been proven effective in various cardiomyopathy models. Here, we aimed to define a previously unrevealed cardioprotective mechanism of mesaconine-mediated restoration of obstructive mitophagy. The functional implications of mesaconine were evaluated in doxorubicin (DOX)-induced heart failure models. DOX-treated mice showed characteristic cardiac dysfunction, ectopic myocardial energy disorder, and impaired mitophagy in cardiomyocytes, which could be remarkably reversed by mesaconine. The cardioprotective effect of mesaconine was primarily attributed to its ability to promote the restoration of mitophagy in cardiomyocytes, as evidenced by elevated expression of PINK1, a key mediator of mitophagy induction. Silencing PINK1 or deactivating mitophagy could completely abolish the protective effects of mesaconine. Together, our findings suggest that the cardioprotective effects of mesaconine appear to be dependent on the activation of PINK1-induced mitophagy and that mesaconine may constitute a promising therapeutic agent for the treatment of heart failure.

Autophagic Flux Detection: Significance and Methods Involved.

Macroautophagy is an important biological process in eukaryotic cells by which longevity proteins, misfolded proteins, and damaged organelles are degraded. The autophagy process consists of three key steps: (1) the formation of autophagosomes; (2) the fusion of the autophagosomes with lysosomes; and (3) the degradation of the contents of autolysosomes. If any of the three steps is impaired, autophagy will not be able to complete its biological function. Dysfunctional or blocked autophagy is closely involved in the pathogenesis of a variety of diseases. The accurate determination of the autophagy activity in vivo and in vitro has become a challenge in the field of autophagy research. At present, the most widely used detection method to determine autophagy activity in mammalian cells is to quantify LC3B in the cells by Western blot, or to observe the formation and changes of autophagosomes and autolysosomes by immunofluorescence and electron microscopy. However, ignoring the dynamic characteristics of autophagy and only evaluating the number of autophagosomes or the presence of LC3B cannot completely reflect the activation or a blockage of the autophagy system, and objectively analyze its real role in the occurrence and development of a disease. For example, the accumulation of autophagosomes and autolysosomes can occur through an increase in substrate to be degraded after the activation of autophagy, or it may be caused by the partial obstruction or blockage of autophagy. In this chapter, new and familiar ways to detect the autophagic flux are methodically summarized to provide researchers with a multi-angled viewpoint.

Disrupting the TRIB3-SQSTM1 interaction reduces liver fibrosis by restoring autophagy and suppressing exosome-mediated HSC activation.

Impaired macroautophagy/autophagy is involved in the pathogenesis of hepatic fibrosis. However, how aberrant autophagy promotes fibrosis is far from understood. Here, we aimed to define a previously unrevealed pro-fibrotic mechanism for the stress protein TRIB3 (tribbles pseudokinase 3)-mediated autophagy dysfunction. Human fibrotic liver tissues were obtained from patients with cirrhosis who underwent an open surgical repair process. The functional implications of TRIB3 were evaluated in mouse models of hepatic fibrosis induced by bile duct ligation (BDL) or thioacetamide (TAA) injection. Human fibrotic liver tissues expressed higher levels of TRIB3 and selective autophagic receptor SQSTM1/p62 (sequestosome 1) than nonfibrotic tissues and the elevated expression of TRIB3 and SQSTM1 was positively correlated in the fibrotic tissues. Silencing Trib3 protected against experimentally induced hepatic fibrosis, accompanied by restored autophagy activity in fibrotic liver tissues. Furthermore, TRIB3 interacted with SQSTM1 and hindered its binding to MAP1LC3/LC3, which caused the accumulation of SQSTM1 aggregates and obstructed autophagic flux. The TRIB3-mediated autophagy impairment not only suppressed autophagic degradation of late endosomes but also promoted hepatocellular secretion of INHBA/Activin A-enriched exosomes which caused migration, proliferation and activation of hepatic stellate cells (HSCs), the effector cells of liver fibrosis. Disrupting the TRIB3-SQSTM1 interaction with a specific helical peptide exerted potent protective effects against hepatic fibrosis by restoring autophagic flux in hepatocytes and HSCs. Together, stress-elevated TRIB3 expression promotes hepatic fibrosis by interacting with SQSTM1 and interfering with its functions in liver-parenchymal cells and activating HSCs. Targeting this interaction is a promising strategy for treating fibroproliferative liver diseases.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTA2/α-SMA: actin, alpha 2, smooth muscle, aorta; BDL: bile duct ligation; BECN1/Beclin 1: beclin 1, autophagy related; CHX: cycloheximide; CQ: chloroquine; Edu: 5-ethynyl-2-deoxyuridine; ESCRT: endosomal sorting complexes required for transport; HSC: hepatic stellate cell; ILV: intralumenal vesicle; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MVB: multivesicular body; PIK3C3: phosphatidylinositol 3-kinase, catalytic subunit type 3; PPI: protein-protein interaction; SQSTM1/p62: sequestosome 1; TAA: thioacetamide; TEM: transmission electron microscopy; TGFB1/TGFß1: transforming growth factor, beta 1; TLR2: toll-like receptor 2; TRIB3: tribbles pseudokinase 3.

Targeting Extracellular Heat Shock Protein 70 Ameliorates Doxorubicin-Induced Heart Failure Through Resolution of Toll-Like Receptor 2-Mediated Myocardial Inflammation.

Background Heart failure (HF) is one of the most significant causes of morbidity and mortality for the cardiovascular risk population. We found previously that extracellular HSP70 (heat shock protein) is an important trigger in cardiac hypertrophy and fibrosis, which are associated with the development of heart dysfunction. However, the potential role of HSP70 in response to HF and whether it could be a target for the therapy of HF remain unknown. Methods and Results An HF mouse model was generated by a single IP injection of doxorubicin at a dose of 15 mg/kg. Ten days later, these mice were treated with an HSP70 neutralizing antibody for 5 times. We observed that doxorubicin treatment increased circulating HSP70 and expression of HSP70 in myocardium and promoted its extracellular release in the heart. Blocking extracellular HSP70 activity by its antibody significantly ameliorated doxorubicin-induced left ventricular dilation and dysfunction, which was accompanied by a significant inhibition of cardiac fibrosis. The cardioprotective effect of the anti-HSP70 antibody was largely attributed to its ability to promote the resolution of myocardial inflammation, as evidenced by its suppression of the toll-like receptor 2-associated signaling cascade and modulation of the intracellular distribution of the p50 and p65 subunits of nuclear factor-κB. Conclusions Extracellular HSP70 serves as a noninfectious inflammatory factor in the development of HF, and blocking extracellular HSP70 activity may provide potential therapeutic benefits for the treatment of HF.