Role of AMP deaminase in diabetic cardiomyopathy.

Diabetes mellitus is one of the major causes of ischemic and nonischemic heart failure. While hypertension and coronary artery disease are frequent comorbidities in patients with diabetes, cardiac contractile dysfunction and remodeling occur in diabetic patients even without comorbidities, which is referred to as diabetic cardiomyopathy. Investigations in recent decades have demonstrated that the production of reactive oxygen species (ROS), impaired handling of intracellular Ca2+, and alterations in energy metabolism are involved in the development of diabetic cardiomyopathy. AMP deaminase (AMPD) directly regulates adenine nucleotide metabolism and energy transfer by adenylate kinase and indirectly modulates xanthine oxidoreductase-mediated pathways and AMP-activated protein kinase-mediated signaling. Upregulation of AMPD in diabetic hearts was first reported more than 30 years ago, and subsequent studies showed similar upregulation in the liver and skeletal muscle. Evidence for the roles of AMPD in diabetes-induced fatty liver, sarcopenia, and heart failure has been accumulating. A series of our recent studies showed that AMPD localizes in the mitochondria-associated endoplasmic reticulum membrane as well as the sarcoplasmic reticulum and cytosol and participates in the regulation of mitochondrial Ca2+ and suggested that upregulated AMPD contributes to contractile dysfunction in diabetic cardiomyopathy via increased generation of ROS, adenine nucleotide depletion, and impaired mitochondrial respiration. The detrimental effects of AMPD were manifested at times of increased cardiac workload by pressure loading. In this review, we briefly summarize the expression and functions of AMPD in the heart and discuss the roles of AMPD in diabetic cardiomyopathy, mainly focusing on contractile dysfunction caused by this disorder.

Downregulation of Mitochondrial Fusion Protein Expression Affords Protection from Canonical Necroptosis in H9c2 Cardiomyoblasts.

Necroptosis, a form of necrosis, and alterations in mitochondrial dynamics, a coordinated process of mitochondrial fission and fusion, have been implicated in the pathogenesis of cardiovascular diseases. This study aimed to determine the role of mitochondrial morphology in canonical necroptosis induced by a combination of TNFα and zVAD (TNF/zVAD) in H9c2 cells, rat cardiomyoblasts. Time-course analyses of mitochondrial morphology showed that mitochondria were initially shortened after the addition of TNF/zVAD and then their length was restored, and the proportion of cells with elongated mitochondria at 12 h was larger in TNF/zVAD-treated cells than in non-treated cells (16.3 ± 0.9% vs. 8.0 ± 1.2%). The knockdown of dynamin-related protein 1 (Drp1) and fission 1, fission promoters, and treatment with Mdivi-1, a Drp-1 inhibitor, had no effect on TNF/zVAD-induced necroptosis. In contrast, TNF/zVAD-induced necroptosis was attenuated by the knockdown of mitofusin 1/2 (Mfn1/2) and optic atrophy-1 (Opa1), proteins that are indispensable for mitochondrial fusion, and the attenuation of necroptosis was not canceled by treatment with Mdivi-1. The expression of TGFß-activated kinase (TAK1), a negative regulator of RIP1 activity, was upregulated and the TNF/zVAD-induced RIP1-Ser166 phosphorylation, an index of RIP1 activity, was mitigated by the knockdown of Mfn1/2 or Opa1. Pharmacological TAK1 inhibition attenuated the protection afforded by Mfn1/2 and Opa1 knockdown. In conclusion, the inhibition of mitochondrial fusion increases TAK1 expression, leading to the attenuation of canonical necroptosis through the suppression of RIP1 activity.

Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts.

Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.

Early Commencement and Long-Term Continuation of Tolvaptan Treatment Attenuate Functional Impairment of Renal Vasopressin V2 Receptors and Improve Clinical Outcomes in Patients with Heart Failure.

Preserved urinary excretion of aquaporin 2, an index for the function of vasopressin V2 receptor (V2-R), has been reported to predict a favorable response of heart failure patients to treatment with tolvaptan. In this study, we investigated the long-term effects of tolvaptan treatment on clinical outcomes and V2-R function in patients with acute decompensated heart failure (ADHF). We enrolled 90 consecutive patients who were hospitalized in Sapporo Medical University Hospital for ADHF and treated with tolvaptan in the BOREAS-ADHF registry and analyzed patients who continued taking tolvaptan after discharge. The effect of tolvaptan treatment on rehospitalization for HF or death was investigated according to whether the V2-R function was preserved (first morning urine osmolarity ≥ 352 mOsm/L, High-Uosm) or impaired (Uosm < 352 mOsm/L, Low-Uosm). During a median follow-up period of 443 days, significantly fewer patients in the High-Uosm group experienced adverse events than did patients in the Low-Uosm group (P < 0.001). Among the patients with High-Uosm, early commencement of tolvaptan administration (on or before day 7 of hospitalization, Early/High-Uosm) significantly reduced adverse events compared to late administration (after day 7 of hospitalization, Late/High-Uosm). Uosm measured during the long-term follow-up period after discharge was significantly reduced compared to that before commencement of tolvaptan administration in the Late/High-Uosm group (from 468 ± 88 to 395 ± 108 mOsm, -18.3 ± 19.6%, P < 0.05) but not in the Early/High-Uosm group (from 478 ± 115 to 455 ± 133 mOsm, -0.50 ± 35.3%, P = 0.66). These findings indicate that early commencement and long-term continuation of tolvaptan treatment attenuate functional impairment of V2-R and improve clinical outcomes in ADHF patients with preserved V2-R function.

Xanthine oxidoreductase-mediated injury is amplified by upregulated AMP deaminase in type 2 diabetic rat hearts under the condition of pressure overload.

BACKGROUND: We previously reported that upregulated AMP deaminase (AMPD) contributes to diastolic ventricular dysfunction via depletion of the adenine nucleotide pool in a rat model of type 2 diabetes (T2DM), Otsuka Long-Evans-Tokushima Fatty rats (OLETF). Meanwhile, AMPD promotes the formation of substrates of xanthine oxidoreductase (XOR), which produces ROS as a byproduct. Here, we tested the hypothesis that a functional link between upregulated AMPD and XOR is involved in ventricular dysfunction in T2DM rats. METHODS AND RESULTS: Pressure-volume loop analysis revealed that pressure overloading by phenylephrine infusion induced severer left ventricular diastolic dysfunction (tau: 14.7 ± 0.8 vs 12.5 ± 0.7 msec, left ventricular end-diastolic pressure: 18.3 ± 1.5 vs 12.2 ± 1.3 mmHg, p < 0.05) and ventricular-arterial uncoupling in OLETF than in LETO, non-diabetic rats, though the baseline parameters were comparable in the two groups. While the pressure overload did not affect AMPD activity, it increased XOR activity both in OLETF and LETO, with OLETF showing significantly higher XOR activity than that in LETO (347.2 ± 17.9 vs 243.2 ± 6.1 µg/min/mg). Under the condition of pressure overload, myocardial ATP level was lower, and levels of xanthine and uric acid were higher in OLETF than in LETO. Addition of exogenous inosine, a product of AMP deamination, to the heart homogenates augmented XOR activity. OLETF showed 68% higher tissue ROS levels and 47% reduction in mitochondrial state 3 respiration compared with those in LETO. Overexpression of AMPD3 in H9c2 cells elevated levels of hypoxanthine and ROS and reduced the level of ATP. Inhibition of XOR suppressed the production of tissue ROS and mitochondrial dysfunction and improved ventricular function under the condition of pressure overload in OLETF. CONCLUSIONS: The results suggest that increases in the activity of XOR and the formation of XOR substrates by upregulated AMPD contribute to ROS-mediated diastolic ventricular dysfunction at the time of increased cardiac workload in diabetic hearts.

Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

Roles of the renin-angiotensin system in autophagy and ischemia/reperfusion (I/R) injury in the kidney have not been fully characterized. Here we examined the hypothesis that modest activation of the angiotensin II (Ang II) receptor upregulates autophagy and increases renal tolerance to I/R injury. Sprague-Dawley rats were assigned to treatment with a vehicle or a non-pressor dose of Ang II (200 ng/kg/min) for 72 h before 30-min renal I/R. LC3-immunohistochemistry showed that Ang II treatment increased autophagosomes in proximal tubular cells by 2.7 fold. In Ang II-pretreated rats, autophagosomes were increased by 2.5 fold compared to those in vehicle-treated rats at 4 h after I/R, when phosphorylation of Akt and S6 was suppressed and ULK1-Ser555 phosphorylation was increased. Serum creatinine and urea nitrogen levels, incidence of oliguria, and histological score of tubular necrosis at 24 h after I/R were attenuated by Ang II-pretreatment. In NRK-52E cells, Ang II induced LC3-II upregulation, which was inhibited by losartan but not by A779. The results indicate that a non-pressor dose of Ang-II promotes autophagy via ULK1-mediated signaling in renal tubular cells and attenuates renal I/R injury. The AT1 receptor, but not the Mas receptor, contributes to Ang-II-induced autophagy and presumably also to the renoprotection.

Involvement of necroptosis in contrast-induced nephropathy in a rat CKD model.

BACKGROUND: The risk of contrast-induced nephropathy (CIN) is high in patients with chronic kidney disease (CKD). However, the mechanism of CIN in CKD is not fully understood. Here, we prepared a clinically relevant model of CIN and examined the role of necroptosis, which potentially cross-talks with autophagy, in CIN. METHODS: In Sprague-Dawley rats, CKD was induced by subtotal nephrectomy (SNx, 5/6 nephrectomy) 4 weeks before induction of CIN. CIN was induced by administration of a contrast medium (CM), iohexol, following administration of indomethacin and N-omega-Nitro-L-arginine methyl ester. Renal function and tissue injuries were assessed 48 h after CM injection. RESULTS: Serum creatinine (s-Cre) and BUN were increased from 0.28 ± 0.01 to 0.52 ± 0.02 mg/dl and from 15.1 ± 0.7 to 29.2 ± 1.2 mg/dl, respectively, after SNx alone. CM further increased s-Cre and BUN to 0.69 ± 0.03 and 37.2 ± 2.1, respectively. In the renal tissue after CM injection, protein levels of receptor-interacting serine/threonine-protein kinase (RIP) 1, RIP3, cleaved caspase 3, and caspase 8 were increased by 64 ~ 212%, while there was reduction in LC3-II and accumulation of p62. Necrostatin-1, an RIP1 inhibitor, administered before and 24 h after CM injection significantly suppressed elevation of s-Cre, BUN and urinary albumin levels, kidney injury molecule-1 expression and infiltration of CD68-positive macrophages in renal tissues after CM injection. CONCLUSION: The results suggest that necroptosis of proximal tubular cells contributes to CIN in CKD and that suppression of protective autophagy by pro-necroptotic signaling may also be involved.

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.

Translational regulation by miR-301b upregulates AMP deaminase in diabetic hearts.

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3'UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3'UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts.

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.

Diabetes increases the susceptibility to acute kidney injury after myocardial infarction through augmented activation of renal Toll-like receptors in rats.

Acute kidney injury (AKI) after acute myocardial infarction (MI) worsens the prognosis of MI patients. Although type 2 diabetes mellitus (DM) is a major risk factor of AKI after MI, the underlying mechanism remains unclear. Here, we examined the roles of renal Toll-like receptors (TLRs) in the impact of DM on AKI after MI. MI was induced by coronary artery ligation in Otsuka-Long-Evans-Tokushima fatty (OLETF) rats, a rat DM model, and Long-Evans-Tokushima-Otsuka (LETO) rats, nondiabetic controls. Sham-operated rats served as no-MI controls. Renal mRNA levels of TLR2 and myeloid differentiation factor 88 (MyD88) were significantly higher in sham-operated OLETF rats than in sham-operated LETO rats, although levels of TLR1, TLR3, and TLR4 were similar. At 12 h after MI, protein levels of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) in the kidney were elevated by 5.3- and 4.0-fold, respectively, and their mRNA levels were increased in OLETF but not LETO rats. The increased KIM-1 and NGAL expression levels after MI in the OLETF kidney were associated with upregulated expression of TLR1, TLR2, TLR4, MyD88, IL-6, TNF-α, chemokine (C-C motif) ligand 2, and transforming growth factor-ß1 and also with activation of p38 MAPK, JNK, and NF-κB. Cu-CPT22, a TLR1/TLR2 antagonist, administered before MI significantly suppressed MI-induced upregulation of KIM-1, TLR2, TLR4, MyD88, and chemokine (C-C motif) ligand 2 levels and activation of NF-κB, whereas NGAL levels and IL-6 and TNF-α expression levels were unchanged. The results suggest that DM increases the susceptibility to AKI after acute MI by augmented activation of renal TLRs and that TLR1/TLR2-mediated signaling mediates KIM-1 upregulation after MI.NEW & NOTEWORTHY This is the first report to demonstrate the involvement of Toll-like recpetors (TLRs) in diabetes-induced susceptibility to acute kidney injury after acute myocardial infarction. We propose that the TLR1/TLR2 heterodimer may be a new therapeutic target for the prevention of acute kidney injury in diabetic patients.

Insufficient activation of Akt upon reperfusion because of its novel modification by reduced PP2A-B55α contributes to enlargement of infarct size by chronic kidney disease.

Chronic kidney disease (CKD) increases myocardial infarct size by an unknown mechanism. Here we examined the hypothesis that impairment of protective PI3K-PDK1-Akt and/or mTORC-Akt signaling upon reperfusion contributes to CKD-induced enlargement of infarct size. CKD was induced in rats by 5/6 nephrectomy (SNx group) 4 weeks before myocardial infarction experiments, and sham-operated rats served as controls (Sham group). Infarct size as a percentage of area at risk after ischemia/reperfusion was significantly larger in the SNx group than in the Sham group (56.3 ± 4.6 vs. 41.4 ± 2.0%). In SNx group, myocardial p-Akt-Thr308 level at baseline was elevated, and reperfusion-induced phosphorylation of p-Akt-Ser473, p-p70s6K and p-GSK-3ß was significantly suppressed. Inhibition of Akt-Ser473 phosphorylation upon reperfusion by Ku-0063794 significantly increased infarct size in the Sham group but not in the SNx group. There was no difference between the two groups in activities of mTORC2 and PDK1 and protein level of PTEN. However, the PP2A regulatory subunit B55α, which specifically targets Akt-Thr308, was reduced by 24% in the SNx group. Knockdown of B55α by siRNA increased baseline p-Akt-Thr308 and blunted Akt-Ser473 phosphorylation in response to insulin-like growth factor-1 (IGF-1) in H9c2 cells. A blunted response of Akt-Ser473 to IGF-1 was also observed in HEK293 cells transfected with a p-Thr308-mimetic Akt mutant (T308D). These results indicate that increased Akt-Thr308 phosphorylation by down-regulation of B55α inhibits Akt-Ser473 phosphorylation upon reperfusion in CKD and that the impaired Akt activation by insufficient Ser473 phosphorylation upon reperfusion contributes to infarct size enlargement by CKD.

Clinical impact of myocardial mTORC1 activation in nonischemic dilated cardiomyopathy.

BACKGROUND: Activity of mTOR complex 1 (mTORC1) has been shown to be up-regulated in animal models of heart failure. Here, we investigated the change and role of mTORC1 in human nonischemic dilated cardiomyopathy (NICM). METHODS: Endomyocardial biopsy specimens were obtained from patients with NICM (n=52) and from Brugada syndrome patients with normal LVEF as controls (n=10). The specimens were stained for phospho-ribosomal protein S6 (p-Rps6) and phospho-p70S6K (p-p70S6K), and the area with p-Rps6 signal was used as an index of mTORC1 activity. Using median mTORC1 activity, patients were divided into a high mTORC1 activity (H-mTOR) group and a low mTORC1 activity (L-mTOR) group. RESULTS: The ratio of p-Rps6-positive area in biopsy samples was 10-fold larger in patients with NICM than in controls (2.0±2.2% vs. 0.2±0.2%, p<0.01). p-p70S6K signal level was higher in the H-mTOR group than in the L-mTOR group. The proportion of patients with a family history of cardiomyopathy was higher and the proportion of patients on ACE inhibitors or angiotensin receptor blockers was lower in the H-mTOR group than in the L-mTOR group. The p-Rps6-positive area was correlated with extent of myocardial fibrosis (r=0.46, p<0.01). The cardiac event-free survival rate during a 5-year follow-up period tended to be lower in the H-mTOR group than in the L-mTOR group (52.9% vs. 81.6%, P=0.10). CONCLUSION: Aberrant activation of mTORC1 in cardiomyocytes was associated with myocardial fibrosis and a trend for worse prognosis in patients with NICM, indicating that persistently activated mTORC1 contributes to progression of human heart failure.

Excessive degradation of adenine nucleotides by up-regulated AMP deaminase underlies afterload-induced diastolic dysfunction in the type 2 diabetic heart.

Type 2 diabetes mellitus (T2DM) is often complicated with diastolic heart failure, which decompensates under increased afterload. Focusing on cardiac metabolomes, we examined mechanisms by which T2DM augments ventricular diastolic stiffness in response to pressure overloading. Pressure-volume relationships (PVRs) and myocardial metabolomes were determined at baseline and during elevation of aortic pressure by phenylephrine infusion in a model of T2DM, OLETF, and its non-diabetic control, LETO. Pressure overloading augmented diastolic stiffness without change in systolic reserve in OLETF as indicated by a left-upward shift of end-diastolic PVR. In contrast, PVRs under cardioplegic arrest in buffer-perfused isolated hearts were similar in OLETF and LETO, indicating that extracellular matrix or titin remodeling does not contribute to the afterload-induced increase in stiffness of the beating ventricle of OLETF. Metabolome analyses revealed impaired glycolysis and facilitation of the pentose phosphate pathway in OLETF. Pressure overloading significantly reduced ATP and total adenine nucleotides by 34% and 40%, respectively, in OLETF but not in LETO, while NADH-to-NAD(+) ratios were similar in the two groups. The decline in ATP by pressure overloading in OLETF was associated with increased inosine 5-monophosphate and decreased adenosine levels, being consistent with the 2.5-times higher activity of cardiac AMP deaminase in OLETF. Tissue ATP level was negatively correlated with tau of LV pressure and LVEDP. These results suggest that ATP depletion due to excessive degradation of adenine nucleotides by up-regulated AMP deaminase underlies ventricular stiffening during acute pressure overloading in T2DM hearts.

Translocation of glycogen synthase kinase-3ß (GSK-3ß), a trigger of permeability transition, is kinase activity-dependent and mediated by interaction with voltage-dependent anion channel 2 (VDAC2).

Glycogen synthase kinase-3ß (GSK-3ß) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3ß translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3ß on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3ß (WT), a mutant GSK-3ß insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3ß (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3ß-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3ß and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3ß was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3ß was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3ß was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3ß inhibitor. These results demonstrate that GSK-3ß translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3ß may function as a mitochondrial targeting sequence.

Inhibition of DPP-4 reduces acute mortality after myocardial infarction with restoration of autophagic response in type 2 diabetic rats.

BACKGROUND: Type 2 diabetes mellitus (T2DM) worsens the outcome after myocardial infarction (MI). Here, we hypothesized that inhibition of dipeptidyl peptidase-4 (DPP-4) improves survival after MI in T2DM by modifying autophagy in the non-infarcted region of the heart. METHODS AND RESULTS: Under baseline conditions, there was no significant difference between levels of myocardial autophagy marker proteins in OLETF, a rat model of T2DM, and in LETO, a non-diabetic control. However, in contrast to the response in LETO, LC3-II protein and LC3-positive autophagosomes in the non-infarcted region of the myocardium were not increased after MI in OLETF. The altered autophagic response in OLETF was associated with lack of AMPK/ULK-1 activation, attenuated response of Akt/mTOR/S6 signaling and increased Beclin-1-Bcl-2 interaction after MI. Treatment with vildagliptin (10 mg/kg/day s.c.), a DPP-4 inhibitor, suppressed Beclin-1-Bcl-2 interaction and increased both LC3-II protein level and autophagosomes in the non-infarcted region in OLETF, though it did not normalize AMPK/ULK-1 or mTOR/S6 signaling. Plasma insulin level, but not glucose level, was significantly reduced by vildagliptin at the dose used in this study. Survival rate at 48 h after MI was significantly lower in OLETF than in LETO (32 vs. 82%), despite similar infarct sizes. Vildagliptin improved the survival rate in OLETF to 80%, the benefit of which was abrogated by chloroquine, an autophagy inhibitor. CONCLUSIONS: The results indicate that vildagliptin reduces T2DM-induced increase in post-MI acute mortality possibly by restoring the autophagic response through attenuation of Bcl-2-Beclin-1 interaction.

Resveratrol improves cardiomyopathy in dystrophin-deficient mice through SIRT1 protein-mediated modulation of p300 protein.

Cardiomyopathy is the main cause of death in Duchenne muscular dystrophy. Here, we show that oral administration of resveratrol, which leads to activation of an NAD(+)-dependent protein deacetylase SIRT1, suppresses cardiac hypertrophy and fibrosis and restores cardiac diastolic function in dystrophin-deficient mdx mice. The pro-hypertrophic co-activator p300 protein but not p300 mRNA was up-regulated in the mdx heart, and resveratrol administration down-regulated the p300 protein level. In cultured cardiomyocytes, cardiomyocyte hypertrophy induced by the α(1)-agonist phenylephrine was inhibited by the overexpression of SIRT1 as well as resveratrol, both of which down-regulated p300 protein levels but not p300 mRNA levels. In addition, activation of atrial natriuretic peptide promoter by p300 was inhibited by SIRT1. We found that SIRT1 induced p300 down-regulation via the ubiquitin-proteasome pathway by deacetylation of lysine residues for ubiquitination. These findings indicate the pathological significance of p300 up-regulation in the dystrophic heart and indicate that SIRT1 activation has therapeutic potential for dystrophic cardiomyopathy.

Type 2 diabetes induces subendocardium-predominant reduction in transient outward K+ current with downregulation of Kv4.2 and KChIP2.

In the present study, we examined if and how cardiac ion channels are modified by type 2 diabetes mellitus (T2DM). Subendocardial (Endo) myocytes and subepicardial (Epi) myocytes were isolated from left ventricles of Otsuka-Long-Evans-Tokushima Fatty rats (OLETF) rats, a rat model of T2DM, and Otsuka-Long-Evans-Tokushima (LETO) rats (nondiabetic control rats). Endo and Epi myocytes were used for whole cell patch-clamp recordings and for protein and mRNA analyses. Action potential durations in Endo and Epi myocytes were longer in OLETF rats than in LETO rats, and the difference was larger in Endo myocytes. Steady-state transient outward K+ current (Ito) density was reduced in Endo but not Epi myocytes of OLETF rats compared with LETO rats, although the contribution of the fast component of Ito recovery from inactivation was smaller in both Endo and Epi myocytes of OLETF rats than in LETO rats. Kv4.2 protein was reduced only in Endo myocytes in OLETF rats, although voltage-gated K+ channel-interacting protein 2 (KChIP2) protein levels in both Endo and Epi myocytes were lower in OLETF rats than in LETO rats. Corresponding regional differences in mRNA levels of KChIP2 and Kv4.2 were observed between OLETF and LETO rats. mRNA levels of Iroquois homeobox 5 in Endo myocytes were 53% higher in OLETF rats than in LETO rats. Densities of inward rectifier K+ current and L-type Ca2+ current and mRNA levels of Kv4.3 and Kv1.4 were similar in OLETF and LETO rats. In conclusion, T2DM induces Endo-predominant prolongation of the action potential duration via a reduction of the fast component of Ito recovery from inactivation and reduced steady-state Ito, in which downregulation of Kv4.2 and KChIP2 may be involved. Increased Iroquois homeobox 5 expression may underlie Kv4.2 downregulation in T2DM.

Does glycemic control reverse dispersion of ventricular repolarization in type 2 diabetes?

BACKGROUND: Abnormal ventricular repolarization is a predictor of cardiovascular mortality. In this study, we tested the hypothesis that glycemic control reverses abnormal ventricular repolarization in patients with type 2 diabetes. METHODS: We analyzed longitudinal changes in repolarization indices of electrocardiograms in retrospectively enrolled 44 patients with type 2 diabetes and 44 age-matched healthy subjects. RESULTS: In the diabetic group, BMI was greater, levels of HbA1c (10.0 ± 1.6 vs. 5.6 ± 0.3%) and triglyceride were higher and level of HDL cholesterol was lower than those in the control group. Although mean QTc intervals were similar (413.6 ± 18.5 vs. 408.3 ± 22.7 ms), QT dispersion (41.8 ± 15.4 vs. 28.7 ± 7.7 ms) and Tpeak-Tend in lead V5 (83.6 ± 13.6 vs. 71.3 ± 10.3 ms) were significantly longer in the diabetic group than in the control group, indicating increased heterogeneity of ventricular repolarization in type 2 diabetes. During follow-up of 36 patients in the diabetic group for 787 ± 301 days, HbA1c level decreased to 7.3 ± 1.6%, while BMI did not significantly change. In contrast to HbA1c, QT dispersion (45.8 ± 15.0 ms) and Tpeak-Tend in lead V5 (83.6 ± 10.6 ms) were not significantly reduced during the follow-up period. There was no correlation between the change in HbA1c and the change in QT dispersion or Tpeak-Tend. CONCLUSIONS: Increased heterogeneity of ventricular repolarization in type 2 diabetic patients was not reduced during the relatively short follow-up period despite significantly improved glycemic control.