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.

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.

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.

Calpain protects the heart from hemodynamic stress.

Calpains make up a family of Ca(2+)-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 Ca(2+) 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.

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.

Activation of MTK1/MEKK4 induces cardiomyocyte death and heart failure.

MTK1 (MEKK4) is a mitogen-activated protein kinase kinase kinase that regulates the activity of its downstream mitogen-activated kinases, p38, and c-Jun N-terminal kinase (JNK). However, the physiological function of MTK1 in the heart remains to be determined. Here, we attempted to elucidate the function of MTK1 in the heart using in vitro and in vivo models. MTK1 was activated in the hearts of mice subjected to pressure overload-induced heart failure. Overexpression of a constitutively active mutant of MTK1 (MTK1DeltaN) induced apoptosis in isolated neonatal rat cardiomyocytes, whereas a kinase domain-deleted form of MTK1 attenuated H(2)O(2)-induced apoptosis. Specific inhibitors of p38 or JNK effectively protected cardiomyocytes from MTK1DeltaN-induced cell death. In mice, cardiac-specific overexpression of MTK1DeltaN resulted in early mortality compared with the lifespan of littermate controls. Echocardiographic analysis revealed increases in end-diastolic and end-systolic left ventricular internal dimensions and a decrease in fractional shortening in MTK1DeltaN transgenic mice. In addition, the mice showed characteristic phenotypes of heart failure such as an increase in lung weight. The number of TUNEL-positive myocytes and the level of cleaved caspase 3 protein were both increased in MTK1DeltaN transgenic mice. Thus, MTK1 plays an important role in the regulation of cell death and is also involved in the pathogenesis of heart failure.

The I{kappa}B kinase {beta}/nuclear factor {kappa}B 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)-kappaB signaling pathway regulates cell death, however, the effect of NF-kappaB 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-kappaB 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-kappaB pathway in response to pressure overload. IkappaB kinase (IKK) and NF-kappaB were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKbeta-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 IKKbeta/NF-kappaB 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.

Atherosclerotic and thrombogenic neointima formed over sirolimus drug-eluting stent: an angioscopic study.

OBJECTIVES: We sought to examine by angioscopy the neointima formation and thrombogenic potential of the neointima after deployment of a drug-eluting stent (DES). BACKGROUND: Late stent thrombosis after DES implantation, a major safety concern, has been associated with poor strut coverage by neointima. Intracoronary angioscopy provides a method for visual evaluation of stent coverage by neointima and detection of thrombus in the stented coronary segment. METHODS: Patients undergoing implantation of a sirolimus DES (n = 57) were serially examined by angioscopy immediately after (baseline) and again at 10 months (follow-up) after implantation. The angioscopic color grade of the neointima from white to yellow was assessed in a semiquantitative manner. Stent coverage was classified into not covered (Grade 0), covered by a thin layer (Grade 1), or buried under neointima (Grade 2). The thrombogenic potential of the neointima was evaluated by the prevalence of thrombus on the neointima. RESULTS: The maximum yellow color grade of the neointima within DES-implanted lesions increased significantly from baseline to follow-up (1.4 +/- 1.1 vs. 1.9 +/- 0.6, p = 0.0008). Even among lesions without yellow color at baseline, yellow color was detected in 94% (17 of 18) of lesions at follow-up. The prevalence of thrombus was significantly higher on the yellow than on the white neointimal areas. Thrombus was detected on yellow and/or Grade-0/1 neointima, but never on the white Grade-2 neointima. CONCLUSIONS: Sirolimus DES promoted formation of atherosclerotic yellow neointima in the stent-implanted lesion at 10-month follow-up. Thrombus was detected more often on the yellow area than on the white area and was never detected where a stent was buried under white neointima. These data suggest that the increased potential risk of late stent thrombosis in DES lesions may be due to the newly formed yellow neotima and cholesterol-laden plaque.

Plaque color analysis by the conventional yellow-color grading system and quantitative measurement using LCH color space.

BACKGROUND: Yellow plaques of higher color grades are regarded as more vulnerable. We tried to elucidate the characteristics of yellow color that are regarded as the sign of vulnerable plaques by an objective and quantitative plaque color analysis. METHODS: The color of yellow plaques was quantitatively analyzed using LCH color space and was presented by the maximum values of lightness (Lmax), chroma (Cmax), and hue (Hmax). Effect of light intensity on these parameters was experimentally examined (five plaque models, six light intensities). Relation between conventional yellow-color grade and LCH parameters was examined (31 plaques). Color analysis with LCH color space was applied (1) to compare the culprit lesions of unstable angina (eight patients) and of stable effort angina (nine patients) and (2) to evaluate the regression of yellow plaque by 80-week statin treatment (four patients, 19 plaques). RESULTS: Cmax and Hmax were not influenced by light intensity so far as it was within appropriate range. Cmax (13 +/- 3, 25 +/- 5, and 28 +/- 4) became higher and Hmax (178 +/- 26, 134 +/- 10, and 91 +/- 16) became lower as the grade became higher (P < 0.0001). Culprit lesions of unstable angina had higher Cmax (22.8 +/- 7.9 vs 14.8 +/- 7.5, P = 0.04) and lower Hmax (73.3 +/- 19.7 vs 168.0 +/- 71.5, P = 0.002) than stable effort angina. Statin treatment reduced Cmax (21.3 +/- 9.6 vs 14.6 +/- 3.1, P = 0.004) and increased Hmax (82.6 +/- 25.8 vs 142.3 +/- 54.1, P = 0.0005). CONCLUSIONS: Plaque color was consistently and quantitatively measurable using LCH color space uninfluenced by light intensity of appropriate range. Vulnerable yellow color had high chroma and low hue, indicating vivid and reddish-yellow color.

Acute myocardial infarction without disrupted yellow plaque in young patients below 50 years old.

OBJECTIVE: Thrombosis caused by disrupted yellow plaque is regarded as a cause of acute myocardial infarction (MI). However, it has not been clarified if young patients have the same pathophysiology as older ones. Therefore, we elucidated clinical and angioscopic characteristics of young patients. METHODS: Among a series of patients (n = 893) who received catheterization for acute MI, clinical characteristics were compared between patients <50 years (n = 66) and the rest of patients. Angioscopic appearance of culprit lesions was evaluated in 20 young patients in whom angioscopic examination was successfully performed. It was determined if culprit lesions had disrupted yellow plaque with thrombus (DYP&T). RESULTS: Patients <50 years had higher prevalence of smoking (68% vs. 48%, P = 0.001), obesity (42% vs. 15%, P < 0.0001), and hypercholesterolemia (56% vs. 35%, P = 0.0005) than those >or=50 years. DYP&T was detected at culprit lesions in 14 (70%) patients. Prevalence of DYP&T was lower in patients <40 years (44% vs. 91%, P = 0.02) than those between 40 and 50 years. Patients <40 years had a trend for higher prevalence of smoking (88% vs. 62%, P = 0.05) than those between 40 and 50 years. CONCLUSIONS: Patients with acute MI < 50 years, especially <40 years, had lower prevalence of DYP&T but higher prevalence of smoking, obesity, and hypercholesterolemia. Smoking may play an important role for thrombotic occlusion at lesions with relatively low thrombogenic potential.

Angioscopic evaluation of neointima coverage: sirolimus drug-eluting stent versus bare metal stent.

BACKGROUND: The process of neointima formation after bare metal stent (BMS) implantation has been previously elucidated by angioscopic observations; however, that after drug-eluting stent (DES) implantation has not been clarified. Therefore, we compared the angioscopic appearance of neointima over DESs with that over BMSs 6 months after implantation. METHODS AND RESULTS: Patients who received an implantation of a BMS (n = 13) or a sirolimus DES (n = 24) were included in this study. Angiographic and angioscopic examinations were performed at 6 months. The color of the stented lesion (white or yellow), coverage of stent by neointima (not covered, covered by a thin layer, or buried under neointima), and thrombus at the stented lesion (presence or absence) were angioscopically evaluated. Of the 24 lesions in which a DES was implanted, 11 (46%) had a part where the stent strut had no coverage, 21 (88%) had a part where it was covered by a thin layer, and 11 (46%) had a part where it was buried under neointima. Of the 13 lesions in which a BMS was implanted, 1 (8%) lesion had a part where the stent strut had no coverage, 4 (31%) lesions had a part where it was covered by a thin layer, and 13 (100%) lesions had a part where it was buried under neointima. The prevalence of a stent buried under neointima (46% vs 100%, P = .001) was lower and that of thrombus (42% vs 8%, P = .03) was higher in DES-implanted lesions as compared with BMS-implanted lesions. The prevalence of thrombus (64% vs 17%, P = .005) was higher in the yellow area than in the white area when a DES was implanted. CONCLUSION: Sirolimus DESs, as compared with BMSs, were poorly covered by neointima and were accompanied by thrombus especially when there was a yellow plaque under the stents. Thus, the thrombogenic potential in DES-implanted lesions may be sustained by the inhibition of neointima formation over thrombogenic plaques.

Number of yellow plaques detected in a coronary artery is associated with future risk of acute coronary syndrome: detection of vulnerable patients by angioscopy.

OBJECTIVES: We sought to test whether the risk of acute coronary syndrome (ACS) can be estimated by angioscopy. BACKGROUND: Disruption of vulnerable plaque and subsequent thrombosis is regarded as a major mechanism of ACS. Although yellow plaques are supposedly vulnerable, the association between angioscopically determined extent of coronary atherosclerosis and risk of ACS events has not been reported. METHODS: Patients (n = 552) who received catheterization and angioscopic examination for the diagnosis of coronary artery diseases were prospectively included and followed up for new onset of ACS events. Yellow color intensities of all detected yellow plaques were graded as 1, 2, or 3 according to the standard colors. Number of yellow plaques (NYP) in a coronary artery and maximum color grade of detected yellow plaques (maxYP) were determined. Association between the incidence of ACS events and angioscopic findings were analyzed. RESULTS: Follow-up interval was 57.3 +/- 22.1 months. Acute coronary syndrome events were detected in 39 patients (7.1%). Although maxYP was not statistically different (2.0 +/- 0.7 vs. 1.8 +/- 0.9; p = 0.18), NYP was higher in the patients with an ACS event than those without the event (3.1 +/- 1.8 vs. 2.2 +/- 1.5; p = 0.008). Patients with NYP > or =2 and those with NYP > or =5 had 2.2- and 3.8-fold higher event rates, respectively, than those with NYP 0 or 1 (9.0% and 15.6%, respectively, vs. 4.1%; p = 0.02). Multivariate logistic regression analysis revealed NYP and multivessel disease as the independent risk factors of ACS events. CONCLUSIONS: Patients with multiple yellow plaques per vessel have a higher risk of suffering ACS events than those with NYP 0 or 1. Angioscopy would be useful to detect vulnerable patients.

Angioscopically-determined extent of coronary atherosclerosis is associated with severity of acute coronary syndrome.

OBJECTIVE: Some patients with acute coronary syndrome (ACS) have large myocardial infarction but others have small or no infarction. However, what makes this difference has not been clarified. We compared the angioscopic findings between those two categories of ACS patients and examined the association between the severity of ACS and the morphology of both culprit lesion and nonculprit coronary segments. METHODS: Prospectively and consecutively enrolled patients with ACS were classified as CK-elevation-ACS (CKE-ACS; n = 54) or non-CK-elevation-ACS (NCKE-ACS; n = 22). Patients were diagnosed as CKE-ACS when the elevation (greater than twice the normal upper limit) of CK-MB was detected; otherwise, patients were diagnosed as NCKE-ACS. They all underwent emergent catheterization and PCI of the culprit lesion. The entire culprit artery was observed by angioscopy, and the prevalence of thrombus and the color grade of yellow plaques were evaluated. The color grade of yellow plaques were classified as 0 (white), 1 (slight yellow), 2 (yellow), or 3 (intense yellow) according to the standard colors. The color grade of culprit plaque (CC), number (NP) and maximum (MC) color grade of yellow plaques in the nonculprit segments, plaque index (PI = N x MC), and prevalence of thrombus at the culprit lesion (CT) and in the nonculprit segments (NT) were compared between CKE-ACS and NCKE-ACS patients. RESULTS: CC (1.9 +/- 0.9 vs. 1.7 +/- 0.8; p = 0.3) and CT (93% vs. 77%; p = 0.06) were not significantly different between CKE-ACS and NCKE-ACS patients, however, NP (2.2 +/- 1.6 vs. 1.4 +/- 1.2; p = 0.03), MC (1.8 +/- 0.9 vs. 1.2 +/- 0.9; p = 0.008), PI (4.8 +/- 4.4 vs. 2.4 +/- 3.1; p = 0.03), and NT (39% vs. 11%; p = 0.02) were significantly higher in CKE-ACS than in NCKE-ACS patients. CONCLUSION: Although the culprit lesions of CKE- and NCKE-ACS had similar yellow color grades, the culprit lesions of CKE-ACS showed a trend towards a higher prevalence of thrombus. A greater number of yellow plaques of higher color grades and a higher prevalence of thrombosis in the nonculprit segments were detected in CKE-ACS compared to NCKE-ACS patients. The angioscopically-determined extent of coronary atherosclerosis appeared advanced in CKE-ACS patients compared to NCKE-ACS patients.