Makoto Sunamori

Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation.

Abstract— Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

Stress response gene ATF3 is a target of c-myc in serum-induced cell proliferation.

The c‐myc proto‐oncogene encodes a transcription factor that promotes cell cycle progression and cell proliferation, and its deficiency results in severely retarded proliferation rates. The ATF3 stress response gene encodes a transcription factor that plays a role in determining cell fate under stress conditions. Its biological significance in the control of cell proliferation and its crosstalk regulation, however, are not well understood. Here, we report that the serum response of the ATF3 gene expression depends on c‐myc gene and that the c‐Myc complex at ATF/CREB site of the gene promoter plays a role in mediating the serum response. Intriguingly, ectopic expression of ATF3 promotes proliferation of c‐myc‐deficient cells, mostly by alleviating the impeded G1‐phase progression observed in these cells, whereas ATF3 knockdown significantly suppresses proliferation of wild‐type cells. Our study demonstrates that ATF3 is downstream of the c‐Myc signaling pathway and plays a role in mediating the cell proliferation function of c‐Myc. Our results provide a novel insight into the functional link of the stress response gene ATF3 and the proto‐oncogene c‐myc.

Synthetic Salusins as Cardiac Depressors in Rat

Using bioinformatic analyses of full-length, enriched human cDNA libraries, we recently identified salusins, multifunctional related peptides ubiquitously expressed in major human tissues. Salusins cause transient and profound hypotension when injected intravenously to rats, the hypotensive effect of salusin-&bgr; being especially striking. However, the mechanisms of this hypotensive action remain elusive. To determine whether salusins modulate cardiac function in rats, we studied serial changes of systemic hemodynamics and functions of isolated perfused working and nonworking hearts before and after salusin administration. Intravenous salusin-&bgr; administration to intact anesthetized rats caused a temporary rapid, profound decrease in aortic blood flow concomitantly with hypotension and bradycardia without affecting systemic vascular resistance. Salusin-&bgr;–induced hypotension and bradycardia were completely blocked by pretreatment with atropine, a muscarinic receptor antagonist, but not by propranolol. In isolated perfused working rat hearts, salusin-&bgr; significantly decreased cardiac output, aortic flow, and stroke work. However, it did not affect coronary flow in isolated working and nonworking hearts. Our results indicate that salusins induce potent hypotension via negative inotropic and chronotropic actions. Salusin-&bgr; promotes its actions by facilitating vagal outflows to the heart, whereas the negative inotropism of salusin-&bgr; is also mediated via a direct myotropic effect.

Stented elephant trunk procedure combined with ascending aorta and arch replacement for acute type A aortic dissection

OBJECTIVES Despite steadily improving outcomes, surgery for acute type A aortic dissection has several unresolved problems such as expansion of the residual false lumen in the descending aorta. We performed transaortic stented graft implantation into the descending aorta combined with the ascending aorta and aortic arch replacement for acute type A aortic dissection. We review the efficacy and outcomes of this procedure with respect to the residual false lumen and postoperative neurologic complications we encountered. METHODS Nine consecutive patients with acute type A aortic dissection underwent this procedure. The stented elephant trunk graft was implanted through the aortic arch under hypothermic circulatory arrest. The stented graft was 15 cm long in six patients, and 10 cm long in three patients. Enhanced computed tomography (CT) was performed 1 month after surgery and once each year after discharge to evaluate the postoperative time course of the residual false lumen. RESULTS Cardiopulmonary bypass (CPB) time was quite long because of slow cooling and re-warming [352+/-92 (mean+/-SD) min], and average lower-body arrest time was 54+/-10min. The intima in one patient was injured at the time of implantation, and a small leak was created. One patient died of multiorgan failure postoperatively. One patient suffered cerebral injury, and two suffered spinal cord injury perioperatively. Average follow-up time was 40.4 months (range, 13-66 months). One patient died of cerebral infarction during follow up, and the other seven survived and remain well. Postoperative enhanced CT scans showed that the dissected descending aortas attached to the stented grafts and the aortas near the stented grafts returned to normal. In one patient with no re-entry, the false lumen completely closed with thrombi and the entire aorta returned to normal. The diameter of the descending aorta decreased or did not change in six of the seven patients (85.8%) and increased by only 2mm in one of them (14.2%) during follow up. CONCLUSIONS Implantation of a stented elephant trunk into the descending aorta combined with replacement of the ascending aorta and total arch for acute type A aortic dissection is effective in closing the residual false lumen of the descending aorta and in preventing expansion of the descending aorta. However, further technical modifications, such as using a short stented elephant trunk, eliminating aortic clamping, shortening CPB and spinal cord ischemic time, and reconstruction of left subclavian artery, are needed to prevent neurologic complications.

Effect of aprotinin to improve myocardial viability in myocardial preservation followed by reperfusion

Isolated canine hearts were preserved at 4 degrees C with multi-dose cardioplegic solution every hour for 6 hours. Reperfusion was observed for 2 hours under cross-circulation without cardiotonic drugs. The aprotinin group (n = 8), which received cardioplegic solution with added aprotinin (150 KIU/mL), was compared with the control group (n = 6). The increase in tissue adenosine triphosphate and total adenine nucleotide content during reperfusion was significant in the aprotinin group; there was no change in the control group, and the levels at the end of reperfusion tended to be higher in the aprotinin group than in the control group. Tissue adenosine diphosphate levels remained unchanged in both groups. Tissue adenosine monophosphate levels declined during reperfusion in both groups and were slightly lower in the control group. Tissue levels of cyclic adenosine monophosphate remained unchanged in the aprotinin group whereas they increased during ischemia and declined significantly during reperfusion in the control group. Tissue levels of cyclic guanosine monophosphate declined during reperfusion in both groups without difference. Creatine phosphate levels recovered in both groups without difference. Serum cyclic guanosine monophosphate concentration tended to be lower in the aprotinin group than in the control group. Serum creatine kinase-MB level increased slightly during reperfusion in both groups without difference. N-acetyl-beta-D-glucosaminidase levels were significantly suppressed during reperfusion in the aprotinin group as compared with the control group. These results suggest that aprotinin is effective in preserving adenine nucleotide and adenosine triphosphate levels and in stabilizing tissue cyclic adenosine monophosphate levels in prolonged hypothermic cardioplegic preservation followed by reperfusion.

Cytokinetic study of aortocoronary bypass vein grafts in place for less than six months

To evaluate the initial mechanism involving atherosclerotic changes of the saphenous vein graft implanted for coronary artery revascularization in the early stage, immunocytochemical analysis was performed to determine the cell components and kinetics of saphenous vein grafts. Specimens of saphenous vein grafts were obtained from 7 necropsy patients who died at 4 days to 6 months after surgery. Monoclonal antibodies specific for smooth muscle cells (HHF35) and macrophages (HAM56) were used for analysis of the cell components. Migration of macrophages into the intima and the media was observed on the fourth postoperative day. Intimal thickening was characterized by the proliferation of smooth muscle cells, and scattered macrophages were present in the subendothelial layer 1 month after surgery. At 2 months, intimal thickening became prominent and macrophages were recognized circumferentially throughout the layer. At 5 to 6 months, some of the saphenous vein grafts were almost occluded by severe intimal thickening due to proliferation of the smooth muscle cells. Macrophages were also observed both inside and outside of the internal elastic lamina; these are rarely found in the artery. These results suggest that compared with the arterial graft, cytokinesia of the saphenous vein graft contributes to the development of early graft failure because of its rapidity in progression and severity.

L-arginine, a nitric oxide precursor, attenuates ischemia-reperfusion injury by inhibiting inositol-1,4,5-triphosphate.

OBJECTIVE We evaluated the effect of pretreatment with nitric oxide precursor before ischemia on recovery with reperfusion in rat hearts. METHODS Isolated rat hearts were perfused with Krebs-Henseleit buffer without (C group) or with 3 mmol/L L-arginine (A group) before 30 minutes of ischemia. The left ventricular function, including heart rate, developed pressure, maximal dp/dt, and coronary flow, were measured before pretreatment and after 10 and 30 minutes of reperfusion. Cyclic guanosine monophosphate (by radioimmunoassay), calcium (by absorption spectrophotometry), and inositol 1,4,5-triphosphate synthesized from tritiated myo-inositol (by ion-exchange chromatography preceding counting) were measured at the same times and immediately after ischemia. RESULTS Recovery of ventricular function was significantly greater in the A group than in the C group. Pretreatment increased postischemic cyclic guanosine monophosphate content compared with the preischemic level (from 1.06 +/- 0.12 to 1.94 +/- 0.09 pmol/mg protein, p < 0.05). No change in cyclic guanosine monophosphate was evident in the C group. In the C group, inositol triphosphate content increased after 10 minutes of reperfusion beyond the preischemic level (from 0.53 +/- 0.023 to 1.15 +/- 0.045 cpm x 10(-3)/gm, p < 0.05) as did calcium at 30 minutes (from 4.12 +/- 0.164 to 6.86 +/- 0.544 mmol/gm dry weight). In the A group, both of these increases were significantly attenuated. CONCLUSION These data suggest that L-arginine pretreatment may reduce calcium overload by increasing cyclic guanosine monophosphate production, which in turn downregulates inositol triphosphate synthesis during reperfusion.

Adrenomedullin Receptor Antagonism by Calcitonin Gene-Related Peptide(8-37) Inhibits Carotid Artery Neointimal Hyperplasia After Balloon Injury

Intimal injury by angioplasty results in a series of changes, including smooth muscle cell hyperplasia, that lead to vascular restenosis. Adrenomedullin, a potent vasodilator peptide, has natriuretic effects, and its plasma concentration is elevated in cardiovascular diseases. Adrenomedullin is secreted by endothelial and vascular smooth muscle cells, but its role in neointimal hyperplasia after balloon injury has not been previously described. We investigated the role of endogenous adrenomedullin in neointimal hyperplasia using an in vivo rat model of postinjury vascular restenosis. In the injured rats, bromodeoxyuridine-labeled nuclei in the media of untreated common carotid arteries were increased 2 days after injury, which were suppressed by in vivo treatment with the adrenomedullin receptor antagonist calcitonin gene-related peptide (CGRP)(8-37). Inhibition of neointimal hyperplasia by CGRP(8-37) was distinct at 7 and 14 days, whereas CGRP(1-37) had no effect. The expression of adrenomedullin in the media of both untreated and treated common carotid arteries was elevated at 2 days and further enhanced in hyperplastic intima of untreated common carotid arteries at 7 days. Our findings suggest a novel role for endogenous adrenomedullin in balloon injury-induced restenosis and indicate that CGRP(8-37) may be useful for the prevention of vascular restenosis.

Clinical experience of coenzyme Q10 to enhance intraoperative myocardial protection in coronary artery revascularization

SummarySeventy-eight patients undergoing coronary artery bypass grafting (CABG) were compared retrospectively to evaluate whether pretreatment with coenzyme Q10 (CoQ) is effective in preventing left ventricular depression in early reperfusion following CABG. CoQ (5 mg/kg, intravenously) was given to 60 patients, 2 hours prior to the onset of cardiopulmonary bypass (CPB). CABG was performed using saphenous vein under CPB associated with cold cardioplegia in the standard fashion. Heart rate, mean arterial pressure, and cardiac index showed no significant difference between the CoQ and control groups. However, left ventricular stroke work index was significantly elevated at 6 and 10 hours of reperfusion following CABG in the CoQ-treated group compared with the controls. Serum MB-CK was lower at 0 and 6 hours of reperfusion in the CoQ group compared with the controls. These results suggest that pretreatment with intravenous CoQ is effective in preventing left ventricular depression in early reperfusion and in minimizing myocardial cellular injury during CABG followed by reperfusion.

Radial Artery Graft for Coronary Artery Bypass Surgery: Biological Characteristics and Clinical Outcome

Abstract  The radial artery (RA) is gaining popularity as a bypass conduit for coronary artery bypass grafting, and its impact on clinical practice has been extensively explored. In the present article, we provide a review of postoperative hand circulation, vascular biological characteristics of the RA graft, the efficacy of vasodilator therapies, and mid‐term clinical results of use of the RA graft. Fundamental studies revealed excellent vascular biological characteristics of the RA graft as a living arterial conduit, making it almost equivalent to the internal thoracic artery (ITA) graft. Clinical studies have yielded encouraging mid‐term results. Most studies reported in favor of the RA graft over the saphenous vein graft with regard to patency rate, freedom from cardiac events, and survival. However, superiority of either the RA or right ITA graft has not been conclusively determined. The long‐term results of RA grafts remain unknown, but at present, supplementary use of an RA graft with a left ITA graft appears feasible for CABG.