Oleg Bogachev

The impacts of age and frailty on heart rate and sinoatrial node function

Sinoatrial node (SAN) function declines with age; however, not all individuals age at the same rate and health status can vary from fit to frail. Frailty was quantified in young and aged mice using a non‐invasive frailty index so that the impacts of age and frailty on heart rate and SAN function could be assessed. SAN function was impaired in aged mice due to alterations in electrical conduction, changes in SAN action potential morphology and fibrosis in the SAN. Changes in SAN function, electrical conduction, action potential morphology and fibrosis were correlated with, and graded by, frailty. This study shows that mice of the same chronological age have quantifiable differences in health status that impact heart rate and SAN function and that these differences in health status can be identified using our frailty index.

Impaired sinoatrial node function and increased susceptibility to atrial fibrillation in mice lacking natriuretic peptide receptor C

Natriuretic peptides (NPs) elicit their effects via multiple NP receptors (including NPR‐A, NPR‐B and NPR‐C, with NPR‐C being relatively poorly understood). We have studied the effects of NPR‐C ablation on cardiac structure, function and arrhythmogenesis using NPR‐C knockout (NPR‐C−/−) mice. NPR‐C−/− mice are characterized by sinoatrial node (SAN) dysfunction and a profound increase in susceptibility to atrial fibrillation. Increased susceptibility to arrhythmias in NPR‐C−/− mice was associated with slowed electrical conduction in the SAN as well as the right and left atria due to enhanced collagen expression and deposition in the atria (structural remodelling), but without changes in action potential morphology (electrical remodelling) in isolated SAN or atrial myocytes. This study demonstrates a critical protective role for NPR‐C in the heart.

Adipocyte enhancer-binding protein 1 (AEBP1) (a novel macrophage proinflammatory mediator) overexpression promotes and ablation attenuates atherosclerosis in ApoE (-/-) and LDLR (-/-) mice.

Atherogenesis is a long-term process that involves inflammatory response coupled with metabolic dysfunction. Foam cell formation and macrophage inflammatory response are two key events in atherogenesis. Adipocyte enhancer-binding protein 1 (AEBP1) has been shown to impede macrophage cholesterol efflux, promoting foam cell formation, via peroxisome proliferator-activated receptor (PPAR)-γl and liver X receptor α (LXRα) downregulation. Moreover, AEBP1 has been shown to promote macrophage inflammatory responsiveness by inducing nuclear factor (NF)-κB activity via IκBα downregulation. Lipopolysaccharide (LPS)-induced suppression of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, has been shown to be mediated by AEBP1. Herein, we showed that AEBP1-transgenic mice (AEBP1TG) with macrophage-specific AEBP1 overexpression exhibit hyperlipidemia and develop atherosclerotic lesions in their proximal aortas. Consistently, ablation of AEBP1 results in significant attenuation of atherosclerosis (males: 3.2-fold, P = 0.001 (en face)), 2.7-fold, P = 0.0004 (aortic roots); females: 2.1-fold, P = 0.0026 (en face), 1.7-fold, P = 0.0126 (aortic roots)) in the AEBP1−/−/low-density lipoprotein receptor (LDLR)−/− double-knockout (KO) mice. Bone marrow (BM) transplantation experiments further revealed that LDLR−/− mice reconstituted with AEBP1−/−/LDLR−/− BM cells (LDLR−/−/KO-BM chimera) display significant reduction of atherosclerosis lesions (en face: 2.0-fold, P = 0.0268; aortic roots: 1.7-fold, P = 0.05) compared with control mice reconstituted with AEBP1+/+/LDLR−/− BM cells (LDLR−/−/WT-BM chimera). Furthermore, transplantation of AEBP1TG BM cells with the normal apolipoprotein E (ApoE) gene into ApoE−/− mice (ApoE−/−/TG-BM chimera) leads to significant development of atherosclerosis (males: 2.5-fold, P = 0.0001 (en face), 4.7-fold, P = 0.0001 [aortic roots]; females: 1.8-fold, P = 0.0001 (en face), 3.0-fold, P = 0.0001 [aortic roots]) despite the restoration of ApoE expression. Macrophages from ApoE−/−/TG-BM chimeric mice express reduced levels of PPARγ1, LXRα, ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) and increased levels of the inflammatory mediators interleukin (IL)-6 and tumor necrosis factor (TNF)-α compared with macrophages of control chimeric mice (ApoE−/−/NT-BM) that received AEBP1 nontransgenic (AEBP1NT) BM cells. Our in vivo experimental data strongly suggest that macrophage AEBP1 plays critical regulatory roles in atherogenesis, and it may serve as a potential therapeutic target for the prevention or treatment of atherosclerosis.

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling

Background: Stromal-epithelial interactions regulate mammary gland development and tumorigenesis. Results: Targeted overexpression of adipocyte enhancer-binding protein (AEBP1) in stromal macrophages induces alveolar hyperplasia via up-regulation of NF-κB, TNFα, and hedgehog pathway components. Conclusion: AEBP1 orchestrates the stromal-epithelial interactions via proinflammatory and hedgehog signaling. Significance: This is a first report implicating AEBP1 in mammary gland hyperplasia with possible association to tumorigenesis. Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1TG bone marrow cells into non-transgenic (AEBP1NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1TG mammary macrophages and epithelium. Shh expression was induced in AEBP1TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1TG peritoneal macrophages. The conditioned AEBP1TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.

Atrial structure, function and arrhythmogenesis in aged and frail mice

Atrial fibrillation (AF) is prevalent in aging populations; however not all individuals age at the same rate. Instead, individuals of the same chronological age can vary in health status from fit to frail. Our objective was to determine the impacts of age and frailty on atrial function and arrhythmogenesis in mice using a frailty index (FI). Aged mice were more frail and demonstrated longer lasting AF compared to young mice. Consistent with this, aged mice showed longer P wave duration and PR intervals; however, both parameters showed substantial variability suggesting differences in health status among mice of similar chronological age. In agreement with this, P wave duration and PR interval were highly correlated with FI score. High resolution optical mapping of the atria demonstrated reduced conduction velocity and action potential duration in aged hearts that were also graded by FI score. Furthermore, aged mice had increased interstitial fibrosis along with changes in regulators of extracellular matrix remodelling, which also correlated with frailty. These experiments demonstrate that aging results in changes in atrial structure and function that create a substrate for atrial arrhythmias. Importantly, these changes were heterogeneous due to differences in health status, which could be identified using an FI.

Effects of Wild-Type and Mutant Forms of Atrial Natriuretic Peptide on Atrial Electrophysiology and Arrhythmogenesis

Background—Atrial natriuretic peptide (ANP) is a hormone with numerous beneficial cardiovascular effects. Recently, a mutation in the ANP gene, which results in the generation of a mutant form of ANP (mANP), was identified and shown to cause atrial fibrillation in people. The mechanism(s) through which mANP causes atrial fibrillation is unknown. Our objective was to compare the effects of wild-type ANP and mANP on atrial electrophysiology in mice and humans. Methods and Results—Action potentials (APs), L-type Ca2+ currents (ICa,L), and Na+ current were recorded in atrial myocytes from wild-type or natriuretic peptide receptor C knockout (NPR-C−/−) mice. In mice, ANP and mANP (10–100 nmol/L) had opposing effects on atrial myocyte AP morphology and ICa,L. ANP increased AP upstroke velocity (Vmax), AP duration, and ICa,L similarly in wild-type and NPR-C−/− myocytes. In contrast, mANP decreased Vmax, AP duration, and ICa,L, and these effects were completely absent in NPR-C−/− myocytes. ANP and mANP also had opposing effects on ICa,L in human atrial myocytes. In contrast, neither ANP nor mANP had any effect on Na+ current in mouse atrial myocytes. Optical mapping studies in mice demonstrate that ANP sped electric conduction in the atria, whereas mANP did the opposite and slowed atrial conduction. Atrial pacing in the presence of mANP induced arrhythmias in 62.5% of hearts, whereas treatment with ANP completely prevented the occurrence of arrhythmias. Conclusions—These findings provide mechanistic insight into how mANP causes atrial fibrillation and demonstrate that wild-type ANP is antiarrhythmic.

Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice

Cardiovascular autonomic neuropathy (CAN) is a serious complication of diabetes mellitus that impairs autonomic regulation of heart rate (HR). This has been attributed to damage to the nerves that modulate spontaneous pacemaker activity in the sinoatrial node (SAN). Our objective was to test the hypothesis that impaired parasympathetic regulation of HR in diabetes is due to reduced responsiveness of the SAN to parasympathetic agonists. We used the Akita mouse model of type 1 diabetes to study the effects of the parasympathetic agonist carbachol (CCh) on SAN function using intracardiac programmed stimulation, high resolution optical mapping and patch-clamping of SAN myocytes. CCh decreased HR by 30% and increased corrected SAN recovery time (cSNRT) by 123% in wildtype mice. In contrast, CCh only decreased HR by 12%, and only increased cSNRT by 37% in Akita mice. These alterations were due to smaller effects of CCh on SAN electrical conduction and spontaneous action potential firing in isolated SAN myocytes. Voltage clamp experiments demonstrate that the acetylcholine-activated K(+) current (IKACh) is reduced in Akita SAN myocytes due to enhanced desensitization and faster deactivation kinetics. These IKACh alterations were normalized by treating Akita SAN myocytes with PI(3,4,5)P3 or an inhibitor of regulator of G-protein signaling 4 (RGS4). There was no difference in the effects of CCh on the hyperpolarization-activated current (If) between wildtype and Akita mice. Our study demonstrates that Akita diabetic mice demonstrate impaired parasympathetic regulation of HR and SAN function due to reduced responses of the SAN to parasympathetic agonists. Our experiments demonstrate a key role for insulin-dependent phosphoinositide 3-kinase (PI3K) signaling in the parasympathetic dysfunction seen in the SAN in diabetes.

Lactation defect with impaired secretory activation in AEBP1-null mice.

Adipocyte enhancer binding protein 1 (AEBP1) is a multifunctional protein that negatively regulates the tumor suppressor PTEN and IκBα, the inhibitor of NF-κB, through protein-protein interaction, thereby promoting cell survival and inflammation. Mice homozygous for a disrupted AEBP1 gene developed to term but showed defects in growth after birth. AEBP1 −/− females display lactation defect, which results in the death of 100% of the litters nursed by AEBP1 −/− dams. Mammary gland development during pregnancy appears normal in AEBP1 −/− dams; however these mice exhibit expansion of the luminal space and the appearance of large cytoplasmic lipid droplets (CLDs) in the mammary epithelial cells at late pregnancy and parturition, which is a clear sign of failed secretory activation, and accumulation of milk proteins in the mammary gland, presumably reflecting milk stasis following failed secretory activation. Eventually, AEBP1 −/− mammary gland rapidly undergoes involution at postpartum. Stromal restoration of AEBP1 expression by transplanting wild-type bone marrow (BM) cells is sufficient to rescue the mammary gland defect. Our studies suggest that AEBP1 is critical in the maintenance of normal tissue architecture and function of the mammary gland tissue and controls stromal-epithelial crosstalk in mammary gland development.

Altered heart rate regulation by the autonomic nervous system in mice lacking natriuretic peptide receptor C (NPR-C)

Natriuretic peptides (NPs) play essential roles in the regulation of cardiovascular function. NP effects are mediated by receptors known as NPR-A, NPR-B or NPR-C. NPs have potent effects on regulation of heart rate (HR) by the autonomic nervous system (ANS), but the role of NPR-C in these effects has not been investigated. Accordingly, we have used telemetric ECG recordings in awake, freely moving wildtype and NPR-C knockout (NPR-C−/−) mice and performed heart rate variability (HRV) analysis to assess alterations in sympatho-vagal balance on the heart following loss of NPR-C. Our novel data demonstrate that NPR-C−/− mice are characterized by elevations in HR, reductions in circadian changes in HR and enhanced occurrence of sinus pauses, indicating increased arrhythmogenesis and a loss of HRV. Time domain and frequency domain analyses further demonstrate that HRV is reduced in NPR-C−/− mice in association with a reduction in parasympathetic activity. Importantly, the low frequency to high frequency ratio was increased in NPR-C−/− mice indicating that sympathetic activity is also enhanced. These changes in autonomic regulation were confirmed using atropine and propranolol to antagonize the ANS. These findings illustrate that loss of NPR-C reduces HRV due to perturbations in the regulation of the heart by the ANS.

The impact of ovariectomy on cardiac excitation-contraction coupling is mediated through cAMP/PKA-dependent mechanisms

Ovariectomy (OVX) promotes sarcoplasmic reticulum (SR) Ca2+ overload in ventricular myocytes. We hypothesized that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway contributes to this Ca2+ dysregulation. Myocytes were isolated from adult female C57BL/6 mice following either OVX or sham surgery (surgery at ≈1mos). Contractions, Ca2+ concentrations (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in voltage-clamped myocytes. Intracellular cAMP levels were determined with an enzyme immunoassay; phosphodiesterase (PDE) and adenylyl cyclase (AC) isoform expression was examined with qPCR. Ca2+ currents were similar in myocytes from sham and OVX mice but Ca2+ transients, excitation-contraction (EC)-coupling gain, SR content and contractions were larger in OVX than sham cells. To determine if the cAMP/PKA pathway mediated OVX-induced alterations in EC-coupling, cardiomyocytes were incubated with the PKA inhibitor H-89 (2μM), which abolished baseline differences. While basal intracellular cAMP did not differ, levels were higher in OVX than sham in the presence of a non-selective PDE inhibitor (300μM IBMX), or an AC activator (10μM forskolin). This suggests the production of cAMP by AC and its breakdown by PDE were enhanced by OVX. Consistent with this, mRNA levels for both AC5 and PDE4A were higher in OVX in comparison to sham. Differences in Ca2+ homeostasis and contractions were abolished when sham and OVX cells were dialyzed with patch pipettes containing the same concentration of 8-bromoadenosine-cAMP (50μM). Interestingly, selective inhibition of PDE4 increased Ca2+ current only in OVX cells. Together, these findings suggest that estrogen suppresses SR Ca2+ release and that this is regulated, at least in part, by the cAMP/PKA pathway. These changes in the cAMP/PKA pathway may promote Ca2+ dysregulation and cardiovascular disease when ovarian estrogen levels fall. These results advance our understanding of female-specific cardiomyocyte mechanisms that may affect responses to therapeutic interventions in older women.