Heart failure is a leading cause of morbidity and mortality in industrialized countries. Although infection with microorganisms is not involved in the development of heart failure in most cases, inflammation has been implicated in the pathogenesis of heart failure1. However, the mechanisms responsible for initiating and integrating inflammatory responses within the heart remain poorly defined. Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA2,3,4. Mitochondria damaged by external hemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes5. Here, we show that mitochondrial DNA that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated inflammatory responses in cardiomyocytes and is capable of inducing myocarditis, and dilated cardiomyopathy. Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions, but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload. Early in the pathogenesis, DNase II-deficient hearts exhibited infiltration of inflammatory cells and increased mRNA expression of inflammatory cytokines, with accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium. Administration of the inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9 attenuated the development of cardiomyopathy in DNase II-deficient mice. Furthermore, Tlr9-ablation improved pressure overload-induced cardiac dysfunction and inflammation even in mice with wild-type Dnase2a alleles. These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.

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Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells.

Abstract-Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-B signaling pathway regulates cell death, however, the effect of NF-B pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-B pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-B pathway in response to pressure overload. IB kinase (IKK) and NF-B were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKK␤-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKK␤/NF-B signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload. Key Words: heart failure Ⅲ apoptosis Ⅲ NF-B C ardiac remodeling is generally accepted as a determinant of the clinical course of heart failure. Cardiomyocyte apoptotic death plays an important role in the progression of cardiac remodeling. [1][2][3][4] The loss of cardiomyocytes caused by apoptosis is predicted to reduce contractility and promote slippage of muscle bundles, wall thinning, and dilatation, which are commonly observed during heart failure. Neurohumoral factors and cytokines that are induced by mechanical stress on cardiomyocytes activate various intracellular signaling pathways, which regulate apoptotic cell death.The nuclear factor (NF)-B transcription factors (p50, p52, RelA, c-Rel, and RelB) play important roles in many physiological and pathological conditions. These transcriptional factors are kept inactive in the cytoplasm by binding of inhibitory proteins, the IB (inhibitor of NF-B) family. On stimulation, IBs are phosphorylated at serine residues, leading to their ubiquitination and degradation by the 26S proteasome. The freed NF-B components dimerize and translocate to nucleus, where they bind to specific sequences in either the promoter or enhancer regions of target genes. 5 Activation process is dependent on phosphorylation of IB proteins, which is mediated by the IKK complex. The IKK complex is comp...

SummaryDegradation of mitochondria by selective autophagy, termed mitophagy, contributes to the control of mitochondrial quality. Bcl2-L-13 is a mammalian homolog of Atg32, which is an essential mitophagy receptor in yeast. However, the molecular machinery involved in Bcl2-L-13-mediated mitophagy remains to be elucidated. Here, we show that the ULK1 (unc-51-like kinase) complex is required for Bcl2-L-13 to process mitophagy. Screening of a series of yeast Atg mutants revealed that a different set of ATG genes is used for Bcl2-L-13- and Atg32-mediated mitophagy in yeast. The components of the Atg1 complex essential for starvation-induced autophagy were indispensable in Bcl2-L-13-, but not Atg32-mediated, mitophagy. The ULK1 complex, a counterpart of the Atg1 complex, is necessary for Bcl2-L-13-mediated mitophagy in mammalian cells. We propose a model where, upon mitophagy induction, Bcl2-L-13 recruits the ULK1 complex to process mitophagy and the interaction of LC3B with ULK1, as well as Bcl2-L-13, is important for the mitophagy.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC) 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/-) mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts.

Heart failure is a major public health problem, and abnormal iron metabolism is common in patients with heart failure. Although iron is necessary for metabolic homeostasis, it induces a programmed necrosis. Iron release from ferritin storage is through nuclear receptor coactivator 4 (NCOA4)-mediated autophagic degradation, known as ferritinophagy. However, the role of ferritinophagy in the stressed heart remains unclear. Deletion of Ncoa4 in mouse hearts reduced left ventricular chamber size and improved cardiac function along with the attenuation of the upregulation of ferritinophagy-mediated ferritin degradation 4 weeks after pressure overload. Free ferrous iron overload and increased lipid peroxidation were suppressed in NCOA4-deficient hearts. A potent inhibitor of lipid peroxidation, ferrostatin-1, significantly mitigated the development of pressure overload-induced dilated cardiomyopathy in wild-type mice. Thus, the activation of ferritinophagy results in the development of heart failure, whereas inhibition of this process protects the heart against hemodynamic stress.

Background: Rheb (Ras homologue enriched in brain) regulates mammalian target of rapamycin complex 1 (mTORC1). Results: mTORC1 activity and cardiac hypertrophy are attenuated in Rheb-deficient hearts after the early postnatal period. Conclusion: Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after the early postnatal period. Significance: The findings provide insight into the regulatory mechanism of mTORC1 in postnatal heart development.