Susumu Sakata

Reversal of Cardiac Dysfunction After Long-Term Expression of SERCA2a by Gene Transfer in a Pre-Clinical Model of Heart Failure

OBJECTIVES The aim of this study was to examine the effects of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) gene transfer in a swine heart failure (HF) model. BACKGROUND Reduced expression and activity of SERCA2a have been documented in HF. Prior studies have reported the beneficial effects of short-term SERCA2a overexpression in rodent models. However, the effects of long-term expression of SERCA2a in pre-clinical large animal models are not known. METHODS Yorkshire-Landrace pigs were used (n = 16) to create volume overload by percutaneously severing chordae tendinae of the mitral apparatus with a bioptome to induce mitral regurgitation. At 2 months, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 1 (rAAV1) carrying SERCA2a under a cytomegalovirus promoter (rAAV1.SERCA2a) (n = 10; group 1) or saline (n = 6; group 2). RESULTS At 2 months, study animals were found to be in a compensated state of volume-overload HF (increased left ventricular internal diastolic and systolic diameters [LVIDd and LVIDs]). At 4 months, gene transfer resulted in: 1) positive left ventricular (LV) inotropic effects (adjusted peak left ventricular pressure rate of rise (dP/dt)max/P, 21.2 +/- 3.2 s(-1) group 1 vs. 15.5 +/- 3.0 s(-1) group 2; p < 0.01); 2) improvement in LV remodeling (% change in LVIDs -3.0 +/- 10% vs. +15 +/- 11%, respectively; p < 0.01). At follow-up, brain natriuretic peptide levels remained stable in group 1 after gene transfer, in contrast to rising levels in group 2. Further, cardiac SERCA2a expression was significantly decreased in group 2 whereas in group 1 it was restored to normal levels. There was no histopathological evidence of acute myocardial inflammation or necrosis. CONCLUSIONS Using a large-animal, volume-overload model of HF, we report that long-term overexpression of SERCA2a by in vivo rAAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction, and improved ventricular remodeling.

Role of resistin in cardiac contractility and hypertrophy

Cardiovascular sequelae including diabetic cardiomyopathy constitute the major cause of death in diabetic patients. Although several factors may contribute to the development of this cardiomyopathy, the underlying molecular/cellular mechanisms leading to cardiac dysfunction are still partially understood. Recently, a novel paradigm for the role of the adipocytokine resistin in diabetes has emerged. Resistin has been proposed to be a link between obesity, insulin resistance and diabetes. Using microarray analysis, we have recently found that cardiomyocytes isolated from type 2 diabetic hearts express high levels of resistin. However, the function of resistin with respect to cardiac function is unknown. In this study we show that resistin is not only expressed in the heart, but also promotes cardiac hypertrophy. Adenovirus-mediated overexpression of resistin in cultured neonatal rat ventricular myocytes (NRVM) significantly increased sarcomere organization and cell size, increased protein synthesis and increased the expression of atrial natriuretic factor and beta-myosin heavy chain. Overexpression of resistin in NRVM was also associated with activation of the mitogen-activated protein (MAP) kinases, ERK1/2 and p38, as well as increased Ser-636 phosphorylation of insulin receptor substrate-1 (IRS-1), indicating that IRS-1/MAPK pathway may be involved in the observed hypertrophic response. Overexpression of resistin in adult cultured cardiomyocytes significantly altered myocyte mechanics by depressing cell contractility as well as contraction and relaxation velocities. Intracellular Ca(2+) measurements showed slower Ca(2+) transients decay in resistin-transduced myocytes compared to controls, suggesting impaired cytoplasmic Ca(2+) clearing or alterations in myofilament activation. We conclude that resistin overexpression alters cardiac contractility, confers to primary cardiomyocytes all the features of the hypertrophic phenotype and promotes cardiac hypertrophy possibly via the IRS-1/MAPK pathway.

PICOT Inhibits Cardiac Hypertrophy and Enhances Ventricular Function and Cardiomyocyte Contractility

Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca2+ handing analysis revealed that increases in myofilament Ca2+ responsiveness, together with increased rate of sarcoplasmic reticulum Ca2+ reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.

Gene Remodeling in Type 2 Diabetic Cardiomyopathy and Its Phenotypic Rescue with SERCA2a

Background/Aim Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression. Methods/Results Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling. Conclusion This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role of SERCA2a normalization in multiple pathways in diabetic cardiomyopathy.

Development of a preclinical model of ischemic cardiomyopathy in swine

A number of promising therapies for ischemic cardiomyopathy are emerging, and the role of translational research in testing the efficacy and safety of these agents in relevant clinical models has become important. The goal of this study was to develop a chronic model of ischemic cardiomyopathy in a large animal model. In this study, 40 consecutive pigs were initially enrolled. To induce progressive stenosis, a plastic occluder with a fixed diameter of 1.0 mm fitted with an 18-gauge copper wire was placed around the proximal left anterior descending (LAD) coronary artery. Coronary angiography, hemodynamic measurements, and echocardiography were performed at 2 wk and 1, 2, and 3 mo. Overall mortality was 26% at 3 mo, and up to 80% of the pigs showed total occlusion of LAD at 1 mo. A significant depression of peak LV pressure rate of rise (+dP/dt(max)) was observed in the animals showing total artery occlusion throughout the study. Left ventricular ejection fraction was also impaired, and the left ventricular volumes tended to be larger in the pigs with occlusion. Approximately 10% of scar tissue was found in the LAD occluded pigs, whereas the coronary flow pattern in the rest of the area took the pattern of hibernating myocardium. At the same time, histological and protein analysis established the presence of fibrosis and ongoing apoptosis in the ischemic area. In this model, the timing and incidence of total occlusion and low mortality offer significant advantages over other ischemic cardiomyopathy models in conducting preclinical studies.

Correlation between a sandwich ELISA and an in-vitro bioassay for erythropoietin in human plasma.

Summary. A sandwich‐type enzyme‐linked immunosorbent assay (ELISA) for human erythropoietin (EPO) using two anti‐EPO monoclonal antibodies has been compared with an in‐vitro EPO bioassay based on CFU‐E colony formation in fetal mouse liver cell cultures. In normal subjects and non‐uraemic anaemic patients the plasma EPO values estimated with the ELISA correlated well with those by the bioassay, and also inversely with the values of blood Hb, PCV and RBC counts. Dose‐response curves for plasma and standard recombinant human EPO in the ELISA were parallel to each other. These results further confirm the validity for the ELISA in measuring circulating human EPO.

In vitro erythropoietin assay based on erythroid colony formation in fetal mouse liver cell culture

Summary. Critical studies were made on erythroid colony formation from cultured fetal mouse liver cells in an attempt to develop a simple and sensitive erythropoietin (Epo) assay procedure. The maximum colony formation was observed 24 h after plating of the cells when an evident dose–response relation was found for Epo added. The colony forming ability decreased steadily as the gestational age of the fetus advanced and was gradually lost by postnatal days 10–11. By morphological and cytochemical criteria almost all the colonies were found to be erythroid. 59Fe‐labelling experiments revealed a fairly good correlation between the colony number and 59Fe incorporation into both cells and haem. Dose–response curves for plasma were parallel to the Epo standard curve. Based on these findings we developed a procedure which could measure as little as 0·4 mU of Epo without requiring 59Fe. Using this method, plasma Epo titres were determined in 16 normal and 69 anaemic subjects.

Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics

We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross‐circulated, excised normal rat hearts and even in Ca2+ overload‐induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after β‐adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end‐systolic pressure and systolic pressure–volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat intercept, which is composed of for the total Ca2+ handling in excitation–contraction coupling and basal metabolism, of the and PVA linear relation was also significantly (P < 0.05) decreased (n= 8). The mean slope of the linear relation was unchanged in such hearts. Post‐dobutamine basal metabolism was unchanged (n= 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, α‐fodrin was identified (n= 7 of the 8 hearts with the decreased intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post‐β‐adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation–contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, α‐fodrin.

HB hope, β136(H14)Gly→Asp, in a Diabetic Japanese Female and its Functional Characterization

A beta-variant hemoglobin, first misjudged as a marked elevation of Hb A1, was found in a 68-year-old Japanese female with diabetes mellitus. This hemoglobin was isolated by Bio-Rex 70 chromatography combined with chromatofocusing, and was found to be Hb Hope, beta 136(H14)Gly----Asp, by classical and high performance liquid chromatographic peptide mapping techniques. Intrinsic oxygen affinity of this hemoglobin was approximately one-third as compared with that of Hb A0. This property was still observed in the constituent beta subunits isolated. Effects of such allosteric effectors as H+ (at a fixed concentration of Cl-), anion (Cl-), 2,3-diphosphoglycerate and carbon dioxide were more or less depressed. Among others, a marked reduction in the carbamate effect should be noted in a structural interpretation of the functional modifications. Subunit cooperativity, on the contrary, was not different from that in Hb A0 (n = 2.8-2.9). Explanation of these altered functions were attempted on the basis of the altered structure. The reduced stability of Hb Hope is also described.

Wide Variation of Myoglobin Contents in Gizzard Smooth Muscles of Various Avian Species

We determined myoglobin contents of gizzards (muscular stomach) and breast muscles in 34 avian species by a modification of Reynafarjes spectrophotometric procedure. The birds were apparently differentiated into two groups in respect of the gizzard, one with a high myoglobin content (7.74 +/- 1.81 mg/g muscle) and the other with a low (1.54 +/- 0.41 mg/g). In the former group of 15 species all but one were herbivorous, and all but one were carnivorous or else omnivorous in the latter group of 19 species. The myoglobin level was considered to closely correlate with mechanical performance and therefore oxygen demands of the gizzards. It might also be relevant to a circulatory situation during the tonic contractions of this organs.