Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice

2013 ◽  
Vol 91 (12) ◽  
pp. 1135-1142 ◽  
Author(s):  
Luca Pacini ◽  
Silvia Suffredini ◽  
Donatella Ponti ◽  
Raffaele Coppini ◽  
Giacomo Frati ◽  
...  
Keyword(s):  
Heart Development ◽  
Calcium Content ◽  
Calcium Transient ◽  
Early Response ◽  
Left Ventricular ◽  
Patch Clamp Technique ◽  
Knock Out ◽  
Early Growth Response ◽  
Knock Out Mice ◽  
Early Growth Response 1

Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1−/−) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1−/− mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca2+-channel, and PLB mRNA expression were reduced in Egr-1−/− mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1−/− hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-β) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1−/− mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation–contraction coupling in cardiac myocytes.

scholarly journals Intercalated disc protein Xinβ is required for Hippo-YAP signaling in the heart

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Haipeng Guo ◽  
Yao Wei Lu ◽  
Zhiqiang Lin ◽  
Zhan-Peng Huang ◽  
Jianming Liu ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Cardiac Function ◽  
Heart Development ◽  
Genetic Interaction ◽  
Specific Cell ◽  
Cardiomyocyte Proliferation ◽  
Intercalated Disc ◽  
Knock Out ◽  
Genes Encoding ◽  
Knock Out Mice

Abstract Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinβ, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinβ in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinβ mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinβ knock-out mice—indicating a functional and genetic interaction between Xinβ and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinβ modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.

SCN10A-knock-out improves survival and proarrhythmia in a transgenic heart failure mouse model

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
P.R.F Bengel ◽  
C Krekeler ◽  
S Ahmad ◽  
N Hartmann ◽  
P Tirilomis ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mouse Model ◽  
Sodium Channel ◽  
Structural Parameters ◽  
Left Ventricular ◽  
Heart Weight ◽  
Na Channel ◽  
Funding Source ◽  
Patch Clamp Technique ◽  
Knock Out

Abstract Background In heart failure (HF) both Ca2+/Calmodulin-dependent protein-kinase II (CaMKII) and late sodium current (INaL) are known to contribute to arrhythmogenesis as they contribute to action-potential (AP) prolongation and the occurrence of early- (EADs) and delayed afterdepolarizations (DADs). Further, augmented CaMKII and INaL maintain a vicious cycle as they both can activate each other. We recently found that the sodium channel isoform NaV1.8 is upregulated in HF and hypertrophy and that it is involved in INaL-generation. In the current study we investigated the effects of NaV1.8-knock-out (KO) on HF-progression and arrhythmogenesis in a CaMKII-overexpressing HF mouse model. Methods/Results CaMKII overexpressing mice (CaMKII+/T) were crossbred with NaV1.8-KO mice (SCN10A−/−). To our surprise knock-out of NaV1.8 in CaMKII+/T mice (SCN10A−/−/CaMKII+/T) significantly improved survival (median survival 103 days vs 74.5 CaMKII+/T, p<0.01). CaMKII+/T mice exhibited a strong HF phenotype compared to WT with increased heart-weight to tibia length ratio as well as reduced ejection fraction and left-ventricular end-diastolic diameter obtained by echocardiography. However, these structural parameters did not differ between SCN10A−/−/CaMKII+/T and CaMKII+/T. Therefore, cellular electrophysiology experiments were performed in isolated cardiomyocytes for a better understanding of the observed improvement in survival. INaL, measured by patch-clamp technique, was significantly augmented in CaMKII+/T vs WT and SCN10A−/−, while SCN10A−/−/CaMKII+/T showed significantly less INaL than CaMKII+/T alone. Further, AP-duration (APD) was significantly reduced in SCN10A−/−/CaMKII+/T vs CaMKII+/T while AP-amplitude, resting membrane-potential and upstroke velocity (dv/dtmax) remained unchanged. In addition, the occurrence of afterdepolarizations was significantly lower in SCN10A−/−/ CaMKII+/T vs CaMKII+/T. Confocal microscopy using the dye Fluo-4AM was performed and significantly less diastolic Ca2+-waves occurred in SCN10A−/−/CaMKII+/T compared to CaMKII+/T. In order to analyze an organ-specific SCN10A-KO, we generated homozygous SCN10A-KO lines of induced pluripotent stem cells by using CRISPR/Cas9 technology. 2-month old iPSC-cardiomyocytes lacking NaV1.8 were treated with low dose isoprenaline and showed significantly less INaL, thereby serving as a final proof of the relevant role of this Na+-channel on INaL-generation in the cardiomyocyte. Conclusion We found a survival benefit by selective knock-out of the neuronal sodium channel isoform NaV1.8 in a proarrhythmic HF mouse model with augmented CaMKII expression. However, in our model NaV1.8-knock-out showed no effects on HF progression, while cellular proarrhythmic triggers were attenuated. Taken together with our findings in IPS-cardiomyocytes treated with the CRSIPR/Cas9 technology NaV1.8 plays a significant role for the generation of INaL and cellular arrhythmogenic triggers in the cardiomyocyte. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Stiftung für Herzforschung

Initial Contractile Response of Isolated Rat Heart Cells to Halothane, Enflurane, and Isoflurane

1997 ◽  
Vol 86 (1) ◽  
pp. 137-146 ◽  
Author(s):  
David M. Wheeler ◽  
Todd R. Rice ◽  
William H. duBell ◽  
Harold A. Spurgeon
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Calcium Content ◽  
Calcium Transient ◽  
Temporary Increase ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Intracellular Calcium Transient ◽  
Sarcoplasmic Reticulum Calcium ◽  
In Cells

Background In several beating cardiac muscle preparations, a short-lived increase in twitch tension or amplitude has been observed when they were exposed abruptly to solutions containing halothane or enflurane. As exposure to the anesthetics was continued, the expected negative inotropic effect became evident after the short-lived increase in twitch. No such increase in twitch has been reported during exposure to isoflurane. It has been hypothesized that this short-lived increase in twitch is caused by an enhancement of calcium release from the sarcoplasmic reticulum, but other mechanisms have not been excluded. Methods Freshly isolated, single rat ventricular cells were stimulated to beat at room temperature and abruptly exposed to solutions containing halothane (0.25-0.64 mM), enflurane (0.69-1 mM), or isoflurane (0.31-0.54 mM). During these exposures, twitch amplitude was measured and intracellular calcium concentration was followed using the calcium-sensitive dye indo-1. In some experiments, the whole-cell patch-clamp technique was used to measure membrane current. In addition, in several cells the sarcoplasmic reticulum calcium content was assessed through the response to brief pulses of caffeine. Results Both the twitch amplitude and the intracellular calcium transient were increased temporarily in cells abruptly exposed to halothane or enflurane. No such behavior was found with isoflurane. After continued exposure to all three agents, both the twitch amplitude and the calcium transient were less than control. During the beats exhibiting an increase in twitch, no alteration in the relation between cell length (twitch amplitude) and the intracellular calcium transient was found compared with control conditions. In addition, the temporary increase in twitch amplitude occurred in cells contracting under voltage-clamp control when halothane was introduced, and it was not associated with any increase in the calcium current. The sarcoplasmic reticulum calcium content at the time of the halothane-induced increase in twitch also was not increased. Conclusions The short-lived increase in twitch after abrupt exposure to halothane or enflurane is related to increased intracellular calcium during the beat and not to any changes in myofilament sensitivity to calcium. Because these results eliminate most alternative explanations for this phenomenon, the authors conclude that halothane, and probably also enflurane, increases the fraction of calcium released from the sarcoplasmic reticulum with each heart beat. Isoflurane appears to lack this action.

scholarly journals Sex differences in cardio-oncology: utilizing a genetic variant as a therapeutic target doxorubicin-induced cardiomyopathy

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
K Bruno ◽  
A C Morales-Lara ◽  
D N Diflorio ◽  
Z Anastasiadis ◽  
C Landolfo ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ejection Fraction ◽  
Therapeutic Target ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Male Mice ◽  
Wild Type ◽  
Knock Out ◽  
Increased Risk ◽  
Knock Out Mice

Abstract Introduction Doxorubicin is an anthracycline used as a chemotherapeutic drug for the treatment of a wide range of adult and pediatric cancers. Doxorubicin is associated with an increased risk of cardiomyopathy and heart failure with up to 10% of patients developing cardiac complications. A GWAS conducted of 1,191 patients from the N9831 clinical trial identified that cardiac gene expression and genetic variants of TRPC6 were associated with a decline in left ventricular ejection fraction (LVEF) (p=0.005 and p=1.6x10–6, respectively). TRPC6 is a non-selective cation channel expressed in heart and vascular tissue. TRPC6 participates in the pathogenesis of cardiac hypertrophy as a pathological response to chronic mechanical stress. Chronic activation has been found to promote cardiac fibrosis leading to heart failure. Purpose TRPC6 variants could be associated with increased risk of doxorubicin-induced cardiotoxicity. Data/tests to determine which patients may progress to cardiomyopathy and heart failure are currently lacking and there are no targeted treatments to prevent cardiomyopathy in these patients. Methods In preliminary in vivo data, B6.129 wild-type mice or TRPC6 knockout mice were treated with either 6x intraperitoneal saline or 4mg/kg doxorubicin injections (cumulative dose of 24mg/kg). Results We found doxorubicin increased cardiac vacuolation (male, p≤0.001 and female, p≤0.05) in WT mice compared to controls. Higher HW/BW ratio was observed in male TRPC6 knock out mice compared to wild-type mice when both were treated with doxorubicin (males, p=0.005 and females, p=0.19). Additionally, we found that doxorubicin-induced cardiac injury was significantly reduced in TRPC6 knock-out mice compared to wild-type mice based on reduced vacuolation (p=0.0004 males, p=0.03 females), with the effect being greater in male mice than female mice. Furthermore, a significant decrease in stroke volume (p=0.007), diastolic volume (p=0.01) and cardiac output (p=0.004) in wild-type male mice treated with doxorubicin compared to control and TRPC6 knock-out mice. Our in vitro preliminary data show that inhibition of TRPC6 using the TRPC6 inhibitor GsMTx-4 in human iPSC-derived cardiomyocytes significantly reduced doxorubicin-induced apoptosis (p<0.0001). Further we treated male mice with the TRPC6 inhibitor, GsMTx4, and found that they had less global longitudinal cardiac strain (p=0.04) and higher ejection fraction (p=0.01) compared to mice who were only treated with doxorubicin. Histologically we found that mice given the TRPC6 inhibitor had less fibrosis as measured by Trichrome stained heart sections (p=0.004) which could account for improvements in cardiac function. Conclusion TRPC6 could be a novel therapeutic target in the prevention of chemotherapy-induced cardiomyopathy and heart failure. Additionally genetic mapping of TRCP6 functional variants may provide a new screening tool to determine increased risk of developing heart failure. FUNDunding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Mayo Clinic

Increased glyoxalase-1 levels in Fkbp5 knock-out mice caused by Glo1 gene duplication

2013 ◽  
Vol 46 (06) ◽  
Author(s):  
LK Kollmannsberger ◽  
NC Gassen ◽  
A Bultmann ◽  
J Hartmann ◽  
P Weber ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Knock Out ◽  
Glyoxalase 1 ◽  
Knock Out Mice
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