Toshitaka Yajima

An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice

The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress-induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Galphaq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.

Viral Myocarditis From the Perspective of the Virus

Viral myocarditis has been recognized as a cause of congestive heart failure for >50 years, but it is still a challenging disease to diagnose and treat.1,2 The history and clinical features are often nonspecific, and practical serological markers are not available during the acute phase of the disease. Even after proper diagnosis, no clinically proven treatment exists to inhibit the development of subsequent dilated cardiomyopathy (DCM) and, in some cases, death. Accordingly, to facilitate future scientific work into this difficult clinical entity, this review proposes a clinical paradigm that focuses on the phases of viral infection and the molecular insights that are important for these phases of the infectious process with a focus on interactions between the virus and the cardiac myocyte. Myocarditis is defined as inflammation of the heart muscle. The gold standard for diagnosis has been the Dallas criteria based on histopathology from an endomyocardial biopsy.3 It is now recognized that the Dallas criteria are not sensitive for myocarditis because they do not consider the presence of viral genome in the heart.4 Furthermore, an invasive procedure is required to obtain a sample of the myocardium. Many viruses have been implicated as causes of myocarditis. These most commonly include adenoviruses and enteroviruses such as the coxsackieviruses. Recently, parvovirus B19 has been associated with a significant percentage of patients diagnosed with myocarditis and DCM.5 However, a growing body of data indicates that parvovirus is present in a large percentage of patients who do not have myocarditis.6–8 Of the viruses that cause myocarditis, the cellular and molecular mechanisms associated with coxsackieviral infection of the heart have been most thoroughly investigated with murine models. Therefore, the mechanistic experiments described in this review focus primarily, but not exclusively, on coxsackieviral myocarditis. It is important to note that coxsackieviruses …

The suppressor of cytokine signaling-1 (SOCS1) is a novel therapeutic target for enterovirus-induced cardiac injury.

Enteroviral infections of the heart are among the most commonly identified causes of acute myocarditis in children and adults and have been implicated in dilated cardiomyopathy. Although there is considerable information regarding the cellular immune response in myocarditis, little is known about innate signaling mechanisms within the infected cardiac myocyte that contribute to the host defense against viral infection. Here we show the essential role of Janus kinase (JAK) signaling in cardiac myocyte antiviral defense and a negative role of an intrinsic JAK inhibitor, the suppressor of cytokine signaling (SOCS), in the early disease process. Cardiac myocyte-specific transgenic expression of SOCS1 inhibited enterovirus-induced signaling of JAK and the signal transducers and activators of transcription (STAT), with accompanying increases in viral replication, cardiomyopathy, and mortality in coxsackievirus-infected mice. Furthermore, the inhibition of SOCS in the cardiac myocyte through adeno-associated virus-mediated (AAV-mediated) expression of a dominant-negative SOCS1 increased the myocyte resistance to the acute cardiac injury caused by enteroviral infection. These results indicate that strategies directed at inhibition of SOCS in the heart and perhaps other organs can augment the host-cell antiviral system, thus preventing viral-mediated end-organ damage during the early stages of infection.

Coxsackievirus and adenovirus receptor (CAR) mediates atrioventricular-node function and connexin 45 localization in the murine heart

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that belongs to the family of adhesion molecules. In the postnatal heart, it is localized predominantly at the intercalated disc, where its function is not known. Here, we demonstrate that a first degree or complete block of atrioventricular (AV) conduction developed in the absence of CAR in the adult mouse heart and that prolongation of AV conduction occurred in the embryonic heart of the global CAR-KO mouse. In the cardiac-specific CAR-KO (CAR-cKO) mouse, we observed the loss of connexin 45 localization to the cell-cell junctions of the AV node but preservation of connexin 40 and 43 in contracting myocardial cells and connexin 30.2 in the AV node. There was also a marked decrease in beta-catenin and zonula occludens-1 (ZO-1) localization to the intercalated discs of CAR-cKO mouse hearts at 8 weeks before the mice developed cardiomyopathy at 21 weeks of age. We also found that CAR formed a complex with connexin 45 via its PSD-95/DigA/ZO-1-binding (PDZ-binding) motifs. We conclude that CAR expression is required for normal AV-node conduction and cardiac function. Furthermore, localization of connexin 45 at the AV-node cell-cell junction and of beta-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.

Small proline‐rich protein 1A is a gp130 pathway‐ and stress‐inducible cardioprotective protein

The interleukin‐6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline‐rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte‐restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF‐IL6 (C/EBPβ) and c‐Jun to the SPRR1A promoter was observed after CT‐1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress‐inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.

Inducible Cardiac-Restricted Expression of Enteroviral Protease 2A Is Sufficient to Induce Dilated Cardiomyopathy

Background— Enterovirus infection is a cause of cardiomyopathy. We previously demonstrated that enteroviral protease 2A directly cleaves the cytoskeletal protein dystrophin. However, the direct effect of protease 2A in enteroviral cardiomyopathy is less clear because other viral proteins are also expressed with viral infection. Methods and Results— A transgenic mouse with inducible cardiac-restricted expression of enteroviral protease 2A was generated. In the transgenic mouse, a tamoxifen-regulated Cre-loxP system, MerCreMer (MCM), was used to induce genetic recombination in cardiac myocytes, which led to protease 2A expression. Protease 2A and MCM double transgenic (2AxMCM) mice were treated with tamoxifen; the controls included 2AxMCM mice treated with diluents for tamoxifen and tamoxifen-treated MCM littermates. Protease 2A activity was significantly induced after tamoxifen in the 2AxMCM mice compared with controls. Echocardiographic analysis demonstrated an increase in left ventricular end-diastolic and end-systolic chamber size, with decreased fractional shortening in tamoxifen-treated 2AxMCM mice. There was an increase in heart weight-to-body weight ratio in 2AxMCM mice treated with tamoxifen. Only a small increase in interstitial fibrosis and inflammation was found in tamoxifen-treated 2AxMCM mice; however, ultrastructural analysis demonstrated myofibrillar collapse with abnormalities of intercalated discs and sarcolemmal membranes. Evans blue dye–positive myocytes with disruption of dystrophin were present in 2AxMCM mice treated with tamoxifen. Disruption of dystrophin was also found in cultured myocytes isolated from 2AxMCM mice with Cre in the nucleus. Conclusions— Protease 2A has a significant role in enteroviral cardiomyopathy and alone is sufficient to induce dilated cardiomyopathy, which is associated with disruption of the sarcolemmal membrane and cleavage of dystrophin with protease 2A expression.

Innate defense mechanism against virus infection within the cardiac myocyte requiring gp130-STAT3 signaling.

Background— Little is known about innate immune mechanisms within the cardiac myocyte that determine susceptibility to enterovirus infection, an important cause of myocarditis and subsequent heart failure. Although interferon (IFN) generally plays a key role in innate immunity, ablation of IFN receptors has little or no effect on acute coxsackievirus B3 infection in the heart. Interestingly, gp130-cytokine–mediated stimulation of neonatal ventricular myocytes has a cytoprotective effect against virus infection in culture that can be inhibited by suppressors of cytokine signaling (SOCS)-3, a physiological inhibitor of gp130 signaling that does not affect IFN signaling. Therefore, we hypothesized that inhibition of gp130 signaling by SOCS3 would change cardiac myocyte susceptibility to virus infection without affecting IFN signaling. Methods and Results— We generated cardiac-specific SOCS3 transgenic mice. Despite an intact IFN-mediated antiviral response in adult transgenic myocytes, there was a marked increase in susceptibility to viral infection in the SOCS3 transgenic mouse hearts. This indicated the presence of IFN-independent innate defense mechanisms within the cardiac myocyte. Subsequently, we demonstrated that cardiac-specific gp130-knockout mice also had increased susceptibility to viral infection. Furthermore, we demonstrated that the gp130-mediated increase in survival of infected myocytes occurred through a signal transducers and activators of transcription-3–dependent mechanism that did not affect viral replication. This was accompanied by a persistent expression of full-length dystrophin after coxsackievirus B3 infection. In addition, we found that both SOCS3 transgenic and gp130-deficient mice had a decrease in α-sarcoglycan. Conclusions— SOCS3-mediated regulation of gp130 signaling can affect susceptibility to viral infection in the heart. Increased cardiac cell survival through gp130–signal transducers and activators of transcription-3 signaling appears to play an important role in preserving nondividing cardiac myocytes until specific immune responses begin to clear the virus.

Prion-Induced Amyloid Heart Disease with High Blood Infectivity in Transgenic Mice

We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 107 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.

Cardiac-specific deletion of SOCS-3 prevents development of left ventricular remodeling after acute myocardial infarction.

OBJECTIVES The study investigated the role of myocardial suppressor of cytokine signaling-3 (SOCS3), an intrinsic negative feedback regulator of the janus kinase and signal transducer and activator of transcription (JAK-STAT) signaling pathway, in the development of left ventricular (LV) remodeling after acute myocardial infarction (AMI). BACKGROUND LV remodeling after AMI results in poor cardiac performance leading to heart failure. Although it has been shown that JAK-STAT-activating cytokines prevent LV remodeling after AMI in animals, little is known about the role of SOCS3 in this process. METHODS Cardiac-specific SOCS3 knockout mice (SOCS3-CKO) were generated and subjected to AMI induced by permanent ligation of the left anterior descending coronary artery. RESULTS Although the initial infarct size after coronary occlusion measured by triphenyltetrazolium chloride staining was comparable between SOCS3-CKO and control mice, the infarct size 14 days after AMI was remarkably inhibited in SOCS3-CKO, indicating that progression of LV remodeling after AMI was prevented in SOCS3-CKO hearts. Prompt and marked up-regulations of multiple JAK-STAT-activating cytokines including leukemia inhibitory factor and granulocyte colony-stimulating factor (G-CSF) were observed within the heart following AMI. Cardiac-specific SOCS3 deletion enhanced multiple cardioprotective signaling pathways including STAT3, AKT, and extracellular signal-regulated kinase (ERK)-1/2, while inhibiting myocardial apoptosis and fibrosis as well as augmenting antioxidant expression. CONCLUSIONS Enhanced activation of cardioprotective signaling pathways by inhibiting myocardial SOCS3 expression prevented LV remodeling after AMI. Our data suggest that myocardial SOCS3 may be a key molecule in the development of LV remodeling after AMI.

Adenylyl Cyclase Type VI Increases Akt Activity and Phospholamban Phosphorylation in Cardiac Myocytes

Increased expression of adenylyl cyclase VI has beneficial effects on the heart, but strategies that increase cAMP production in cardiac myocytes usually are harmful. Might adenylyl cyclase VI have beneficial effects unrelated to increased β-adrenergic receptor-mediated signaling? We previously reported that adenylyl cyclase VI reduces cardiac phospholamban expression. Our focus in the current studies is how adenylyl cyclase VI influences phospholamban phosphorylation. In cultured cardiac myocytes, increased expression of adenylyl cyclase VI activates Akt by phosphorylation at serine 473 and threonine 308 and is associated with increased nuclear phospho-Akt. Activated Akt phosphorylates phospholamban, a process that does not require β-adrenergic receptor stimulation or protein kinase A activation. These previously unrecognized signaling events would be predicted to promote calcium handling and increase contractile function of the intact heart independently of β-adrenergic receptor activation. We speculate that phospholamban phosphorylation, through activation of Akt, may be an important mechanism by which adenylyl cyclase VI increases the function of the failing heart.