Osamu Tsukamoto

Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5

Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.

Higd1a is a positive regulator of cytochrome c oxidase.

Significance We identified hypoxia-inducible domain family, member 1A (Higd1a) as a positive regulator of cytochrome c oxidase (CcO). CcO, the terminal component of the mitochondrial electron transfer system, reductively converts molecular oxygen to water coupled to pump protons across the inner mitochondrial membrane. Higd1a is transiently induced under hypoxic conditions and increases CcO activity by directly interacting with CcO in the vicinity of its active center. Induction of Higd1a leads to increased oxygen consumption and subsequent mitochondrial ATP synthesis, thereby improving cell viability under hypoxia. Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.

PDE5 inhibitor efficacy is estrogen dependent in female heart disease

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormones involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

Long-term biopersistence of tangled oxidized carbon nanotubes inside and outside macrophages in rat subcutaneous tissue

Because of their mechanical strength, chemical stability, and low molecular weight, carbon nanotubes (CNTs) are attractive biological implant materials. Biomaterials are typically implanted into subcutaneous tissue or bone; however, the long-term biopersistence of CNTs in these tissues is unknown. Here, tangled oxidized multi-walled CNTs (t-ox-MWCNTs) were implanted into rat subcutaneous tissues and structural changes in the t-ox-MWCNTs located inside and outside of macrophages were studied for 2 years post-implantation. The majority of the large agglomerates were present in the intercellular space, maintained a layered structure, and did not undergo degradation. By contrast, small agglomerates were found inside macrophages, where they were gradually degraded in lysosomes. None of the rats displayed symptoms of cancer or severe inflammatory reactions such as necrosis. These results indicate that t-ox-MWCNTs have high biopersistence and do not evoke adverse events in rat subcutaneous tissue in vivo, demonstrating their potential utility as implantable biomaterials.

A Development of Nucleic Chromatin Measurements as a New Prognostic Marker for Severe Chronic Heart Failure.

Background Accurate prediction of both mortality and morbidity is of significant importance, but it is challenging in patients with severe heart failure. It is especially difficult to detect the optimal time for implanting mechanical circulatory support devices in such patients. We aimed to analyze the morphometric ultrastructure of nuclear chromatin in cardiomyocytes by developing an original clinical histopathological method. Using this method, we developed a biomarker to predict poor outcome in patients with dilated cardiomyopathy (DCM). Methods and Results As a part of their diagnostic evaluation, 171 patients underwent endomyocardial biopsy (EMB). Of these, 63 patients diagnosed with DCM were included in this study. We used electron microscopic imaging of cardiomyocyte nuclei and an automated image analysis software program to assess whether it was possible to detect discontinuity of the nuclear periphery. Twelve months after EMB, all patients with a discontinuous nuclear periphery (Group A, n = 11) died from heart failure or underwent left ventricular assist device (VAD) implantation. In contrast, in patients with a continuous nuclear periphery (Group N, n = 52) only 7 patients (13%) underwent VAD implantation and there were no deaths (p<0.01). We then evaluated chromatin particle density (Nuc-CS) and chromatin thickness in the nuclear periphery (Per-CS) in Group N patients; these new parameters were able to identify patients with poor prognosis. Conclusions We developed novel morphometric methods based on cardiomyocyte nuclear chromatin that may provide pivotal information for early prediction of poor prognosis in patients with DCM.

Btg2 is a Negative Regulator of Cardiomyocyte Hypertrophy through a Decrease in Cytosolic RNA

Under hypertrophic stimulation, cardiomyocytes enter a hypermetabolic state and accelerate biomass accumulation. Although the molecular pathways that regulate protein levels are well-studied, the functional implications of RNA accumulation and its regulatory mechanisms in cardiomyocytes remain elusive. Here, we have elucidated the quantitative kinetics of RNA in cardiomyocytes through single cell imaging and c-Myc (Myc)-mediated hypermetabolic analytical model using cultured cardiomyocytes. Nascent RNA labeling combined with single cell imaging demonstrated that Myc protein significantly increased the amount of global RNA production per cardiomyocyte. Chromatin immunoprecipitation with high-throughput sequencing clarified that overexpressed Myc bound to a specific set of genes and recruits RNA polymerase II. Among these genes, we identified Btg2 as a novel target of Myc. Btg2 overexpression significantly reduced cardiomyocyte surface area. Conversely, shRNA-mediated knockdown of Btg2 accelerated adrenergic stimulus-induced hypertrophy. Using mass spectrometry analysis, we determined that Btg2 binds a series of proteins that comprise mRNA deadenylation complexes. Intriguingly, Btg2 specifically suppresses cytosolic, but not nuclear, RNA levels. Btg2 knockdown further enhances cytosolic RNA accumulation in cardiomyocytes under adrenergic stimulation, suggesting that Btg2 negatively regulates reactive hypertrophy by negatively regulating RNA accumulation. Our findings provide insight into the functional significance of the mechanisms regulating RNA levels in cardiomyocytes.

Different Implications of Functional Tricuspid Regurgitation Between Heart Failure (HF) With Reduced Ejection Fraction (EF) and HF With Preserved EF.

In left-sided HF, elevated left atrial pressure (LAP) is transmitted through the lungs as pulmonary hypertension (PH), classified as group 2 PH.4 In the early stage of group 2 PH, pulmonary artery pressure (PAP) is elevated by passive downstream elevation in LAP with normal pulmonary vascular resistance (PVR), known as passive PH.4 When functional and structural abnormalities occur in the pulmonary vasculature, PVR is elevated and causes an increased transpulmonary gradient (TPG), called reactive PH.4 When the right ventricle (RV) is subjected to elevated afterload by PH, it initially dilates and may chronically adapt to PH with hypertrophy. The dilatation of the RV eventually leads to tricuspid annular dilatation, resulting in impaired tricuspid annular contraction and functional TR. Once the TV is dilated, it continues to dilate the RV further and worsen the TR, which eventually results in RV dysfunction.4 Thus, elevated LAP induces passive and subsequent he clinical significance of tricuspid regurgitation (TR) has long been ignored, because it is a common echocardiographic finding observed in 80–90% of healthy individuals1 and TR may have been tolerated for years. In addition, attention has not been paid to the right-side of the heart compared with the left side. However, TR has received a lot of attention in recent years as a predictor and/or a contributor of clinical outcomes in patients with heart failure (HF).1 TR has primary and secondary etiologies. Primary TR is uncommon and attributed to intrinsic lesions of the tricuspid valve (TV). Secondary TR, also known as functional TR, is the most common form, which is mainly caused by left-sided HF2 and mitral valve surgery.3

Targeting Lysosomal Ca2+ to reduce Reperfusion Injury

This editorial refers to ‘Inhibition of NAADP signalling on reperfusion protects the heart by preventing lethal calcium oscillations via two-pore channel 1 and opening of the mitochondrial permeability transition pore’ by S.M. Davidson et al. , pp. 357–366. The use of percutaneous coronary intervention (PCI) in patients with acute myocardial infarction (AMI) has successfully reduced the incidence of associated mortality and/or morbidity. However, despite widespread use of PCI, there has been an increase in the prevalence of ischaemic heart failure, which is mainly attributable to the fact that this treatment has not been as successful as expected in limiting the size of infarcts. Because reducing the infarct size is essential to decrease the development of heart failure, PCI alone is not sufficient to reduce the incidence of ischaemic heart failure following AMI. To overcome this challenge, many researchers have investigated the major causes of ischaemia and reperfusion injury and methods to attenuate the damage caused by them.1 Many lines of evidence for the causes of ischaemia and reperfusion injury implicate mitochondrial dysfunction. Myocardial ATP depletion through mitochondrial dysfunction decreases Ca2+ uptake into the sarcoplasmic reticulum (SR) in ischaemic/reperfused myocardium, leading to an increase in intracellular Ca2+ [Ca2+]c levels. These findings suggest that the phenomena of ATP depletion and Ca2+ overload may play a central role in ischaemia and reperfusion injury; in contrast, Ca2+ is also important for maintaining normal cardiac performance, thus demonstrating that [Ca2+]c has both a physiological and a pathological role in the heart. The levels of cyclic ADP-ribose (cADPR), one of the secondary messengers for Ca2+ signalling, are increased by Ca2+ influx …

Recent Progress and Next Challenges in the Treatment of Symptomatic Heart Failure in Japan

diseases (hypertension, dyslipidemia, diabetes mellitus, and obesity), ischemic etiology, and HF with preserved ejection fraction (HFpEF). We can recognize that recent epidemiological trends in patients with symptomatic HF in Japan are being affected by multiple complex and diverse factors: westernization of diet, marked increase in lifestyle-related diseases and ischemic heart diseases, advances in medical and surgical treatment, and aging of society. In the United States, the incidence of HF approaches 10 per 1,000 population after 65 years of age4 and the prevalence of HF is expected to increase 46% from 2012 to 2030.5 The prevalence of ischemic HF in Japan has already reached the levels in Western countries. In addition, Japan has already entered an era of the aging/super-aging society. Accordingly, it is expected that the number of geriatric HF patients will rapidly and continuously increase in Japan in the foreseeable future, because lifestyle-related diseases, ischemic heart disease and older age are risk factors for HF.5 This report elucidates what needs to be done next (Figure). First of all, the reduction of ischemic events is important. The incidence of ischemic HF can be reduced by implementing existing preventive approaches to lifestyle-associated diseases. Secondly, further improvement in the use of existing evidencebased medications for HF treatment is also important, which can prevent disease progression. Thirdly, it is also crucial to develop a strategy against the increased prevalence of HFpEF. Although no medicines have been proved to have beneficial effects on HFpEF according to prospective randomized clinical study so far, recent observational studies showed a potenhronic heart failure (CHF) remains a major cause of mortality and morbidity in the Western and developed countries, while developing countries in Asia are also in the process of the epidemiologic transition (ie, an etiological shift from infectious diseases and nutritional deficiencies to lifestyle-related diseases including cardiovascular diseases).1,2 Thus, CHF has become a major public health concern with increasing incidence and prevalence worldwide. Japan has become the leading country for longevity mainly via improvements in public health, advances of medicine and a unique dietary culture. However, modernization and westernization of the Japanese diet has recently changed the epidemiological trend, especially a marked increase in lifestyle-related diseases, subsequent cardiovascular diseases and CHF. On the other hand, recent widespread use of evidence-based medications in the treatment of cardiovascular diseases is thought to have modified the recent trends in the epidemiology of CHF in Japan.