Transcription factor CHF1/Hey2 regulates EC coupling and heart failure in mice through regulation of FKBP12.6

Heart failure is a leading cause of morbidity and mortality in Western society. The cardiovascular transcription factor CHF1/Hey2 has been linked to experimental heart failure in mice, but the mechanisms by which it regulates myocardial function remain incompletely understood. The objective of this study was to determine how CHF1/Hey2 affects development of heart failure through examination of contractility in a myocardial knockout mouse model. We generated myocardial-specific knockout mice. At baseline, cardiac function was normal, but, after aortic banding, the conditional knockout mice demonstrated a greater increase in ventricular weight-to-body weight ratio compared with control mice (5.526 vs. 4.664 mg/g) and a significantly decreased ejection fraction (47.8 vs. 72.0% control). Isolated cardiac myocytes from these mice showed decreased calcium transients and fractional shortening after electrical stimulation. To determine the molecular basis for these alterations in excitation-contraction coupling, we first measured total sarcoplasmic reticulum calcium stores and calcium-dependent force generation in isolated muscle fibers, which were normal, suggesting a defect in calcium cycling. Analysis of gene expression demonstrated normal expression of most genes known to be involved in myocardial calcium cycling, with the exception of the ryanodine receptor binding protein FKBP12.6, which was expressed at increased levels in the conditional knockout hearts. Treatment of the isolated knockout myocytes with FK506, which inhibits the association of FKBP12.6 with the ryanodine receptor, restored contractile function. These findings demonstrate that conditional deletion of CHF1/Hey2 in the myocardium leads to abnormalities in calcium handling mediated by FKBP12.6 that predispose to pressure overload-induced heart failure.

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ZEB2 regulates a transcriptional network of calcium-handling genes in the injured heart

European Heart Journal ◽

10.1093/ehjci/ehaa946.3633 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

M Gladka ◽

A De Leeuw ◽

A Kohela ◽

B Molenaar ◽

D Versteeg ◽

...

Keyword(s):

Heart Failure ◽

Transcription Factor ◽

Cardiac Function ◽

Cardiac Dysfunction ◽

Molecular Mechanisms ◽

Critical Role ◽

Calcium Handling ◽

Funding Source ◽

Post Injury ◽

Mesenchymal Transition

Abstract Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable progress in managing the progression of the disease, the development of effective therapies for heart failure (HF) remains challenging. Therefore, it is of great importance to understand the molecular mechanisms that maintain calcium level and contractility in homeostatic conditions. Here we identified a transcription factor ZEB2 that regulates the expression of numerous contractile and calcium-related genes. Zinc finger E-box-binding homeobox2 (ZEB2) is a transcription factor that plays a role during early fetal development and epithelial-to-mesenchymal transition (EMT); however, its function in the heart remains to be determined. Recently, we found that ZEB2 is upregulated in murine cardiomyocytes shortly after an ischemic event, but returns to baseline levels as the disease progresses. Gain- and loss-of-function genetic mouse models revealed the necessity and sufficiency of ZEB2 to maintain proper cardiac function after ischemic injury. We show that cardiomyocyte-specific ZEB2 overexpression (Zeb2 cTG) protected from ischemia-induced diastolic dysfunction and attenuated the structural remodeling of the heart. Moreover, RNA-sequencing of Zeb2 cTG hearts post-injury implicated ZEB2 in the regulation of numerous calcium-handling and contractile-related genes when compared to wildtype mice. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), thereby augmenting contractility. Improved cardiac function in ZEB2-overexpressing hearts correlated with higher expression of several sarcomeric proteins like myosin-binding protein C3 (MYBPC3), desmin (DES) and myosin regulatory light chain 2 (MYL2) further contributing to the observed protective phenotype. Furthermore, we observed a decrease in the activity of Ca2+-depended calcineurin/NFAT signaling, which is the main driver of pathological cardiac remodeling. Conversely to Zeb2 cTg mice, loss of ZEB2 from cardiomyocytes perturbed the expression of calcium- and contractile-related proteins and increased the activity of calcineurin/NFAT pathway, exacerbating cardiac dysfunction. Together, we show that ZEB2 is a central regulator of contractile and calcium-handling components, consequently mediating contractility in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in the regulation of calcium homeostasis in cardiomyocytes is a critical step towards the development of improved therapies for various forms of heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): DR. E. Dekker from Dutch Heart Foundation

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NAD(P)H oxidase subunit p47phox is elevated, and p47phox knockout prevents diaphragm contractile dysfunction in heart failure

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00176.2015 ◽

2015 ◽

Vol 309 (5) ◽

pp. L497-L505 ◽

Cited By ~ 22

Author(s):

Bumsoo Ahn ◽

Adam W. Beharry ◽

Gregory S. Frye ◽

Andrew R. Judge ◽

Leonardo F. Ferreira

Keyword(s):

Heart Failure ◽

Knockout Mice ◽

Contractile Function ◽

Isometric Force ◽

Exercise Intolerance ◽

Contractile Dysfunction ◽

Wild Type ◽

Shortening Velocity ◽

Protein Levels ◽

Specific Source

Patients with chronic heart failure (CHF) have dyspnea and exercise intolerance, which are caused in part by diaphragm abnormalities. Oxidants impair diaphragm contractile function, and CHF increases diaphragm oxidants. However, the specific source of oxidants and its relevance to diaphragm abnormalities in CHF is unclear. The p47phox-dependent Nox2 isoform of NAD(P)H oxidase is a putative source of diaphragm oxidants. Thus, we conducted our study with the goal of determining the effects of CHF on the diaphragm levels of Nox2 complex subunits and test the hypothesis that p47phox knockout prevents diaphragm contractile dysfunction elicited by CHF. CHF caused a two- to sixfold increase ( P < 0.05) in diaphragm mRNA and protein levels of several Nox2 subunits, with p47phox being upregulated and hyperphosphorylated. CHF increased diaphragm extracellular oxidant emission in wild-type but not p47phox knockout mice. Diaphragm isometric force, shortening velocity, and peak power were decreased by 20–50% in CHF wild-type mice ( P < 0.05), whereas p47phox knockout mice were protected from impairments in diaphragm contractile function elicited by CHF. Our experiments show that p47phox is upregulated and involved in the increased oxidants and contractile dysfunction in CHF diaphragm. These findings suggest that a p47phox-dependent NAD(P)H oxidase mediates the increase in diaphragm oxidants and contractile dysfunction in CHF.

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Cardiac deletion of α-E-catenin leads to redused expression of the main component of desmosomes – γ-catenin

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v21.854 ◽

1970 ◽

Vol 21 ◽

pp. 293-296

Author(s):

V. V. Balatskyi ◽

L. L. Matsevych ◽

O. O. Piven

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Western Blot ◽

Real Time ◽

Knockout Mice ◽

Protein Level ◽

Conditional Knockout ◽

Protein Levels ◽

Main Component ◽

Real Time Qpcr

Aim. In our present work, we have addressed to the γ-catenin, known main component of desmosomes, expression in hearts with heterozygous and homozygous knockout of α-E-catenin gene. Methods. Alpha-E-catenin conditional knockout mice were bred with α-MHC-Cre transgenic mice. We analyze expression of γ-catenin with real time qPCR and Western blot. Results. Cardiac α-E-catenin deletion leads to downregulation of γ-catenin mRNA and protein levels only in homozygous mice, while we not observed any perturbation of γ-catenin expression in heterozygous mice. Conclusions. We have shown that homozygous knockout of α-E-catenin gene in embryonic heart occur reduction of the main component of desmosomes – γ-catenin mRNA and protein level of expression, which can lead to disruption of the desmosomes structure in adult myocardium. Keywords: α-E-catenin, heart failure, γ-catenin.

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Abstract 159: Ablation of the Hypertension Candidate Gene ATP2B1 Leads To Deficient Calcium Cycling, Systolic Dysfunction and Heart Failure Following Pressure Overload

Circulation Research ◽

10.1161/res.115.suppl_1.159 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Nicholas P Stafford ◽

Min Zi ◽

Ludwig Neyses ◽

Elizabeth J Cartwright

Keyword(s):

Heart Failure ◽

Systolic Dysfunction ◽

Pressure Overload ◽

Early Response ◽

Left Ventricular ◽

Calcium Handling ◽

Calcium Cycling ◽

Lung Weight ◽

Hypertrophic Growth ◽

Ventricular Elastance

Mutations in ATP2B1 encoding the ubiquitous calcium extrusion pump Plasma Membrane Calcium ATPase 1 (PMCA1) have recently identified it as having the strongest association of any gene to hypertension, yet the role of PMCA1 in the pressure-overloaded heart is not known. To investigate this we generated a novel mouse line carrying cardiomyocyte-specific deletion of PMCA1 (PMCA1 cko ) and challenged them with transverse aortic constriction (TAC) alongside littermate ‘floxed’ controls (PMCA1 f/f ). After two weeks, echocardiographic analysis revealed signs of systolic dysfunction and left ventricular (LV) dilation in PMCA1 cko hearts as evidenced by reduced fractional shortening and increased diastolic diameter (both p<0.05), whilst function in PMCA1 f/f TAC controls remained preserved. This was accompanied by an increase in normalised lung weight in PMCA1 cko mice compared to sham operated and TAC controls (p<0.05) indicative of pulmonary congestion and a progression into LV failure, despite comparable hypertrophic growth amongst the two TAC cohorts. Hemodynamic analysis following LV catheterisation revealed contractility, as measured by left ventricular elastance (E es ), to be increased in controls after TAC (PMCA1 f/f TAC 12.69 ± 1.63 vs sham 7.02 ± 1.11 mmHg/μl, p<0.05), a change which was not reciprocated in knockout hearts (PMCA1 cko TAC 7.70 ± 1.19 vs sham 7.22 ± 1.55 mmHg/μl). To examine whether altered calcium handling could be the underlying cause of the observed phenotype, cardiomyocytes were isolated following one week TAC and loaded with Indo-1, prior to the onset of failure in PMCA1 cko hearts. Compatible with an increase in E es , systolic calcium levels were higher in PMCA1 f/f myocytes following pressure overload compared to sham controls (p<0.05), whilst PMCA1 cko TAC myocytes displayed equivalent peak calcium levels to their respective sham controls. These results suggest that PMCA1 may play a necessary role in enhancing calcium cycling during the early response to pressure overload, and that disrupting this gene may increase the susceptibility to heart failure under these conditions. This may provide first evidence of a novel genetic basis for the development of heart failure in a proportion of hypertensive patients.

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Phosphorylation of cardiac myosin–binding protein-C contributes to calcium homeostasis

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013296 ◽

2020 ◽

Vol 295 (32) ◽

pp. 11275-11291 ◽

Cited By ~ 1

Author(s):

Mohit Kumar ◽

Kobra Haghighi ◽

Evangelia G. Kranias ◽

Sakthivel Sadayappan

Keyword(s):

Heart Failure ◽

Protein C ◽

Contractile Function ◽

Binding Protein ◽

Stress Conditions ◽

Calcium Handling ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Cardiac myosin–binding protein-C (cMyBP-C) is highly phosphorylated under basal conditions. However, its phosphorylation level is decreased in individuals with heart failure. The necessity of cMyBP-C phosphorylation for proper contractile function is well-established, but the physiological and pathological consequences of decreased cMyBP-C phosphorylation in the heart are not clear. Herein, using intact adult cardiomyocytes from mouse models expressing phospho-ablated (AAA) and phosphomimetic (DDD) cMyBP-C as well as controls, we found that cMyBP-C dephosphorylation is sufficient to reduce contractile parameters and calcium kinetics associated with prolonged decay time of the calcium transient and increased diastolic calcium levels. Isoproterenol stimulation reversed the depressive contractile and Ca2+-kinetic parameters. Moreover, caffeine-induced calcium release yielded no difference between AAA/DDD and controls in calcium content of the sarcoplasmic reticulum. On the other hand, sodium–calcium exchanger function and phosphorylation levels of calcium-handling proteins were significantly decreased in AAA hearts compared with controls. Stress conditions caused increases in both spontaneous aftercontractions in AAA cardiomyocytes and the incidence of arrhythmias in vivo compared with the controls. Treatment with omecamtiv mecarbil, a positive cardiac inotropic drug, rescued the contractile deficit in AAA cardiomyocytes, but not the calcium-handling abnormalities. These findings indicate a cascade effect whereby cMyBP-C dephosphorylation causes contractile defects, which then lead to calcium-cycling abnormalities, resulting in aftercontractions and increased incidence of cardiac arrhythmias under stress conditions. We conclude that improvement of contractile deficits alone without improving calcium handling may be insufficient for effective management of heart failure.

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Conditional knockout mice demonstrate function of Klf5 as a myeloid transcription factor

Blood ◽

10.1182/blood-2015-12-684514 ◽

2016 ◽

Vol 128 (1) ◽

pp. 55-59 ◽

Cited By ~ 17

Author(s):

Nur Hezrin Shahrin ◽

Sonya Diakiw ◽

Lindsay A. Dent ◽

Anna L. Brown ◽

Richard J. D’Andrea

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Knockout Mice ◽

Conditional Knockout ◽

Conditional Knockout Mice ◽

Key Points

Key Points Klf5 functions in hematopoiesis to regulate HSC and progenitor proliferation and localization in the bone marrow. Klf5 is required in the granulocyte lineage and positively affects neutrophil output at the expense of eosinophil production.

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The role of calcium handling in the improved contractile function associated with high‐fat feeding in heart failure

The FASEB Journal ◽

10.1096/fasebj.24.1_supplement.1048.1 ◽

2010 ◽

Vol 24 (S1) ◽

Author(s):

YI‐HSIN CHENG ◽

Tracy A. McElfresh ◽

Xiaoqin Chen ◽

Wei Li ◽

Xin Yu ◽

...

Keyword(s):

Heart Failure ◽

Contractile Function ◽

Calcium Handling ◽

High Fat ◽

Fat Feeding

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Increased ROS-Mediated CaMKII Activation Contributes to Calcium Handling Abnormalities and Impaired Contraction in Barth Syndrome

Circulation ◽

10.1161/circulationaha.120.048698 ◽

2021 ◽

Author(s):

Xujie Liu ◽

Suya Wang ◽

Xiaoling Guo ◽

Yifei Li ◽

Roza Ogurlu ◽

...

Keyword(s):

Genome Editing ◽

Knockout Mice ◽

Contractile Function ◽

Barth Syndrome ◽

Model Systems ◽

Calcium Handling ◽

Inner Mitochondrial Membrane ◽

Cardiomyocyte Contractility ◽

Induced Pluripotent

Background: Mutations in tafazzin ( TAZ ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. Methods: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) and cardiac specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca 2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca 2+ handling and contractility. Results: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared to WT, BTHS iPSC-CMs had increased diastolic Ca 2+ and decreased Ca 2+ transient amplitude. BTHS iPSC-CMs had higher levels of mitochondrial and cellular ROS than WT, which activated Ca 2+ /calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII phosphorylated the cardiac ryanodine receptor (RYR2) on serine 2814, increasing Ca 2+ leak through RYR2. Inhibition of this ROS-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca 2+ handling in BTHS iPSC-CMs and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca 2+ and decreased Ca 2+ transient amplitude. These abnormalities were ameliorated by CaMKII or ROS inhibition. Conclusions: This study identified a molecular pathway that links TAZ mutation to abnormal Ca 2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial ROS production.

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Regulation of myocardial function by histidine-rich, calcium-binding protein

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01211.2003 ◽

2004 ◽

Vol 287 (4) ◽

pp. H1705-H1711 ◽

Cited By ~ 45

Author(s):

Guo-Chang Fan ◽

Kimberly N. Gregory ◽

Wen Zhao ◽

Woo Jin Park ◽

Evangelia G. Kranias

Keyword(s):

Heart Failure ◽

Ryanodine Receptor ◽

Calcium Binding ◽

Recombinant Adenovirus ◽

Contractile Function ◽

Binding Protein ◽

Human Heart Failure ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Protein Levels

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.

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Abstract 389: Alterations of Mitochondrial Calcium Handling in Heart Failure

Circulation Research ◽

10.1161/res.117.suppl_1.389 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

An-Chi Wei ◽

Ting Liu ◽

Brian O’Rourke

Keyword(s):

Heart Failure ◽

Contractile Function ◽

Critical Role ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Therapeutic Interventions ◽

Calcium Handling ◽

Aortic Constriction ◽

Cardiac Contractile Function

Heart failure (HF) and sudden cardiac death (SCD) are major public health concerns that are increasing in incidence, yet the mechanisms underlying SCD in patients with HF are poorly understood. In a novel guinea pig model of HF/SCD, we showed that in vivo treatment with a mitochondrial Na+/Ca2+ exchanger (mNCE) inhibitor attenuates cardiac remodeling, preserves cardiac contractile function, and improves survival, supporting a critical role for altered mitochondrial Ca2+ dynamics in the pathophysiology. Here, we investigate whether the intrinsic mitochondrial Ca2+ transport rates are altered in this HF model. Methods: Ascending aortic constriction, combined with daily i.p. injection of isoproterenol (ISO), were used to induce HF (ACi) with acquired long QT. This group was compared with animals subjected to aortic constriction alone (AC), or sham-operated animals with (SHAMi) or without (SHAM) ISO treatment. Ca2+ Green-5N was used to measure total mitochondrial Ca2+ uptake and to quantify mitochondrial Ca2+ influx and efflux rates in isolated cardiac mitochondria. Results: Both the total mitochondrial Ca2+ load and the Ca2+ capacity prior to triggering permeability transition pore (mPTP) opening were reduced in HF mitochondria (5mM NaCl present). Mitochondrial Ca2+ fluxes, individually measured with sequential additions of 15μM free Ca2+, 10nM Ru360 and 5mM NaCl, showed that initial Ca2+ uptake rate through the mitochondrial Ca2+ uniporter (mCU: 0.55 nmol/sec/mg) was not significantly changed in HF; however, the Ca2+ extrusion rate through mNCE was larger in HF (AC:0.022 nmol/sec/mg; SHAM:0.018; ACi:0.013; SHAMi:0.009), but with a lower affinity for Na+. Interestingly, Na+-independent efflux via mPTP increased in HF (AC:0.0040 nmol/sec/mg; SHAM:0.0022; ACi:0.0013; SHAMi:0.012). Mitochondria from failing hearts also showed decreased respiration and increased ROS emission. Conclusions: The data indicate that an increase of intrinsic Ca2+ efflux and the increase in cytoplasmic Na+ in HF could both contribute to blunted mitochondrial Ca2+ in HF, which will affect cardiac energetics and ROS balance. Inhibitors of mNCE or mPTP are thus proposed to be therapeutic interventions that would improve mitochondrial Ca2+ balance and function in HF.

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