Kazumasa Unno

Impact of a Single Intracoronary Administration of Adiponectin on Myocardial Ischemia/Reperfusion Injury in a Pig Model

Background—Adiponectin plays a protective role in the development of obesity-linked disorders. We demonstrated that adiponectin exerts beneficial actions on acute ischemic injury in mice hearts. However, the effects of adiponectin treatment in large animals and its feasibility in clinical practice have not been investigated. This study investigated the effects of intracoronary administration of adiponectin on myocardial ischemia-reperfusion (I/R) injury in pigs. Methods and Results—The left anterior descending coronary artery was occluded in pigs for 45 minutes and then reperfused for 24 hours. Recombinant adiponectin protein was given as a bolus intracoronary injection during ischemia. Cardiac functional parameters were measured by a manometer-tipped catheter. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. Tumor necrosis factor-α and interleukin-10 transcripts were analyzed by real-time polymerase chain reaction. Serum levels of derivatives of reactive oxygen metabolites and biological antioxidant potential were measured. Adiponectin protein was determined by immunohistochemical and Western blot analyses. Intracoronary administration of adiponectin protein led to a reduction in myocardial infarct size and improvement of left ventricular function in pigs after I/R. Injected adiponectin protein accumulated in the I/R-injured heart. Adiponectin treatment resulted in decreased tumor necrosis factor-α and increased interleukin-10 mRNA levels in the myocardium after I/R. Adiponectin-treated pigs had reduced apoptotic activity in the I/R-injured heart and showed increased biological antioxidant potential levels and decreased derivatives of reactive oxygen metabolite levels in the blood stream after I/R. Conclusions—These data suggest that adiponectin protects against I/R injury in a preclinical pig model through its ability to suppress inflammation, apoptosis, and oxidative stress. Administration of intracoronary adiponectin could be a useful adjunctive therapy for acute myocardial infarction.

MicroRNA-34a Plays a Key Role in Cardiac Repair and Regeneration Following Myocardial Infarction

RATIONALE In response to injury, the rodent heart is capable of virtually full regeneration via cardiomyocyte proliferation early in life. This regenerative capacity, however, is diminished as early as 1 week postnatal and remains lost in adulthood. The mechanisms that dictate postinjury cardiomyocyte proliferation early in life remain unclear. OBJECTIVE To delineate the role of miR-34a, a regulator of age-associated physiology, in regulating cardiac regeneration secondary to myocardial infarction (MI) in neonatal and adult mouse hearts. METHODS AND RESULTS Cardiac injury was induced in neonatal and adult hearts through experimental MI via coronary ligation. Adult hearts demonstrated overt cardiac structural and functional remodeling, whereas neonatal hearts maintained full regenerative capacity and cardiomyocyte proliferation and recovered to normal levels within 1-week time. As early as 1 week postnatal, miR-34a expression was found to have increased and was maintained at high levels throughout the lifespan. Intriguingly, 7 days after MI, miR-34a levels further increased in the adult but not neonatal hearts. Delivery of a miR-34a mimic to neonatal hearts prohibited both cardiomyocyte proliferation and subsequent cardiac recovery post MI. Conversely, locked nucleic acid-based anti-miR-34a treatment diminished post-MI miR-34a upregulation in adult hearts and significantly improved post-MI remodeling. In isolated cardiomyocytes, we found that miR-34a directly regulated cell cycle activity and death via modulation of its targets, including Bcl2, Cyclin D1, and Sirt1. CONCLUSIONS miR-34a is a critical regulator of cardiac repair and regeneration post MI in neonatal hearts. Modulation of miR-34a may be harnessed for cardiac repair in adult myocardium.

Evidence for the Importance of Adiponectin in the Cardioprotective Effects of Pioglitazone

The favorable effects of the peroxisome proliferator-activated receptor-&ggr; ligand pioglitazone on glucose metabolism are associated with an increase in the fat-derived hormone adiponectin in the bloodstream. A recent clinical trial, Prospective Pioglitazone Clinical Trial in Macrovascular Events, demonstrated that pioglitazone improved cardiovascular outcomes in patients with type 2 diabetes mellitus. However, the functional role of adiponectin in cardioprotection by pioglitazone has not been examined experimentally. Here we investigated the effect of pioglitazone on angiotensin II (Ang II)–induced cardiac hypertrophy and assessed the potential contribution of adiponectin to the action of pioglitazone on the heart. Wild-type or adiponectin-deficient mice were treated with pioglitazone as food admixture at a concentration of 0.01% for 1 week followed by 2 weeks of infusion with Ang II at 3.2 mg/kg per day. Ang II infusion in wild-type mice resulted in exacerbated myocyte hypertrophy and increased interstitial fibrosis, which were accompanied by elevated phosphorylation of extracellular signal-regulated kinase and expression of transforming growth factor-&bgr;1 in the heart. Treatment of wild-type mice with pioglitazone attenuated cardiac hypertrophy and fibrosis, extracellular signal-regulated kinase phosphorylation, and transforming growth factor-&bgr;1 expression in response to Ang II. Pioglitazone also increased the plasma adiponectin level and phosphorylation of cardiac AMP-activated protein kinase in wild-type mice in the presence of Ang II. The suppressive effects of pioglitazone on Ang II–induced cardiac hypertrophy and fibrosis were diminished in adiponectin-deficient mice. Furthermore, pioglitazone had no effects on the phosphorylation of extracellular signal-regulated kinase and AMP-activated protein kinase in the Ang II–infused heart of adiponectin-deficient mice. These data provide direct evidence that pioglitazone protects against Ang II–induced pathological cardiac remodeling via an adiponectin-dependent mechanism.

Dobutamine stress testing as a diagnostic tool for evaluation of myocardial contractile reserve in asymptomatic or mildly symptomatic patients with dilated cardiomyopathy.

OBJECTIVES We performed dobutamine stress testing for evaluation of myocardial contractile reserve in asymptomatic or mildly symptomatic patients with dilated cardiomyopathy (DCM). BACKGROUND Catecholamine sensitivity is reduced in failing hearts as a result of myocardial abnormalities in the beta-adrenergic receptor signaling pathway. However, little is known about adrenergic myocardial contractile reserve in asymptomatic or mildly symptomatic patients with DCM. METHODS The maximal first derivative of left ventricular pressure (LV dP/dt(max)) was determined during infusion of dobutamine (10 microg kg(-1) min(-1)) in 46 asymptomatic or mildly symptomatic (New York Heart Association functional class I or II) patients with DCM. The expression of messenger ribonucleic acid (mRNA) for contractile regulatory proteins in endomyocardial biopsy specimens was quantified by reverse transcription and real-time polymerase chain reaction analysis. Plasma norepinephrine levels were measured in all patients and [(123)I]metaiodobenzylguanidine (MIBG) scintigraphy performed. RESULTS Patients were classified into 3 groups based on the percentage increase in LV dP/dt(max) induced by dobutamine (DeltaLV dP/dt(max)) and on LV ejection fraction (LVEF) at baseline: group I (n = 18): DeltaLV dP/dt(max) >100% and LVEF >25%; group IIa (n = 17): DeltaLV dP/dt(max) <or=100% and LVEF > 25%; and group IIb (n = 11): DeltaLV dP/dt(max) <or=100% and LVEF <or=25%. The amounts of beta(1)-adrenergic receptor, sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase, and phospholamban mRNA were significantly smaller in groups IIa and IIb than in group I. The plasma norepinephrine level was increased and the delayed heart/mediastinum count ratio in MIBG scintigraphy was decreased in both groups IIa and IIb. CONCLUSIONS Dobutamine stress testing is a useful diagnostic tool for identifying reduced adrenergic myocardial contractile reserve related to altered myocardial expression of beta(1)-adrenergic receptor, sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase, and phospholamban genes even in asymptomatic or mildly symptomatic patients with DCM.

Cathepsin K-mediated notch1 activation contributes to neovascularization in response to hypoxia

Cysteine proteases play important roles in pathobiology. Here we reveal that cathepsin K (CatK) has a role in ischaemia-induced neovascularization. Femoral artery ligation-induced ischaemia in mice increases CatK expression and activity, and CatK-deficient mice show impaired functional recovery following hindlimb ischaemia. CatK deficiency reduces the levels of cleaved Notch1 (c-Notch1), Hes1 Hey1, Hey2, vascular endothelial growth factor, Flt-1 and phospho-Akt proteins of the ischaemic muscles. In endothelial cells, silencing of CatK mimicked, whereas CatK overexpression enhanced, the levels of c-Notch1 and the expression of Notch downstream signalling molecules, suggesting CatK contributes to Notch1 processing and activates downstream signalling. Moreover, CatK knockdown leads to defective endothelial cell invasion, proliferation and tube formation, and CatK deficiency is associated with decreased circulating endothelial progenitor cells-like CD31(+)/c-Kit(+) cells in mice following hindlimb ischaemia. Transplantation of bone marrow-derived mononuclear cells from CatK(+/+) mice restores the impairment of neovascularization in CatK(-/-) mice. We conclude that CatK may be a potential therapeutic target for ischaemic disease.

Relation of functional and morphological changes in mitochondria to myocardial contractile and relaxation reserves in asymptomatic to mildly symptomatic patients with hypertrophic cardiomyopathy

AIMS To examine the relation between mitochondrial dysfunction and myocardial contractile and relaxation reserves in hypertrophic cardiomyopathy (HCM). METHODS AND RESULTS Thirty HCM patients (LVEF >or=60%) underwent biventricular cardiac catheterization analysis both at rest and during atrial pacing as well as myocardial (99m)Tc-sestamibi scintigraphy at rest to calculate washout rate. Endomyocardial biopsy specimens were obtained for quantitative mRNA analysis and electron microscopy. The HCM patients were divided into two groups-group A: normal force-frequency relation and a pressure half-time (T(1/2)) of <30 ms (n = 15); group B: abnormal force-frequency relation or T(1/2) of >or=30 ms (n = 15). The (99m)Tc-sestamibi washout rate was significantly correlated with T(1/2) for all patients (r = 0.74, P < 0.01) and was also significantly greater in group B (29.2 +/- 6.3%) than in group A (19.3 +/- 3.1%). The abundance of mRNAs for mitochondrial electron transport-related enzymes was significantly higher in group A than in group B. Mitochondria showed a greater variation in size and were more disorganized in group B than in group A. CONCLUSION Mitochondria showed functional impairment and morphological disorganization in the left ventricle of HCM patients without baseline systolic dysfunction. These mitochondrial changes were associated with impaired myocardial contractile and relaxation reserves.

Increased 99mTc-Sestamibi Washout Reflects Impaired Myocardial Contractile and Relaxation Reserve During Dobutamine Stress Due to Mitochondrial Dysfunction in Dilated Cardiomyopathy Patients

OBJECTIVES This study investigated whether the technitium-99m sestamibi (MIBI) washout rate (WR) would predict mitochondrial damage and myocardial dysfunction in patients with dilated cardiomyopathy (DCM). BACKGROUND Myocardial mitochondrial damage reduces adenosine triphosphate production, resulting in myocardial dysfunction. Increased myocardial (99m)Tc-MIBI washout is reportedly caused by mitochondrial dysfunction. METHODS Twenty DCM patients (New York Heart Association functional class I-III) underwent myocardial (99m)Tc-MIBI scintigraphy and cardiac catheterization. Myocardial MIBI uptake was quantified as an early and delayed heart-to-mediastinum ratio, and WR was calculated. Maximum first derivative of left ventricular (LV) pressure (LV dP/dtmax) (an index of myocardial contractility) and LV pressure half-time (T1/2) (an index of myocardial relaxation) were calculated by the left ventricular pressure curve at baseline and during dobutamine infusion (15 μg/kg/min at maximum). Endomyocardial biopsy specimens were obtained for quantitative mRNA analysis and electron microscopy. The patients were divided into two groups as follows: 1) group A of 10 patients showing a WR ≤ 24.3% (median value) and 2) group B of 10 patients showing a WR >24.3%. RESULTS WR was significantly correlated with the percentage changes in LV dP/dtmax (%LV dP/dtmax) (r: -0.59; p = 0.01) and T1/2 (r: -0.57; p = 0.03) from baseline to peak dobutamine stress. The %LV dP/dtmax was significantly greater in group B than in group A. The abundance of mRNAs for mitochondrial electron transport-related enzymes was more significantly reduced in group B than in group A. Electron microscopy revealed significant correlations between WR and the severity of mitochondrial damage (r: 0.88; p = 0.048) and glycogen accumulation (r: 0.90; p = 0.044). CONCLUSIONS Increased (99m)Tc-MIBI washout may predict mitochondrial dysfunction and the impairment of myocardial contractile and relaxation reserves during dobutamine stress in DCM patients.

Mechanism of Diastolic Stiffening of the Failing Myocardium and Its Prevention by Angiotensin Receptor and Calcium Channel Blockers

Objective: To investigate the mechanism responsible for the increased cardiac stiffness associated with hypertensive heart failure in Dahl salt-sensitive (DS) rats and the effects of treatment with the combination of a calcium channel blocker [azelnidipine (AZE)] and angiotensin II type 1 receptor blocker [olmesartan (OLM)]. Methods: DS rats fed a high-salt diet from 7 weeks of age were treated (or not) from 12 to 19 weeks of age with the vasodilator hydralazine, OLM plus AZE, or the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. Rats fed a low-salt diet served as controls. Results: Treatment with OLM plus AZE attenuated changes in the expression of collagen isoforms and a decrease in the ratio of elastin to collagen in the left ventricle and prevented the increase in myocardial stiffness and diastolic dysfunction in DS rats in a manner independent of the hypotensive effect of these drugs. Such treatment also inhibited the expression and activation of elastolytic proteases (including cathepsins S and K and metalloproteinases-2, -9, and -12), NADPH oxidase-dependent superoxide production, and inflammatory changes in the failing myocardium. All these effects were mimicked by treatment with apocynin. Conclusions: The changes in collagen isoform expression and the decrease in the elastin to collagen ratio in the failing myocardium likely account for the increase in diastolic stiffness in this model of hypertensive heart failure. Administration of angiotensin receptor and calcium channel blockers prevented these changes in a manner independent of the hypotensive effect of these drugs by inhibiting the increase in elastolytic activity induced by activation of NADPH oxidase.

Regional cardiac dysfunction and dyssynchrony in a murine model of afterload stress.

Small animal models of afterload stress have contributed much to our present understanding of the progression from hypertension to heart failure. High-sensitivity methods for phenotyping cardiac function in vivo, particular in the setting of compensated cardiac hypertrophy, may add new information regarding alterations in cardiac performance that can occur even during the earliest stages of exposure to pressure overload. We have developed an echocardiographic analytical method, based on speckle-tracking-based strain analyses, and used this tool to rapidly phenotype cardiac changes resulting from afterload stress in a small animal model. Adult mice were subjected to ascending aortic constriction, with and without subsequent reversal of the pressure gradient. In this model of compensated hypertrophic cardiac remodeling, conventional echocardiographic measurements did not detect changes in left ventricular (LV) function at the early time points examined. Strain analyses, however, revealed a decrement in basal longitudinal myofiber shortening that was induced by aortic constriction and improved following relief of the pressure gradient. Furthermore, we observed that pressure overload resulted in LV segmental dyssynchrony that was attenuated with return of the afterload to baseline levels. Herein, we describe the use of echocardiographic strain analyses for cardiac phenotyping in a mouse model of pressure overload. This method provides evidence of dyssynchrony and regional myocardial dysfunction that occurs early with compensatory hypertrophy, and improves following relief of aortic constriction. Importantly, these findings illustrate the utility of a rapid, non-invasive method for characterizing early cardiac dysfunction, not detectable by conventional echocardiography, following afterload stress.

Adiponectin acts as a positive indicator of left ventricular diastolic dysfunction in patients with hypertrophic cardiomyopathy.

Background Adiponectin is an adipose-derived plasma protein that exhibits beneficial actions on the heart. Recently, it was shown that adiponectin levels were elevated in patients with systolic heart failure. Objective To investigate the association between adiponectin levels and left ventricular (LV) diastolic function in patients with hypertrophic cardiomyopathy (HCM), characterised by diastolic dysfunction. Methods Twenty-six patients with HCM showing LV ejection fraction of >60% were enrolled. LV pressure half-time (T1/2) was measured as an index of myocardial relaxation. Patients were divided into two groups on the basis of baseline T1/2 (group A: T1/2< 35 ms, group B: T1/2≥ 35 ms). Blood samples were simultaneously collected from the coronary sinus (CS) and aortic root (Ao) as well as the peripheral vein (PV) for measurement of plasma adiponectin levels. Results Plasma adiponectin levels were significantly higher in group B than in group A. Adiponectin levels in the PV were positively correlated with the baseline T1/2 in patients with HCM. The transcardiac gradient of adiponectin as calculated by the Ao−CS difference was significantly higher in group A than in group B. The transcardiac gradient of adiponectin also inversely correlated with the baseline T1/2 and adiponectin levels in PV in patients with HCM. The expression of AdipoR1 but not AdipoR2 in the heart decreased in group B. The baseline T1/2 was negatively associated with AdipoR1 expression in patients with HCM. Conclusions These data document that adiponectin is an indicator of LV diastolic dysfunction in patients with HCM.