Cardiac MR Characterization of Myocardial Tissue Injury in a Miniature Swine Model of Cancer Therapy-related Cardiovascular Toxicity.

BACKGROUND: Left ventricular ejection fraction (LVEF) is the most commonly clinically used imaging parameter for assessing cancer therapy-related cardiac dysfunction (CTRCD). However, LVEF declines may occur late, after substantial injury. This study sought to investigate cardiovascular magnetic resonance (CMR) imaging markers of subclinical cardiac injury in a miniature swine model. METHODS: Female Yucatan miniature swine (n=14) received doxorubicin (2mg/kg) every 3 weeks for 4 cycles. CMR, including cine, tissue characterization via T1 and T2 mapping, and late gadolinium enhancement (LGE) was performed on the same day as doxorubicin administration and three weeks after the final chemotherapy cycle. In addition, MR spectroscopy (MRS) was performed during the 3 weeks after the final chemotherapy in 7 pigs. A single CMR and MRS exam was also performed in three Yucatan miniature swine that were age- and weight-matched to the final imaging exam of the doxorubicin-treated swine to serve as controls. CTRCD was defined as histological early morphologic changes, including cytoplasmic vacuolization and myofibrillar loss of myocytes, based on post-mortem analysis of humanely euthanized pigs after the final CMR exam. RESULTS: Of 13 swine completing five serial CMR scans, 10 (77%) had histological evidence of CTRCD. Three animals had neither histological evidence nor changes in LVEF from baseline. No absolute LVEF <40% or LGE were observed. Native T1, extracellular volume (ECV), and T2 at 12 weeks were significantly higher in swine with CTRCD than those without CTRCD (1178 ms vs. 1134 ms, p=0.002, 27.4% vs. 24.5%, p=0.03, and 38.1 ms vs. 36.4 ms, p=0.02, respectively). There were no significant changes in strain parameters. The temporal trajectories in native T1, ECV, and T2 in swine with CTRCD showed similar and statistically significant increases. At the same time, there were no differences in their temporal changes between those with and without CTRCD. MRS myocardial triglyceride content substantially differed among controls, swine with and without CTRCD (0.89%, 0.30%, 0.54%, respectively, ANOVA, p=0.01), and associated with the severity of histological findings and incidence of vacuolated cardiomyocytes. CONCLUSIONS: Serial CMR imaging alone has a limited ability to detect histologic CTRCD beyond LVEF. Integrating MRS myocardial triglyceride content may be useful for detection of early potential CTRCD.

Scanner-Independent MyoMapNet for Accelerated Cardiac MRI T1 Mapping Across Vendors and Field Strengths.

BACKGROUND: In cardiac T1 mapping, a series of T1 -weighted (T1 w) images are collected and numerically fitted to a two or three-parameter model of the signal recovery to estimate voxel-wise T1 values. To reduce the scan time, one can collect fewer T1 w images, albeit at the cost of precision or/and accuracy. Recently, the feasibility of using a neural network instead of conventional two- or three-parameter fit modeling has been demonstrated. However, prior studies used data from a single vendor and field strength; therefore, the generalizability of the models has not been established. PURPOSE: To develop and evaluate an accelerated cardiac T1 mapping approach based on MyoMapNet, a convolution neural network T1 estimator that can be used across different vendors and field strengths by incorporating the relevant scanner information as additional inputs to the model. STUDY TYPE: Retrospective, multicenter. POPULATION: A total of 1423 patients with known or suspected cardiac disease (808 male, 57 ± 16 years), from three centers, two vendors (Siemens, Philips), and two field strengths (1.5 T, 3 T). The data were randomly split into 60% training, 20% validation, and 20% testing. FIELD STRENGTH/SEQUENCE: A 1.5 T and 3 T, Modified Look-Locker inversion recovery (MOLLI) for native and postcontrast T1 . ASSESSMENT: Scanner-independent MyoMapNet (SI-MyoMapNet) was developed by altering the deep learning (DL) architecture of MyoMapNet to incorporate scanner vendor and field strength as inputs. Epicardial and endocardial contours and blood pool (by manually drawing a large region of interest in the blood pool) of the left ventricle were manually delineated by three readers, with 2, 8, and 9 years of experience, and SI-MyoMapNet myocardial and blood pool T1 values (calculated from four T1 w images) were compared with conventional MOLLI T1 values (calculated from 8 to 11 T1 w images). STATISTICAL TESTS: Equivalency test with 95% confidence interval (CI), linear regression slope, Pearson correlation coefficient (r), Bland-Altman analysis. RESULTS: The proposed SI-MyoMapNet successfully created T1 maps. Native and postcontrast T1 values measured from SI-MyoMapNet were strongly correlated with MOLLI, despite using only four T1 w images, at both field-strengths and vendors (all r > 0.86). For native T1 , SI-MyoMapNet and MOLLI were in good agreement for myocardial and blood T1 values in institution 1 (myocardium: 5 msec, 95% CI [3, 8]; blood: -10 msec, 95%CI [-16, -4]), in institution 2 (myocardium: 6 msec, 95% CI [0, 11]; blood: 0 msec, [-18, 17]), and in institution 3 (myocardium: 7 msec, 95% CI [-8, 22]; blood: 8 msec, [-14, 30]). Similar results were observed for postcontrast T1 . DATA CONCLUSION: Inclusion of field strength and vendor as additional inputs to the DL architecture allows generalizability of MyoMapNet across different vendors or field strength. EVIDENCE LEVEL: 2. TECHNICAL EFFICACY: Stage 2.

Microvascular Dysfunction in Patients with Idiopathic Dilated Cardiomyopathy: Quantitative Assessment with Phase Contrast Cine MR Imaging of the Coronary Sinus.

PURPOSE: The purposes of this study were to compare global coronary flow reserve (CFR) between patients with idiopathic dilated cardiomyopathy (DCM) and risk-matched controls using cardiac MRI (CMR), and to evaluate the relationship between global CFR and CMR left ventricular (LV) parameters. METHODS: Twenty-six patients with DCM and 26 risk-matched controls who underwent comprehensive CMR examination, including stress-rest coronary sinus flow measurement by phase contrast (PC) cine CMR were retrospectively studied. LV peak global longitudinal, radial, and circumferential strains (GLS, GRS, and GCS) were determined by feature tracking. RESULTS: Patients with DCM had significantly lower global CFR compared with the risk-matched controls (2.87 ± 0.86 vs. 4.03 ± 1.47, P = 0.001). Among the parameters, univariate linear regression analyses revealed significant correlation of global CFR with LV end-diastolic volume index (r = -0.396, P = 0.045), LV mass index (r = -0.461, P = 0.018), GLS (r = -0.558, P = 0.003), and GRS (r = 0.392, P = 0.047). Multiple linear regression analysis revealed GLS as the only independent predictor of global CFR (standardized ß = -0.558, P = 0.003). CONCLUSION: Global CFR was significantly impaired in patients with idiopathic DCM and independently associated with LV GLS, suggesting that microvascular dysfunction may contribute to deterioration of LV function in patients with idiopathic DCM.

Complementary prognostic value of stress perfusion imaging and global coronary flow reserve derived from cardiovascular magnetic resonance: a long-term cohort study.

BACKGROUND: Phase-contrast cine cardiovascular magnetic resonance (CMR) quantifies global coronary flow reserve (CFR) by measuring blood flow in the coronary sinus (CS), allowing assessment of the entire coronary circulation. However, the complementary prognostic value of stress perfusion CMR and global CFR in long-term follow-up has yet to be investigated. This study aimed to investigate the complementary prognostic value of stress myocardial perfusion imaging and global CFR derived from CMR in patients with suspected or known coronary artery disease (CAD) during long-term follow-up. METHODS: Participants comprised 933 patients with suspected or known CAD who underwent comprehensive CMR. Major adverse cardiac events (MACE) comprised cardiac death, non-fatal myocardial infarction, unstable angina, hospitalization for heart failure, stroke, ventricular arrhythmia, and late revascularization. RESULTS: During follow-up (median, 5.3 years), there were 223 MACE. Kaplan-Meier curve analysis revealed a significant difference in event-free survival among tertile groups for global CFR (log-rank, p < 0.001) and between patients with and without ischemia (p < 0.001). The combination of stress perfusion CMR and global CFR enhanced risk stratification (p < 0.001 for overall), and prognoses were comparable between the subgroup with ischemia and no impaired CFR and the subgroup with no ischemia and impaired CFR (p = 0.731). Multivariate Cox proportional hazard regression analysis showed that impaired CFR remained a significant predictor for MACE (hazard ratio, 1.6; p = 0.002) when adjusted for coronary risk factors and CMR predictors, including ischemia. The addition of impaired CFR to coronary risk factors and ischemia significantly increased the global chi-square value from 88 to 109 (p < 0.001). Continuous net reclassification improvement and integrated discrimination with the addition of global CFR to coronary risk factors plus ischemia improved to 0.352 (p < 0.001) and 0.017 (p < 0.001), respectively. CONCLUSIONS: During long-term follow-up, stress perfusion CMR and global CFR derived from CS flow measurement provided complementary prognostic value for prediction of cardiovascular events. Microvascular dysfunction or diffuse atherosclerosis as shown by impaired global CFR may play a role as important as that of ischemia due to epicardial coronary stenosis in the risk stratification of CAD patients.

Comparison of DeepStrain and Feature Tracking for Cardiac MRI Strain Analysis.

BACKGROUND: Myocardial feature tracking (FT) provides a comprehensive analysis of myocardial deformation from cine balanced steady-state free-precession images (bSSFP). However, FT remains time-consuming, precluding its clinical adoption. PURPOSE: To compare left-ventricular global radial strain (GRS) and global circumferential strain (GCS) values measured using automated DeepStrain analysis of short-axis cine images to those calculated using manual commercially available FT analysis. STUDY TYPE: Retrospective, single-center. POPULATION: A total of 30 healthy subjects and 120 patients with cardiac disease for DeepStrain development. For evaluation, 47 healthy subjects (36 male, 53 ± 5 years) and 533 patients who had undergone a clinical cardiac MRI (373 male, 59 ± 14 years). FIELD STRENGTH/SEQUENCE: bSSFP sequence at 1.5 T (Phillips) and 3 T (Siemens). ASSESSMENT: Automated DeepStrain measurements of GRS and GCS were compared to commercially available FT (Circle, cvi42) measures obtained by readers with 1 year and 3 years of experience. Comparisons were performed overall and stratified by scanner manufacturer. STATISTICAL TESTS: Paired t-test, linear regression slope, Pearson correlation coefficient (r). RESULTS: Overall, FT and DeepStrain measurements of GCS were not significantly different (P = 0.207), but measures of GRS were significantly different. Measurements of GRS from Philips (slope = 1.06 [1.03 1.08], r = 0.85) and Siemens (slope = 1.04 [0.99 1.09], r = 0.83) data showed a very strong correlation and agreement between techniques. Measurements of GCS from Philips (slope = 0.98 [0.98 1.01], r = 0.91) and Siemens (slope = 1.0 [0.96 1.03], r = 0.88) data similarly showed a very strong correlation. The average analysis time per subject was 4.1 ± 1.2 minutes for FT and 34.7 ± 3.3 seconds for DeepStrain, representing a 7-fold reduction in analysis time. DATA CONCLUSION: This study demonstrated high correlation of myocardial GCS and GRS measurements between freely available fully automated DeepStrain and commercially available manual FT software, with substantial time-saving in the analysis. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 3.

Validation of Estimated Small Dense Low-Density Lipoprotein Cholesterol Concentration in a Japanese General Population.

AIM: A high level of directly measured small dense low-density lipoprotein cholesterol (sdLDL-C) is a strong risk factor for atherosclerotic cardiovascular disease. A method for estimating sdLDL-C by using Sampson's equation that includes levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), non-HDL-C and triglycerides (TG) has recently been proposed. We investigated the validation and exploration of estimated sdLDL-C level. METHODS: The associations between measured and estimated sdLDL-C levels were investigated in 605 Japanese subjects (men/women: 280/325; mean age: 65±15 years) who received annual health check-ups in the Tanno Sobetsu Study, a population-based cohort. RESULTS: Estimated sdLDL-C level was highly correlated with measured sdLDL-C level in all subjects (R2 =0.701), nondiabetic subjects without any medication (n=254, R 2=0.686) and subjects with diabetes mellitus (n=128, R2=0.721). Multivariable regression analysis showed that levels of non-HDL-C, TG and γ-glutamyl transpeptidase (γGTP) were independent predictors of measured sdLDL-C level. In a stratification of the LDL window, all of the subjects with a combination of high non-HDL-C (≥ 170 mg/dL) and high TG (≥ 150 mg/dL) had high levels of measured and estimated sdLDL-C (≥ 35 mg/dL). Furthermore, machine learning-based estimation of sdLDL-C level by artificial intelligence software, Prediction One, was substantially improved by using components of Sampson's equation (R2=0.803) and by using those components with the addition of γGTP and deletion of TC (R2=0.929). CONCLUSIONS: sdLDL-C level estimated by Sampson's equation can be used instead of measured sdLDL-C level in general practice. By building multiple machine learning models of artificial intelligence, a more accurate and practical estimation of sdLDL-C level might be possible.

Long-term prognostic value of whole-heart coronary magnetic resonance angiography.

BACKGROUND: Coronary magnetic resonance angiography (CMRA) allows non-ionizing visualization of luminal narrowing in coronary artery disease (CAD). Although a prior study showed the usefulness of CMRA for risk stratification in short-term follow-up, the long-term prognostic value of CMRA remains unclear. The purpose of this study was to evaluate the long-term prognostic value of CMRA. METHODS: A total of 506 patients without history of myocardial infarction or prior coronary artery revascularization underwent free-breathing whole-heart CMRA between 2009 and 2015. Images were acquired using a 1.5 T or 3 T scanner and visually evaluated as the consensus decisions of two observers. Obstructive CAD on CMRA was defined as luminal narrowing of ≥ 50% in at least one coronary artery. Major adverse cardiac events (MACE) comprised cardiac death, nonfatal myocardial infarction, and unstable angina. RESULTS: Obstructive CAD on CMRA was observed in 214 patients (42%). During follow-up (median, 5.6 years), 31 MACE occurred. Kaplan-Meier curve analysis revealed a significant difference in event-free survival between patients with and without obstructive CAD for MACE (log-rank, p = 0.003) and cardiac death (p = 0.012). Annualized event rates for MACE in patients with no obstructive CAD, 1-vessel disease, 2-vessel disease, and left-main or 3-vessel disease were 0.6%, 1.5%, 2.3%, and 3.6%, respectively (log-rank, p = 0.003). Cox proportional hazard regression analysis showed that, among obstructive CAD on CMRA and clinical risk factors (age, sex, hypertension, diabetes, dyslipidemia, smoking, and family history of CAD), obstructive CAD and diabetes were significant predictors of MACE (hazard ratios, 2.9 [p = 0.005] and 2.2 [p = 0.034], respectively). In multivariate analysis, obstructive CAD remained an independent predictor (adjusted hazard ratio, 2.6 [p = 0.010]) after adjusting for diabetes. Addition of obstructive CAD to clinical risk factors significantly increased the global chi-square result from 8.3 to 13.8 (p = 0.022). CONCLUSIONS: In long-term follow-up, free breathing whole heart CMRA allows non-invasive risk stratification for MACE and cardiac death and provides incremental prognostic value over conventional risk factors in patients without a history of myocardial infarction or prior coronary artery revascularization. The presence and severity of obstructive CAD detected by CMRA were associated with worse prognosis. Importantly, patients without obstructive CAD on CMRA displayed favorable prognosis.

Serial cardiac magnetic resonance imaging in wet beriberi.

A 69-year-old woman with previous pancreaticoduodenectomy was admitted for evaluation of chest discomfort on effort and leg edema for a few months. Oral flosemide before admission for 1 week failed to relieve her symptoms. Her blood pressure was 105/51 mmHg and heart rate was 76 beats/min. Chest X-ray revealed an enlarged heart and mild pulmonary congestion. Echocardiography demonstrated normal left ventricular ejection fraction and diastolic dysfunction with no left ventricular hypertrophy. Cardiac catheterization showed normal coronary arteries, high cardiac index, and elevated intracardial pressures. Myocardial biopsy from the right ventricular septum revealed nearly normal findings. Cardiac magnetic resonance imaging (CMRI) showed both ventricles enlarged and increased global extracellular volume fraction (ECV) of 37%, but normal native T1 and T2 values. As she had pancreaticoduodenectomy, beriberi was suspected. Vitamin B1 significantly increased urine output and lowered intracardiac pressures and cardiac index. After 3 months of vitamin B1, CMRI exhibited that the right ventricle had decreased in size and the global ECV value had been lowered. Our case highlights that chronic beriberi may be associated with little myocardial damage. The increased ECV suggests that the diffuse expansion of extracellular space unrelated to myocardial edema might have been reversed by vitamin B1treatment. Morphological changes in the ventricles and myocardial damage by wet beriberi can be demonstrated by CMRI. .

mTORC1 inhibition attenuates necroptosis through RIP1 inhibition-mediated TFEB activation.

Accumulating evidence indicates that necroptosis contributes to cardiovascular diseases. We recently reported suppression of autophagy by necroptotic signals in cardiomyocytes and protective action of rapamycin. Here we examined the mechanism by which mTORC1 inhibition protects cardiomyocytes from necroptosis. Necroptosis of H9c2 cells was induced by treatment with tumor necrotic factor-α (TNF) and z-VAD-fmk (zVAD), and the extent of necroptosis was determined as the level of LDH release (as % of total). TNF/zVAD increased RIP1-RIP3 interaction and LDH release from 3.4 ±â€¯1.3% to 46.1 ±â€¯2.3%. The effects of TNF/zVAD were suppressed by an mTORC1 inhibitor, rapamycin, and an mTORC1/2 inhibitor, Ku-0063794, but not by a p70s6K inhibitor, PF-4708671. Protection by rapamycin was not abolished by inhibitors of TAK1, IKKα/ß, and cIAP, endogenous necroptosis suppressors upstream of RIP1. Rapamycin and Ku-0063794 suppressed TNF/zVAD-induced RIP1-Ser166 phosphorylation and increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site, though such an effect on RIP1-Ser320 was not observed for PF-4708671. Protective effects of rapamycin on TNF/zVAD-induced RIP1-RIP3 binding and necroptosis were undetected in cells transfected with RIP1-S320A. In TNF/zVAD-treated cells, rapamycin and a RIP1 inhibitor, necrostatin-1, increased nuclear localization of transcriptional factor EB (TFEB) and promoted autolysosome formation from autophagosomes in a TFEB-dependent manner. Knockdown of TFEB expression attenuated rapamycin-induced protection from necroptosis in TNF/zVAD-treated cells. The results suggest that mTORC1 inhibition promotes autophagy and protects cardiomyocytes from necroptosis by a TFEB-dependent mechanism and that inhibition of RIP1 by increased phosphorylation at Ser320 is crucial in the cardiomyocyte protection afforded by mTORC1 inhibition.

Canagliflozin, a sodium-glucose cotransporter 2 inhibitor, normalizes renal susceptibility to type 1 cardiorenal syndrome through reduction of renal oxidative stress in diabetic rats.

AIMS/INTRODUCTION: Type 2 diabetes mellitus is a risk factor of acute kidney injury after myocardial infarction (MI), a form of cardiorenal syndrome. Recent clinical trials have shown that a sodium-glucose cotransporter 2 (SGLT2) inhibitor improved both cardiac and renal outcomes in patients with type 2 diabetes mellitus, but effects of an SGLT2 inhibitor on cardiorenal syndrome remain unclear. MATERIALS AND METHODS: Type 2 diabetes mellitus (Otsuka Long-Evans Tokushima Fatty rats [OLETF]) and control (Long-Evans Tokushima Otsuka rats [LETO]) were treated with canagliflozin, an SGLT2 inhibitor, for 2 weeks. Renal tissues were analyzed at 12 h after MI with or without preoperative fasting. RESULTS: Canagliflozin reduced blood glucose levels in OLETF, and blood ß-hydroxybutyrate levels were increased by canagliflozin only with fasting. MI increased neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 protein levels in the kidney by 3.2- and 1.6-fold, respectively, in OLETF, but not in LETO. The renal messenger ribonucleic acid level of Toll-like receptor 4 was higher in OLETF than in LETO after MI, whereas messenger ribonucleic acid levels of cytokines/chemokines were not significantly different. Levels of lipid peroxides, nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 and NOX4 proteins after MI were significantly higher in OLETF than in LETO. Canagliflozin with pre-MI fasting suppressed MI-induced renal expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in OLETF, together with reductions in lipid peroxides and NOX proteins in the kidney. Blood ß-hydroxybutyrate levels before MI were inversely correlated with neutrophil gelatinase-associated lipocalin protein levels in OLETF. Pre-incubation with ß-hydroxybutyrate attenuated angiotensin II-induced upregulation of NOX4 in NRK-52E cells. CONCLUSIONS: The findings suggest that SGLT2 inhibitor treatment with a fasting period protects kidneys from MI-induced cardiorenal syndrome, possibly by ß-hydroxybutyrate-mediated reduction of NOXs and oxidative stress, in type 2 diabetic rats.

Accuracy of flash glucose monitoring in insulin-treated patients with type 2 diabetes.

The present study evaluated the accuracy of interstitial glucose measurements by flash glucose monitoring (FGM) and continuous glucose monitoring (CGM). Five diabetes patients simultaneously underwent FGM (FreeStyle Libre Pro) and CGM (iPro™2), and their glucose levels were compared with venous blood and capillary blood glucose levels. The range of daily venous blood glucose levels (30 measurements) was 70-245 mg/dL, with a median of 138 mg/dL. There were good correlations of glucose levels measured by FGM (r2  = 0.90, mean absolute relative difference 8.2 ± 5.6%), CGM (r2  = 0.86, mean absolute relative difference 9.2 ± 9.1%) and capillary blood (r2  = 0.87, mean absolute relative difference 7.2 ± 7.2%) with venous blood glucose levels. The accuracy of FGM measurements was also shown against CGM, with 99.9% of the FGM values (1,279 measurements) being within the Parkes error grid zones A and B. The results suggest that the accuracy of FGM is similar to that of CGM, and that FGM is a useful tool for determining daily glucose profile.

Empagliflozin, an SGLT2 Inhibitor, Reduced the Mortality Rate after Acute Myocardial Infarction with Modification of Cardiac Metabolomes and Antioxidants in Diabetic Rats.

The mechanism by which SGLT2 inhibitors reduce cardiac events in diabetic patients remains unclear. Here, we examined the effects of an SGLT2 inhibitor on the acute survival rate after myocardial infarction (MI) in an animal model of type 2 diabetes mellitus (DM) and the possible involvement of modification of cardiac metabolomes and antioxidative proteins. MI was induced in DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) control rats. Treatment with empagliflozin (10 mg/kg per day, 14 days) before MI reduced blood glucose and increased blood and myocardial ß-hydroxybutyrate (ßOHB) levels in OLETF. Survival rate at 48 hours after MI was significantly lower in OLETF rats than in LETO rats (40% vs. 84%), and empagliflozin significantly improved the survival rate in OLETF rats to 70%, although the sizes of MI were comparable. Patterns of metabolomes and gene expression in the noninfarcted myocardium of OLETF rats were consistent with increased fatty acid oxidation and decreased glucose oxidation. The patterns were modified by empagliflozin, suggesting both increased glucose oxidation and ketone utilization in OLETF rats. Empagliflozin prevented reduction of ATP level in the noninfarcted myocardium after MI and significantly increased myocardial levels of Sirt3 and superoxide dismutase 2 in OLETF rats. Administration of ßOHB partially mimicked the effects of empagliflozin in OLETF rats. The results suggest that empagliflozin prevents DM-induced increase in post-MI mortality, possibly by protective modification of cardiac energy metabolism and antioxidant proteins.

Empagliflozin normalizes the size and number of mitochondria and prevents reduction in mitochondrial size after myocardial infarction in diabetic hearts.

To explore mechanisms by which SGLT2 inhibitors protect diabetic hearts from heart failure, we examined the effect of empagliflozin (Empa) on the ultrastructure of cardiomyocytes in the noninfarcted region of the diabetic heart after myocardial infarction (MI). OLETF, a rat model of type 2 diabetes, and its nondiabetic control, LETO, received a sham operation or left coronary artery ligation 12 h before tissue sampling. Tissues were sampled from the posterior ventricle (i.e., the remote noninfarcted region in rats with MI). The number of mitochondria was larger and small mitochondria were more prevalent in OLETF than in LETO. Fis1 expression level was higher in OLETF than in LETO, while phospho-Ser637-Drp1, total Drp1, Mfn1/2, and OPA1 levels were comparable. MI further reduced the size of mitochondria with increased Drp1-Ser616 phosphorylation in OLETF. The number of autophagic vacuoles was unchanged after MI in LETO but was decreased in OLETF. Lipid droplets in cardiomyocytes and tissue triglycerides were increased in OLETF. Empa administration (10 mg/kg per day) reduced blood glucose and triglycerides and paradoxically increased lipid droplets in cardiomyocytes in OLETF. Empa suppressed Fis1 upregulation, increased Bnip3 expression, and prevented reduction in both mitochondrial size and autophagic vacuole number after MI in OLETF. Together with the results of our parallel study showing upregulation of SOD2 and catalase by Empa, the results indicate that Empa normalizes the size and number of mitochondria in diabetic hearts and that diabetes-induced excessive reduction in mitochondrial size after MI was prevented by Empa via suppression of ROS and restoration of autophagy.

Distinct impacts of sleep-disordered breathing on glycemic variability in patients with and without diabetes mellitus.

BACKGROUND: Sleep-disordered breathing (SDB) is highly prevalent in patients with diabetes mellitus (DM) and heart failure (HF) and contributes to poor cardiovascular outcomes. Enlarged glycemic variability (GV) is a risk factor of cardiac events independently of average blood glucose level, but the influence of SDB on GV is uncertain. In this study, we examined whether the impact of SDB on GV is modified by the presence of DM with or without HF. METHODS AND RESULTS: Two hundred three patients (67.5±14.1 [SD] years old, 132 males) who were admitted to our institute for examination or treatment of DM and/or HF underwent continuous glucose monitoring and polysomnography. Both HbA1c (8.0±2.0 vs. 5.7±0.4%) and mean amplitude of glycemic excursion (MAGE, median: 95.5 vs. 63.5 mg/dl) were significantly higher in a DM group (n = 100) than in a non-DM group (n = 103), but apnea-hypopnea index (AHI: 29.0±22.7 vs. 29.3±21.5) was similar in the two groups. AHI was correlated with log MAGE in the non-DM group but not in the DM group, and multivariate regression analysis revealed that AHI was an independent variable for log MAGE in the non-DM group but not in the DM group. We then divided the non-DM patients into two subgroups according to BNP level (100 pg/ml). AHI was positively correlated with log MAGE (r = 0.74, p<0.001) in the non-DM low-BNP subgroup, but such a correlation was not found in the non-DM high-BNP subgroup. Continuous positive airway pressure (CPAP) reduced MAGE from 75.3 to 53.0 mg/dl in the non-DM group but did not reduce MAGE in the DM group. CONCLUSION: Severity of SDB was associated with higher GV, but DM as well as HF diminished the contribution of SDB to GV. Treatment with CPAP was effective for reduction of GV only in patients without DM.

Suppressed autophagic response underlies augmentation of renal ischemia/reperfusion injury by type 2 diabetes.

Diabetes mellitus is a major risk factor for acute kidney injury (AKI). Here, we hypothesized that suppression of autophagic response underlies aggravation of renal ischemia/reperfusion (I/R) injury by type 2 diabetes mellitus (T2DM). In OLETF, a rat model of T2DM, and its non-diabetic control, LETO, AKI was induced by unilateral nephrectomy and 30-min occlusion and 24-h reperfusion of the renal artery in the contralateral kidney. Levels of serum creatinine and blood urea nitrogen and tubular injury score after I/R were significantly higher in OLETF than in LETO. Administration of chloroquine, a widely used autophagy inhibitor, aggravated I/R-induced renal injury in LETO, but not in OLETF. In contrast to LETO, OLETF exhibited no increase in autophagosomes in the proximal tubules after I/R. Immunoblotting showed that I/R activated the AMPK/ULK1 pathway in LETO but not in OLETF, and mTORC1 activation after I/R was enhanced in OLETF. Treatment of OLETF with rapamycin, an mTORC1 inhibitor, partially restored autophagic activation in response to I/R and significantly attenuated I/R-induced renal injury. Collectively, these findings indicate that suppressed autophagic activation in proximal tubules by impaired AMPK/ULK1 signaling and upregulated mTORC1 activation underlies T2DM-induced worsening of renal I/R injury.

Suppression of autophagic flux contributes to cardiomyocyte death by activation of necroptotic pathways.

BACKGROUND: The role of necroptosis in myocardial injury has not been fully characterized. Here we examined roles of mitochondrial permeability transition pore (mPTP) and autophagy in necroptosis of cardiomyocytes. METHODS AND RESULTS: In H9c2 cells, necroptosis was induced by treatment with TNF-α (TNF) and z-VAD-fmk (zVAD) for 24h, and necroptotic death was determined by LDH release (as % of total). TNF/zVAD increased LDH release from 16.6±4.3% to 60.6±2.7%, and the LDH release was suppressed by necrostatin-1 (29.4±4.0%), a RIP1 inhibitor, and by siRNA-mediated knockdown of RIP3 (27.7±2.0%), confirming RIP1-RIP3-dependent necroptosis. TNF/zVAD-induced necroptosis was not attenuated by mPTP inhibitors or GSK-3ß inhibitors. TNF/zVAD increased LC3-II level, but the change was not further enhanced by bafilomycin A1. The increase of LC3-II by TNF/zVAD was associated with suppression of both autophagic flux and LC3-LAMP1 co-localization. TNF/zVAD did not modify phosphorylation of Akt, p70s6K, AMPK, ULK1 or VASP but significantly increased RIP1-p62 binding and conversely reduced p62-LC3 binding. Rapamycin inhibited RIP1-p62 and RIP1-RIP3 interactions induced by TNF/zVAD and partly restored autophagic flux and suppressed LDH release in TNF/zVAD-treated cells. The effect of rapamycin on LDH release was reduced by knockdown of Atg5 expression. Knockdown of p62 by siRNA augmented LDH release by TNF/zVAD. CONCLUSION: Suppression of autophagic flux contributes to RIP1-RIP3 interaction and necroptosis of cardiomyocytes, and sequestration of p62 from its interaction with LC3-II by p62-RIP1 interaction possibly underlies the suppressed autophagy. The mPTP is unlikely to play a major role in execution of necroptosis in cardiomyocytes.