Isao Shiraishi

Cardiac Stem Cell and Myocyte Aging, Heart Failure, and Insulin-Like Growth Factor-1 Overexpression

Abstract— To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27Kip1, p53, p16INK4a, and p19ARF, were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy.

Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy

The serine/threonine kinase Akt regulates cellular survival, proliferation, gene transcription, protein translation, metabolism, and differentiation. Although Akt substrates are found throughout the cell, activated Akt normally accumulates in the nucleus, suggesting that biologically relevant targets are located there. Consequences of nuclear Akt signaling in cardiomyocytes were explored by using nuclear-targeted Akt (Akt-nuc). Accumulation of Akt-nuc did not provoke hypertrophy, unlike constitutively activated Akt. Instead, Akt-nuc inhibited hypertrophy concurrent with increased atrial natriuretic peptide (ANP) expression that depended upon phosphatidylinositol-3 kinase activity. Akt-nuc antihypertrophic effects were blocked by inhibition of either guanylyl cyclase A receptor or cyclic guanosine monophosphate-dependent protein kinase in cultured cardiomyocytes. Corroborating evidence showed blunted acute hypertrophic remodeling in Akt-nuc transgenic mice after transverse aortic constriction coincident with higher ANP expression and smaller myocyte volume. In addition, Akt-nuc expression improved systolic function and survival in the chronic phase of transverse aortic constriction-induced hypertrophy. Thus, Akt-nuc antagonizes certain aspects of hypertrophy through autocrine/paracrine stimulation of a phosphatidylinositol-3 kinase-dependent signaling cascade that promotes ANP expression, resulting in a unique combination of prosurvival coupled with antihypertrophic signaling.

Quantitative histological analysis of the human sinoatrial node during growth and aging.

BackgroundFibrosis or fatty infiltration of the human sinoatrial (SA) node is generally believed to represent replacement of the SA nodal cells by connective tissue. Quantitative analysis, however, has not been performed precisely to validate the interpretation of such histological changes. Methods and ResultsThe actual volume of the SA node and its components were calculated according to the sum of the pixel number representing the colors of SA nodal cells and connective tissue in serial sections using a digital color image analyzer. Average volume occupied by the total SA nodal cells in adolescents and adults (n=7) was 3.55±0.45 mm3, which was 2.4 times greater than that in infants (n=3). The rate of increase was smaller than that of the total SA node (4.2 times, 16.68±2.56 mm3 in adolescents and adults). The considerable discrepancy in the growth ratio between the SA nodal cells and the total SA node resulted from an increase in the volume of connective tissue (7.4 times). In the elderly (n=9), the volume of total SA node and SA nodal cells actually decreased (13.10±1.85 mm3 and 2.18±0.44 mm3), whereas that of fibrous connective tissue remained unchanged. Constant DNA ploidy patterns of SA nodal cells determined by cytofluorometry indicated that SA nodal cells never synthesize DNA during growth. ConclusionsUntil adulthood, the actual volume of SA nodal cells does not decrease, although the increase in volume ratio of the interstitial tissue to the total SA node has merely given a false impression of involution of SA nodal cells. Atrophy of SA nodal cells, however, occurs during aging together with reduction of the SA node and/or infiltration of fatty tissue.

Simulative operation on congenital heart disease using rubber-like urethane stereolithographic biomodels based on 3D datasets of multislice computed tomography

OBJECTIVE Stereolithographic biomodelling is a technique where photosensitive liquid resin is polymerised with a pinpoint laser beam controlled by three-dimensional (3D) datasets. This study was designed to assess whether a stereolithographic biomodelling technique is applicable for precise anatomical diagnosis and simulation surgery of complicated congenital heart disease. METHODS Twelve stereolithographic biomodels were manufactured with multislice computed tomography (MSCT)-based 3D datasets. They were made of photosensitive liquid epoxy or urethane. RESULTS All the solid epoxy and rubber-like urethane biomodels reproduced the complex anatomical structures of the arteries and veins in congenital heart diseases. Furthermore, the rubber-like urethane biomodels allowed the surgeon to cut and suture, thus facilitating the simulation of the surgical operation. CONCLUSIONS Stereolithographic biomodelling is a promising technique for the preoperative practice and simulation of individual surgery. This technique would be useful in the planning of novel and innovative surgical procedures of congenital heart disease.

3-D observation of N-cadherin expression during cardiac myofibrillogenesis of the chick embryo using a confocal laser scanning microscope.

SummaryIt is not yet understood whether cell adhesion molecules play an active role in early cardiac morphogenesis or not. We present here the spatial and temporal expressions of N-cadherin and its relationships to actin filaments during looping (7- to 13-somite stages) of the chick embryonic heart tube observed by means of a confocal laser scanning microscope. Serial optical tomograms were obtained from the whole-mounted heart tubes stained with antibody to N-cadherin (fluoresceinconjugated) and phalloidin (rhodamine-conjugated). Three patterns of N-cadherin expression were observed during looping; a belt-like pattern, speckled pattern, and clumped pattern, corresponding to adhesion belt, nonjunctional cell contact and early intercalated disks, respectively. At the 7-somite stage, myocytes expressed N-cadherin as adhesion belt and nonjunctional cell contact. At the 8- to 10-somite stages, the clumped pattern of N-cadherin was detected before striated myofibrils appeared. Myofibrils began to develop across the clumps to form transcellular networks in the outer layer, and to form circumferential alignments in the inner layer. These results suggest that N-cadherin is responsible for the connection of myofibrils between the neighboring myocytes, and the alignment of the two layers in the developing heart tube.

Early stage-specific inhibitions of cardiomyocyte differentiation and expression of Csx/Nkx-2.5 and GATA-4 by phosphatidylinositol 3-kinase inhibitor LY294002

Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) has been reported to block cardiomyocyte differentiation. However, at which stage PI3-kinase plays this important role and what its molecular targets are remain unknown. To answer these questions, we induced cardiomyocyte differentiation of P19CL6 mouse embryonal carcinoma cells and investigated the activation of PI3-kinase by analyzing phospho-Akt. We also treated P19CL6 cells with the PI3-kinase-specific inhibitor LY294002 either continuously or at various time points and monitored the expression of cardiac contractile proteins and transcription factors. Most cells differentiated into sarcomeric myosin heavy chain (MHC)-positive cardiomyocytes on day 16 after induction. An increase in phospho-Akt was observed after induction and was maintained throughout the differentiation. LY294002 treatment restricted to the phase from days 0 to 4 was sufficient to inhibit cardiomyocyte differentiation in a dose-dependent manner. In contrast, LY294002 treatment either from days 4 to 8 or from days 8 to 12 did not cause significant changes in sarcomeric MHC expression. LY294002 treatment from days 0 to 4 also suppressed Csx/Nkx-2.5 and GATA-4 expression. These results demonstrate that PI3-kinase becomes activated and plays a pivotal role at a very early stage of cardiomyocyte differentiation, possibly by modulating the expression of the cardiac transcription factors.

Transformation of coronary artery aneurysm to obstructive lesion and the role of collateral vessels in myocardial perfusion in patients with Kawasaki disease

OBJECTIVES The aim of this study was to examine the transformation of coronary artery aneurysms to obstructive lesions and to assess the role of collateral vessels in patients with Kawasaki disease. BACKGROUND Coronary artery aneurysms, especially giant aneurysms, are known to become obstructive lesions in patients with Kawasaki disease. However, the process of transformation is not yet clear. METHODS Thirty patients (average age 9.9 years) with obstructive lesions secondary to Kawasaki disease underwent repeated coronary artery angiography and thallium myocardial scintigraphy over a mean period of 7.7 years after the acute onset of Kawasaki disease. RESULTS In the 27 patients who were enrolled in the acute phase of the disease because of coronary artery aneurysms, the later transformation to obstructive lesions was not significantly different between the 61 large and 6 medium-sized aneurysms. Obstructive transformation of aneurysms was more rapid in the right than in the left coronary artery (p < 0.001). From the last coronary angiogram obtained, the obstructive lesions were classified as localized stenosis > 90% in 10 vessels, occlusions in 6 vessels and segmental stenosis in 26 vessels. Both localized and segmental stenosis occurred significantly more often in the left anterior descending and the right coronary artery than in other vessels (p < 0.05). The incidence of collateral vessels was significantly correlated with a younger age at onset of Kawasaki disease, especially in patients with segmental stenosis (p < 0.001). Collateral vessels did not develop in the presence of localized stenosis regardless of the occurrence of myocardial ischemia. All occluded vessels had collateral development regardless of the presence of myocardial infarction. CONCLUSIONS The treatment of localized stenosis may play an important role in preventing myocardial infarction in the chronic phase of Kawasaki disease.

Three-dimensional observation with a confocal scanning laser microscope of fibronectin immunolabeling during cardiac looping in the chick embryo

From the beginning of cardiac myofibrillogenesis in the chick embryo, developing myofibrils at the bottom of the inner myocardial cell layer facing the cardiac jelly are already aligned circumferentially in the direction of the heart tube. To elucidate the mechanism of this alignment, we investigated the temporal and spatial expression of fibronectin and its relationship to actin filaments before and during looping (4- to 13-somite stages) by using a confocal scanning laser microscope. Serial optical tomograms were obtained from whole-mounted heart tubes stained with fluorescein-conjugated antibody against cellular fibronectin and rhodamine-conjugated phalloidin. Before looping (4- to 7-somite stages), particulate and speckled fibronectin formed loose networks. At the onset of looping (8- to 9-somite stages), fine fibrils of fibronectin appeared. They became dense and were arranged circumferentially in the direction of the heart tube. They were aligned parallel with the thick actin bundles that appeared as an initial stage of developing myofibrils. During looping, (10- to 13-somite stages), fibronectin fibrils were fragmented and showed a speckled pattern, while the number of circumferentially aligned mature striated myofibrils increased. These observations suggest that the temporal arrangement of fibronectin fibrils at the beginning of looping plays a role in the circumferential alignment of developing myofibrils.

Regulation of Embryonic Lung Vascular Development by Vascular Endothelial Growth Factor Receptors, Flk-1 and Flt-1

The biological effects of vascular endothelial growth factor A (VEGF‐A) are mediated by fetal liver kinase‐1 (Flk‐1) and fms‐like tyrosine kinase‐1 (Flt‐1). In lung tissue, VEGF‐A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk‐1 and Flt‐1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk‐1 and Flt‐1 of embryonic mouse lung tissue. When Flk‐1 was predominantly expressed (embryonic day [E] 9.5–E13.5), then vascular endothelial cells actively proliferated. When Flt‐1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk‐1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt‐1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt‐1 promoted Flk‐1 expression. These results suggest that the two VEGF‐A receptors regulate pulmonary vascular development by modulating the VEGF‐A signaling. Anat Rec, 290:958–973, 2007.

Renal function and cardiac angiography.

Objective: To study the effect of non-ionic contrast medium on renal function in children with cardiovascular disease.Methods: Analysis of renal function in 98 children with cardiovascular disease before and after the use of lopamidol, lohexol, and loversol was done for angiography. Serum creatinine (s-Cre), urinary N-acetyl-beta-D-glucosaminase (u-NAG), urinary beta 2-microglobulin (u-BMG), and urinary alpha 1-microglobulin (u-AMG) levels were evaluated.Results: Although s-Cre levels remained unchanged, u-NAG/Cre, u-AMG/Cre and u-BMG/Cre significantly increased 12 hours after angiography. Levels of u-NAG/Cre, u-BMG/Cre, and u-AMG/Cre after angiography were significantly higher in neonates and infants (age<12-Months, n=32) than in children (age>1-year, n=61), in patients with more than 5 ml/kg of contrast medium (n=25) than in those with less than 5 ml/kg (n=70) and in cyanotic patients (n=13) than in non-cyanotic (n=80) patients.Conclusion: Transient renal tubular dysfunction occurred in all of these three non-ionic contrast mediums. Although renal tubular function was intact on a long-term basis, one should be careful of contrast medium-induced nephropathy, especially in neonates and infants, in patients receiving more than 5 ml/kg of contrast mediums in total, and in patients with cyanotic heart disease in using non-ionic contrast mediums.