Takafumi Oka

Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure

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 failure. 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 DNA. Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes. 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 showed infiltration of inflammatory cells and increased messenger RNA expression of inflammatory cytokines, with accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium. Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA, 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.

Inhibition of autophagy in the heart induces age-related cardiomyopathy.

Constitutive autophagy is important for control of the quality of proteins and organelles to maintain cell function. Damaged proteins and organelles accumulate in aged organs. We have previously reported that cardiac-specific Atg5 (autophagy-related gene 5)-deficient mice, in which the gene was floxed out early in embryogenesis, were born normally, and showed normal cardiac function and structure up to 10 weeks old. In the present study, to determine the longer-term consequences of Atg5-deficiency in the heart, we monitored cardiac-specific Atg5-deficient mice for further 12 months. First, we examined the age-associated changes of autophagy in the wild-type mouse heart. The level of autophagy, as indicated by decreased LC3-II (microtubule-associated protein 1 light chain 3-II) levels, in the hearts of 6-, 14- or 26-month-old mice was lower than that of 10-week-old mice. Next, we investigated the cardiac function and life-span in cardiac-specific Atg5-deficient mice. The Atg5-deficient mice began to die after the age of 6 months. Atg5-deficient mice exhibited a significant increase in left ventricular dimension and decrease in fractional shortening of the left ventricle at the age of 10 months, compared to control mice, while they showed similar chamber size and contractile function at the age of 3 months. Ultrastructural analysis revealed a disorganized sarcomere structure and collapsed mitochondria in 3- and 10-month-old Atg5-deficient mice, with decreased mitochondrial respiratory functions. These results suggest that continuous constitutive autophagy has a crucial role in maintaining cardiac structure and function.

Distal Protection Improved Reperfusion and Reduced Left Ventricular Dysfunction in Patients With Acute Myocardial Infarction Who Had Angioscopically Defined Ruptured Plaque

Background—Distal protection, in the Saphenous Vein Graft Angioplasty Free of Emboli (SAFER) trial, is demonstrated to prevent distal embolism in the percutaneous coronary intervention of saphenous vein graft. However, in the Enhanced Myocardial Efficacy and Recovery by Aspiration of Liberated Debris (EMERALD) trial, it was not effective in the percutaneous coronary intervention of native coronary arteries in patients with acute myocardial infarction (AMI). We hypothesized that its effectiveness would be determined by lesion characteristics. Therefore, we classified the type of culprit lesion by angioscopy and examined its influence on the effectiveness of distal protection, comparing patients with AMI treated with and without distal protection. Methods and Results—Consecutive patients with AMI treated without distal protection (n=110) from July 2000 to July 2002 and those treated with distal protection (n=81) from July 2002 to July 2004 were included. Patients in each group were subdivided according to whether or not they had angioscopically defined ruptured plaque at culprit lesion. Among those groups, incidence of no-reflow phenomenon, ST-segment resolution, myocardial blush grade, and left ventricular ejection fraction at 6 months were compared. Aspirated samples by distal protection were semiquantitatively and histologically analyzed and compared between patients with and without ruptured plaque. No-reflow phenomenon was most frequently (P<0.05) observed in patients with ruptured plaque treated without distal protection. ST-segment resolution (68±15% versus 40±21%, P<0.001), myocardial blush grade (2.6±0.5 versus 1.8±0.3, P<0.001), and left ventricular ejection fraction (47.2±6.7% versus 41.0±9.7%, P<0.01) were improved by distal protection among patients with ruptured plaque but not among patients without ruptured plaque. Aspirated samples >1 mm were detected more frequently (97.3% versus 78.5%, P<0.05) in patients with ruptured plaque than those without ruptured plaque. Histologically, aspirated samples contained plaque debris (95.3% versus 31.1%, P<0.05) more frequently in patients with ruptured plaque than in those without ruptured plaque. Conclusions—Distal protection reduced microcirculation damage and left ventricular dysfunction in patients with AMI who had angioscopically defined ruptured plaque. Distal embolization of plaque debris was detected more frequently in patients with ruptured plaque. These results suggest that microcirculation damage and left ventricular dysfunction are increased mainly by distal embolization of plaque debris rather than of thrombus.

Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation.

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.

The IκB Kinase β/Nuclear Factor κB Signaling Pathway Protects the Heart From Hemodynamic Stress Mediated by the Regulation of Manganese Superoxide Dismutase Expression

Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-&kgr;B signaling pathway regulates cell death, however, the effect of NF-&kgr;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-&kgr;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-&kgr;B pathway in response to pressure overload. I&kgr;B kinase (IKK) and NF-&kgr;B were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKβ-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 IKKβ/NF-&kgr;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.

Calpain protects the heart from hemodynamic stress.

Calpains make up a family of Ca2+-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca2+ overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.

Autophagy-mediated degradation is necessary for regression of cardiac hypertrophy during ventricular unloading

Cardiac hypertrophy occurs in response to a variety of stresses as a compensatory mechanism to maintain cardiac output and normalize wall stress. Prevention or regression of cardiac hypertrophy can be a major therapeutic target. Although regression of cardiac hypertrophy occurs after control of etiological factors, the molecular mechanisms remain to be clarified. In the present study, we investigated the role of autophagy in regression of cardiac hypertrophy. Wild-type mice showed cardiac hypertrophy after continuous infusion of angiotensin II for 14 days using osmotic minipumps, and regression of cardiac hypertrophy was observed 7 days after removal of the minipumps. Autophagy was induced during regression of cardiac hypertrophy, as evidenced by an increase in microtubule-associated protein 1 light chain 3 (LC3)-II protein level. Then, we subjected cardiac-specific Atg5-deficient (CKO) and control mice (CTL) to angiotensin II infusion for 14 days. CKO and CTL developed cardiac hypertrophy to a similar degree without contractile dysfunction. Seven days after removal of the minipumps, CKO showed significantly less regression of cardiac hypertrophy compared with CTL. Regression of pressure overload-induced cardiac hypertrophy after unloading was also attenuated in CKO. These results suggest that autophagy is necessary for regression of cardiac hypertrophy during unloading of neurohumoral and hemodynamic stress.

Rheb (Ras homologue enriched in brain)-dependent mammalian target of rapamycin complex 1 (mTORC1) activation becomes indispensable for cardiac hypertrophic growth after early postnatal period

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. Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb−/−). Rheb−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb−/− was lower than that in the control (Rheb+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb+/+ mice but not in Rheb−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb−/− hearts during the neonatal period. Rheb−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Regional difference of optimal contact force to prevent acute pulmonary vein reconnection during radiofrequency catheter ablation for atrial fibrillation.

Regional differences in optimal contact force (CF) to prevent acute pulmonary vein reconnection (APVR) during catheter ablation for atrial fibrillation (AF) remain unclear.

Evaluation of the cavotricuspid isthmus and right atrium by multidetector-row computed tomography in patients with common atrial flutter.

The sizes of the right atrium (RA), cavotricuspid isthmus, and Eustachian valve are predictors of success of radiofrequency catheter ablation for atrial flutter (AFL). We examined the relationship between the sizes of cavotricuspid isthmus as measured by multidetector-row computed tomography (MDCT) and fluoroscopy. We used eight-detector MDCT to measure the tricuspid isthmus of 23 patients prior to linear ablation for common AFL. One patient with a deep pouch in the RA was excluded. Parameters measured were (1) the length of the trace of isthmus (Ti), which was equivalent to the blocking line; (2) the size of the tricuspid isthmus (DTi); and (3) the distance from the tricuspid valve and inferior vena cava (IVC) (LDTi). DTi and LDTi indicate the size of the RA, reflecting the appropriately sized steerable ablation catheter, respectively. Of the 22 patients, 21 were ablated successfully without recurrence of AFL, and clinical success was achieved in one additional patient despite failure to obtain a bidirectional block. Ti, DTi, and LDTi were correlated with fluoroscopy time (r = 0.84, r = 0.88, and r = 0.88, respectively; P < 0.0001), total delivered energy (r = 0.81, r = 0.80, and r = 0.83, respectively; P < 0.0001), and application time (r = 0.84, r = 0.80, and r = 0.87, respectively; P < 0.0001). Measurement of the tricuspid isthmus by MDCT may noninvasively provide important information for successful linear ablation.