Abstract 331: HCN Overexpression in Failing Heart Possibly Contributes to Ventricular Arrhythmias

Accumulating evidence suggests increased ventricular expression of hyperpolarization-activated cation channels (HCNs) in hypertrophied and failing hearts contributes to the induction of arrhythmias. In this study, we addressed the capacity of HCNs blockade to prevent lethal arrhythmias associated with heart failure. Transgenic mice expressing a dominant-negative mutant of neuron-restrictive silencer factor in a cardiac-specific manner (dnNRSF-Tg) exhibited dilated cardiomyopathy and sudden arrythmic death with an increase in ventricular HCNs expression, which are potentially responsible for the observed lethal arrhythmias. Ivabradine (Iva), a specific HCN channel inhibitor, significantly improved the survival among dnNRSF-Tg mice. Though echocardiographic, hemodynamic, and histological analyses showed no significant difference between Iva and control, ECG telemetric monitoring showed the significant reduction of arrhythmias in dnNRSF-Tg mice treated with Iva (VT; Iva 19/h, control 92/h ; p<0.05), suggesting that Iva improved the survival by preventing lethal arrhythmias. We also found that the transgenic mice overexpressing HCN2 specifically in the heart (HCN2-Tg) are susceptible to ventricular arrhythmias induced by chronic isoproterenol infusion. In isolated ventricular myocytes from HCN2-Tg, but not in those from wild type mice, isoproterenol induced abnormal spontaneous action potentials, which were suppressed with Iva. Our findings suggest that increased ventricular expression of HCN channels possibly contributes to the ventricular arrhythmias, and HCN channels blockade may represent a new and effective means of preventing sudden arrhythmic death in patients with heart failure.

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NRSF-GNAO1-CaMK2 axis exacerbates cardiac remodeling and progresses heart failure by impairing Ca2+ homeostasis

European Heart Journal ◽

10.1093/ehjci/ehaa946.3672 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

H Inazumi ◽

K Kuwahara ◽

Y Kuwabara ◽

Y Nakagawa ◽

H Kinoshita ◽

...

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Knockout Mice ◽

Cardiac Remodeling ◽

Cardiac Dysfunction ◽

Intracellular Signaling ◽

Molecular Mechanisms ◽

Systolic Function ◽

Dominant Negative Mutant ◽

Funding Source

Abstract Background In the development of heart failure, pathological intracellular signaling reactivates fetal cardiac genes, which leads to maladaptive remodeling and cardiac dysfunction. We previously reported that a transcriptional repressor, neuron restrictive silencer factor (NRSF) represses fetal cardiac genes and maintains normal cardiac function under normal conditions, while hypertrophic stimuli de-repress this NRSF mediated repression via activation of CaMKII. Molecular mechanisms by which NRSF maintains cardiac systolic function remains to be determined, however. Purpose To elucidate how NRSF maintains normal cardiac homeostasis and identify the novel therapeutic targets for heart failure. Methods and results We generated cardiac-specific NRSF knockout mice (NRSF cKO), and found that these NRSF cKO showed cardiac dysfunction and premature deaths accompanied with lethal arrhythmias, as was observed in our previously reported cardiac-specific dominant-negative mutant of NRSF transgenic mice (dnNRSF-Tg). By cDNA microarray analysis of dnNRSF-Tg and NRSF-cKO, we identified that expression of Gnao1 gene encoding Gαo, a member of inhibitory G proteins, was commonly increased in ventricles of both types of mice. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that NRSF transcriptionally regulates Gnao1 gene expression. Genetic Knockdown of Gαo in dnNRSF-Tg and NRSF-cKO by crossing these mice with Gnao1 knockout mice ameliorated the reduced systolic function, increased arrhythmogenicity and reduced survival rates. Transgenic mice expressing a human GNAO1 in their hearts (GNAO1-Tg) showed progressive cardiac dysfunction with cardiac dilation. Ventricles obtained from GNAO1-Tg have increased phosphorylation level of CaMKII and increased expression level of endogenous mouse Gnao1 gene. These data suggest that increased cardiac expression of Gαo is sufficient to induce pathological Ca2+-dependent signaling and cardiac dysfunction, and that Gαo forms a positive regulatory circuit with CaMKII and NRSF. Electrophysiological analysis in ventricular myocytes of dnNRSF-Tg revealed that impaired Ca2+ handling via alterations in localized L-type calcium channel (LTCC) activities; decreased T-tubular and increased surface sarcolemmal LTCC activities, underlies Gαo-mediated cardiac dysfunction. Furthermore, we also identified increased expression of Gαo in ventricles of two different heart failure mice models, mice with transverse aortic constriction and mice carrying a mutant cardiac troponin T, and confirmed that genetic reduction of Gαo prevented the progression of cardiac dysfunction in both types of mice. Conclusions Increased expression of Gαo, induced by attenuation of NRSF-mediated repression forms a pathological circuit via activation of CaMKII. This circuit exacerbates cardiac remodeling and progresses heart failure by impairing Ca2+ homeostasis. Gαo is a potential therapeutic target for heart failure. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grants-in –Aid for Scientific Research from the Japan Society for the Promotion of Science

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Expression of a Dominant Negative Mutant of Interleukin-1β Converting Enzyme in Transgenic Mice Prevents Neuronal Cell Death Induced by Trophic Factor Withdrawal and Ischemic Brain Injury

Journal of Experimental Medicine ◽

10.1084/jem.185.5.933 ◽

1997 ◽

Vol 185 (5) ◽

pp. 933-940 ◽

Cited By ~ 256

Author(s):

Robert M. Friedlander ◽

Valeria Gagliardini ◽

Hideaki Hara ◽

Klaus B. Fink ◽

Weiwei Li ◽

...

Keyword(s):

Brain Injury ◽

Transgenic Mice ◽

Dorsal Root ◽

Neuron Specific Enolase ◽

Dominant Negative ◽

Trophic Factor ◽

Dominant Negative Mutant ◽

Lacz Gene ◽

Converting Enzyme ◽

Induced Apoptosis

To explore the role of the interleukin (IL)-1β converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal–induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro–IL-1β, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal–induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal–induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1β convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1β production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

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Abstract 037: Transient Cardiac Expression Of Constitutively Active G alpha Q Activates Renin-angiotensin System, Leading To Progressive Heart Failure And Ventricular Arrhythmias In Transgenic Mice

Circulation Research ◽

10.1161/res.113.suppl_1.a037 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Naoko Matsushita ◽

Masamichi hirose ◽

Yasuchika Taeishi ◽

Satoshi Suzuki ◽

Toshihide Kashihara ◽

...

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Cardiac Hypertrophy ◽

Ventricular Arrhythmias ◽

Renin Angiotensin System ◽

Gene Expressions ◽

Angiotensin System ◽

Renin Angiotensin ◽

Progressive Heart Failure ◽

Constitutively Active

Introduction: Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) caused progressive heart failure and ventricular arrhythmias after the initiating stimulus becomes undetectable. However, the mechanisms are still unknown. Renin-angiotensin system plays a critical role in the development of cardiac hypertrophy and heart failure. We examined the effects of chronic administration of olmesartan on ventricular function, the number of premature ventricular contractions (PVC), and ventricular remodeling in Gαq-TG mice. Methods and Results: Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan treatment prevented the severe reduction of left ventricular fractional shortening and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan -treated Gαq-TG. The QT interval was significantly shorter in olmesartan-treated Gαq-TG than vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased in vehicle-treated Gαq-TG. Olmesartan significantly decreased these gene expressions in Gαq-TG. Moreover, protein expressions of canonical transient receptor potential (TRPC) channels 3 and 6 increased in vehicle-treated Gαq-TG hearts. Olmesartan significantly decreased TRPC6 expressions in Gαq-TG. Angiotensin converting enzyme (ACE) 1 and 2 gene expressions were also increased in vehicle-treated Gαq-TG and was not decreased to the control level in olmesartan-treated Gαq-TG. Conclusions: These findings suggest that renin-angiotensin system has an important role in the development of cardiac hypertrophy and heart failure even if the initiating stimulus is different from the activation of renin-angiotensin system.

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Abnormal diastolic currents in ventricular myocytes from spontaneous hypertensive heart failure rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01146.2005 ◽

2006 ◽

Vol 291 (5) ◽

pp. H2192-H2198 ◽

Cited By ~ 14

Author(s):

Arun Sridhar ◽

Spencer J. Dech ◽

Veronique A. Lacombe ◽

Terry S. Elton ◽

Sylvia A. McCune ◽

...

Keyword(s):

Heart Failure ◽

Rat Model ◽

Ventricular Arrhythmias ◽

Ventricular Myocytes ◽

Inward Rectifier ◽

Pacemaker Current ◽

Common Cause ◽

Age Dependent ◽

End Stage Heart Failure ◽

End Stage

Hypertension is a common cause of heart failure, and ventricular arrhythmias are a major cause of death in heart failure. The spontaneous hypertension heart failure (SHHF) rat model was used to study altered ventricular electrophysiology in hypertension and heart failure. We hypothesized that a reduction in the inward rectifier K+ current ( IK1) and expression of pacemaker current ( If) would favor abnormal automaticity in the SHHF ventricle. SHHF ventricular myocytes were isolated at 2 and 8 mo of age and during end-stage heart failure (≥17 mo); myocytes from age-matched rats served as controls. Inward IK1 was significantly reduced at both 8 and ≥17 mo in SHHF rats compared with controls. There was a reduction in inward IK1 due to aging in the controls only at ≥17 mo. We found a significant increase in If at all ages in the SHHF rats, compared with young controls. In controls, there was an age-dependent increase in If. Action potential recordings in the SHHF rats demonstrated abnormal automaticity, which was abolished by the addition of an If blocker (10 μM zatebradine). Increased If during hypertension alone or combined increases in If with reduced IK1 during the progression to hypertensive heart failure contribute to a substrate for arrhythmogenesis.

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Nicorandil Prevents Gαq-Induced Progressive Heart Failure and Ventricular Arrhythmias in Transgenic Mice

PLoS ONE ◽

10.1371/journal.pone.0052667 ◽

2012 ◽

Vol 7 (12) ◽

pp. e52667 ◽

Cited By ~ 8

Author(s):

Masamichi Hirose ◽

Yasuchika Takeishi ◽

Tsutomu Nakada ◽

Hisashi Shimojo ◽

Toshihide Kashihara ◽

...

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Ventricular Arrhythmias ◽

Progressive Heart Failure

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Effects of Mutant Ubiquitin on ts1 Retrovirus-Mediated Neuropathology

Journal of Virology ◽

10.1128/jvi.77.13.7193-7201.2003 ◽

2003 ◽

Vol 77 (13) ◽

pp. 7193-7201 ◽

Cited By ~ 11

Author(s):

Mei Zhang ◽

Sherry Thurig ◽

Maria Tsirigotis ◽

Paul K. Y. Wong ◽

Kenneth R. Reuhl ◽

...

Keyword(s):

Transgenic Mice ◽

Therapeutic Potential ◽

Leukemia Virus ◽

Lewy Bodies ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Spongiform Encephalopathy ◽

Temperature Sensitive ◽

Transgenic Mouse Line

ABSTRACT ts1 is a temperature-sensitive mutant of Moloney murine leukemia virus that induces a rapid spongiform encephalopathy in mice infected as newborns. The pathological features include the formation of ubiquitinated inclusions resembling Lewy bodies. To determine how perturbation of the ubiquitin-proteasome pathway might affect ts1-mediated neurodegeneration, the virus was introduced into transgenic mice in which the assembly of ubiquitin chains was compromised by the expression of dominant-negative mutant ubiquitin. The onset of symptoms was greatly delayed in a transgenic mouse line expressing K48R mutant ubiquitin; no such delay was observed in mice expressing a wild-type ubiquitin transgene or K63R mutant ubiquitin. The extended latency was found to correlate with a delayed increase in viral titers. Pathological findings in K48R transgenic mice at 60 days were found to be similar to those in the other strains at 30 days, suggesting that while delayed, the neurodegenerative process in K48R mice was otherwise similar. These data demonstrate the sensitivity of retroviral replication to the partial disruption of ubiquitin-mediated proteolysis in vivo, a finding that may have therapeutic potential.

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Down regulation of PSD-95 and GluR1 expression in the hippocampus of transgenic mice expressing dominant negative mutant of RAR in forebrain

Neuroscience Research ◽

10.1016/j.neures.2011.07.1214 ◽

2011 ◽

Vol 71 ◽

pp. e278

Author(s):

Koji Mitsuda ◽

Masanori Nomoto ◽

Yohei Takeda ◽

Satoshi Kida

Keyword(s):

Transgenic Mice ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Down Regulation ◽

Negative Mutant

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Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

Blood ◽

10.1182/blood-2004-12-4832 ◽

2005 ◽

Vol 106 (5) ◽

pp. 1808-1816 ◽

Cited By ~ 64

Author(s):

Erwan Le Scolan ◽

Dimitri Pchejetski ◽

Yoshiko Banno ◽

Nicole Denis ◽

Patrick Mayeux ◽

...

Keyword(s):

Transgenic Mice ◽

Molecular Mechanisms ◽

Sphingosine Kinase ◽

Dominant Negative ◽

Recurrent Event ◽

Dominant Negative Mutant ◽

Apoptosis Resistance ◽

Kinase Gene ◽

Extracellular Signal

Abstract The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

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Abstract 16820: CaMKIIδc Transgenic Mice Develop Spontaneous Ventricular Arrhythmias Which Can Be Suppressed by Inhibition of Late Na+ Current

Circulation ◽

10.1161/circ.130.suppl_2.16820 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Maria Arolfo ◽

Liguo Chi ◽

Aliya Zeng ◽

Nesrine El-Bizri ◽

Joan Brown ◽

...

Keyword(s):

Single Dose ◽

Patch Clamp ◽

Transgenic Mice ◽

Ventricular Arrhythmias ◽

Ventricular Myocytes ◽

Na Channels ◽

Wild Type ◽

Na Current ◽

Patch Clamp Technique ◽

Post Surgery

Introduction: It has been shown that activated CaMKII can phosphorylate cardiac Na+ channels that leads to an enhanced late Na current (INaL). We hypothesized that enhanced INaL plays an important role in arrhythmogenesis linked to increased CaMKII activity. To test this hypothesis we determined the effect of a selective INaL inhibitor GS-967 on spontaneously occurring ventricular arrhythmias in mice overexpressing CaMKIIδc (TG). Methods: TG (n=6) and wild type (WT, n=3) mice at age 8 week (wk) were instrumented with telemetry transmitters to record ECG. ECG were recorded continuously starting 1 wk post- surgery (age 9 wk) until age 19 wk. Incidence and burden of arrhythmias were evaluated at ages 9, 11, 13, 15, 17 and 19 wks using DSI ECG Pro software. At age 17 wk, when arrhythmia burden was relatively stable, mice were treated with a single dose of GS-967 (1 mg/kg, i.p.) or vehicle. After 4 days of washout, the treatment was repeated in a cross-over manner. Arrhythmia burden was quantified over a duration of 15-hours post-treatment. Using patch clamp technique, INaL was measured in ventricular myocytes isolated from both TG and WT mice at 17 wk age. Results: Incidence and burden of spontaneous ventricular arrhythmias increased progressively with age in TG mice. Between age 9 and 11 wks, 50% of TG mice had an arrhythmia burden ≥ 5 minutes/24 hours. The incidence increased to 83% at wk 13, 15, 17 and to 100% at 19 wks and the burden increased to 71 ± 35 min/24 hr at 17 wk. A single dose of GS-967 significantly decreased arrhythmia burden from 42.5 ± 9.6 min (treated with vehicle) to 9.4 ± 4.3 min (p<0.05) during a 15-hr period. No arrhythmias were observed in WT mice during the course of the study. INaL was enhanced by more than 2-fold in myocytes isolated from TG mice compared to WT (0.2±0.03 vs. 0.08±0.02 pA/pF, n=16 each, p<0.05). GS-967 caused a concentration-dependent reduction of INaL (50.5±3.4% at 0.3 μM, n=7; and 91±5.2% at 1 μM, n=4). Conclusions: This is the first report to show a reduction of spontaneously occurring ventricular arrhythmias by inhibition of INaL indicating a key role for INaL in CaMKII associated arrhythmogenesis.

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