Yasuhiro Ikeda

Catecholaminergic Polymorphic Ventricular Tachycardia Is Caused by Mutation-Linked Defective Conformational Regulation of the Ryanodine Receptor

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved. Objective: Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. Methods and Results: The R2474S/+ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic ventricular tachycardia was induced after exercise on a treadmill. The interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains of the RyR2 (an intrinsic mechanism to close Ca2+ channels) was weakened (domain unzipping). On protein kinase A–mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca2+ transients. cAMP-induced aberrant Ca2+ release events (Ca2+ sparks/waves) occurred at much lower sarcoplasmic reticulum Ca2+ content as compared to the wild type. Addition of a domain-unzipping peptide, DPc10 (amino acids 2460 to 2495), to the wild type reproduced the aforementioned abnormalities that are characteristic of the R2474S/+ KI mice. Addition of DPc10 to the (cAMP-treated) KI cardiomyocytes produced no further effect. Conclusions: A single point mutation within the RyR2 sensitizes the channel to agonists and reduces the threshold of luminal [Ca2+] for activation, primarily mediated by defective interdomain interaction within the RyR2.

Ischemic Pre-Conditioning Enhances the Mobilization and Recruitment of Bone Marrow Stem Cells to Protect Against Ischemia/Reperfusion Injury in the Late Phase

OBJECTIVESnThe aim of this study was to investigate whether the mobilization and recruitment of bone marrow stem cells (BMSCs) contribute to cardioprotection in the late phase after ischemic pre-conditioning (IPC).nnnBACKGROUNDnIPC is an innate phenomenon in which brief exposure to sublethal ischemia provides tissue protection from subsequent ischemia/reperfusion (I/R) injury. A delayed cardioprotection also occurs after IPC, but the precise mechanism is unclear.nnnMETHODSnIPC was created with 4 cycles of 5-min occlusion and reperfusion of the abdominal aorta in mice. Heart I/R injury was induced by occluding the left anterior descending artery for 30 min immediately (early phase) or 24 h (late phase) after IPC.nnnRESULTSnSerum vascular endothelial growth factor and stromal cell-derived factor-1alpha levels were increased significantly 1 and 3 h after IPC, but CD34+ and CD34+/flk-1+ stem cells in the peripheral blood were increased significantly 12 and 24 h after IPC (p < 0.05). Compared with the control treatment, both the early and late phases of IPC protected the heart against I/R injury. However, the recruitment of BMSCs was significantly greater in the heart when I/R injury was induced in late phase than in the early phase of IPC (p < 0.01). Interestingly, the blockade of the recruitment of BMSCs significantly attenuated the cardioprotective effect of IPC in the late phase (p < 0.01) but did not change in the early phase.nnnCONCLUSIONSnCardioprotection was observed in the early and late phases of IPC; however, the enhanced mobilization and recruitment of BMSCs played an important role in the late phase of IPC.

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction.

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 micromol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca(2+) sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca(2+) leak, and then lethal arrhythmia.

TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca(2+) signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (-/-)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) were increased in MLP (-/-) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (-/-) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (-/-) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM.

Isoform-specific roles of protein phosphatase 1 catalytic subunits in sarcoplasmic reticulum-mediated Ca2+ cycling

AIMSnprotein phosphatase 1 (PP1) is the major isotype of serine/threonine phosphatase in cardiomyocytes, and its activity has been thought to be important for heart failure progression. The PP1 catalytic subunits consist of three distinct genes, PP1α, PP1β/δ, and PP1γ. To date, the function of each PP1 isoform is not well characterized in cardiomyocytes. We sought to determine the functional contribution of each PP1 isoform to sarcoplasmic reticulum (SR)-mediated Ca(2+) cycling in isolated adult rat cardiomyocytes.nnnMETHODS AND RESULTSnadenoviral vectors encoding short hairpin RNA for each PP1 isoform were transfected into isolated rat cardiomyocytes, and this was followed by analysis of cell shortening, Ca(2+) transients, and the phosphorylation levels of Ca(2+) regulatory proteins. Physical interactions between each PP1 isoform and SR Ca(2+) regulatory proteins were characterized in isolated cardiomyocytes expressing green fluorescent protein (GFP)-tagged PP1 catalytic subunits, and also in canine junctional and longitudinal SR preparations. Successful PP1 isoform knockdown was achieved for each isoform without affecting the expression of the other isoforms. PP1β knockdown most significantly enhanced the Ca(2+) transient and cell shortening by augmenting phospholamban (PLN) phosphorylation at baseline and with low-dose isoproterenol stimulation (10 nM). Interestingly, PP1β was preferentially associated with sarco-endoplasmic ATPase and PLN in GFP-PP1-transfected cardiomyocytes, as well as in canine longitudinal SR preparations.nnnCONCLUSIONnthese findings indicate that PP1β is the most significant PP1 isoform involved in regulating SR Ca(2+) cycling in rat cardiomyocytes.

Lysyl oxidase resolves inflammation by reducing monocyte chemoattractant protein-1 in abdominal aortic aneurysm

Lysyl oxidase (LOX) is an enzyme critical for the stability of extracellular matrix and also known to have diverse biological functions. Little is known, however, about the role of LOX in regulating inflammation. Here we demonstrate that LOX suppresses secretion of monocyte chemoattractant protein-1 (MCP-1) in cultured vascular smooth muscle cells. Furthermore, enhancement of LOX activity reduces MCP-1 in a mouse model of abdominal aortic aneurysm (AAA), thereby preventing macrophage infiltration and AAA progression. These findings suggest that LOX has a novel function in resolving inflammation by reducing MCP-1 in AAA.

Heart Failure-Inducible Gene Therapy Targeting Protein Phosphatase 1 Prevents Progressive Left Ventricular Remodeling

BACKGROUNDnThe targeting of Ca(2+) cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca(2+) ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+) uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+) downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice.nnnMETHODSnWe created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×10(11) GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months.nnnRESULTSnIn the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months.nnnCONCLUSIONnHeart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure.

Tenascin-C is expressed in abdominal aortic aneurysm tissue with an active degradation process.

Abdominal aortic aneurysm (AAA) is a common disease caused by segmental weakening of the aortic walls and progressive aortic dilation leading to the eventual rupture of the aorta. Currently no biomarkers have been established to indicate the disease status of AAA. Tenascin‐C (TN‐C) is a matricellular protein that is synthesized under pathological conditions. In the current study, we related TN‐C expression to the clinical course and the histopathology of AAA to investigate whether the pattern of TN‐C expression could indicate the status of AAA. We found that TN‐C and matrix metalloproteinase (MMP)‐9 were highly expressed in human AAA. In individual human AAA TN‐C deposition associated with the tissue destruction, overlapped mainly with the smooth muscle actin‐positive cells, and showed a pattern distinct from macrophages and MMP‐9. In the mouse model of AAA high TN‐C expression was associated with rapid expansion of the AAA diameter. Histological analysis revealed that TN‐C was produced mainly by vascular smooth muscle cells and was deposited in the medial layer of the aorta during tissue inflammation and excessive destructive activities. Our findings suggest that TN‐C may be a useful biomarker for indicating the pathological status of smooth muscle cells and interstitial cells in AAA.

Revision Points and Remaining Issues to be Solved in the Updated Guidelines for Treatment of Chronic Heart Failure

Heart failure (HF) is a leading cause of morbidity and mortality in developed countries and afflicts more than 55 million people in the united states. Patients with chronic HF manifests progressive cardiac dysfunction that is characterized by either reduced systolic and diastolic left ventricular function, or both of it, with ventricular remodeling, arrhythmia, and intracardiac conduction disturbances. A growing body of evidence indicates that combination of pharmacological and non-pharmacological therapies had been significantly contributed to the improved morbidity and mortality of HF in the last decade. Reflecting those advances in HF-therapy, guidelines for treatment of chronic heart failure (JCS2010) has been updated at the end of year 2010. The new version of the guidelines includes updated information for device therapy for cardiac arrhythmia [implantable cardioverter defibllirator (ICD) and ablation therapy], respiratory support therapy for sleep disordered breathing, cardiac resynchronization therapy(CRT) for patients with wide QRS, surgical intervention for severe congestive heart failure, and newly developed left ventricular assist devices. In addition, disease management program and protocols of exercise and Waon therapy for HF patients were revised or newly added into the guideline. In this Symposium, I will review these revision points and remaining issues to be solved for treatment of chronic heart failure.

Regression of Abdominal Aortic Aneurysms through Pharmacologic Therapy

Abdominal aortic aneurysm (AAA) is a common disease that eventually results in aortic rupture with high mortality. Although a pharmacologic therapy for AAA is eagerly awaited, the destruction of the aortic walls in AAA has been considered an irreversible process. Recently, we identified c-Jun N-terminal kinase (JNK) as a key molecule in the pathogenesis of AAA. JNK was highly activated in the aortic walls of AAA patients. In in vitro studies, JNK not only enhanced expression of matrix metalloproteinases (MMPs), but also reduced expression of biosynthetic enzymes for extracellular matrix (ECM). An AAA model induced by CaCl2 in mice was accompanied by activation of JNK and MMP-9. Treatment with a specific JNK inhibitor, SP600125, completely prevented AAA formation. SP600125 also caused a significant reduction in aneurysmal diameter in established AAA. Surprisingly, SP600125 was effective in normalizing tissue architecture. Furthermore, treatment with SP600125 caused significant regression of angiotensin II-induced AAA in ApoE-null mice after its establishment. These data indicated that pharmacologic inhibition of JNK caused healing of aneurysmal tissue and regression of established AAA, providing a novel therapeutic option for treatment of AAA.