Publications by authors named "Giovanni Fajardo"

27 Publications

  • Page 1 of 1

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Proc Natl Acad Sci U S A 2021 Jun;118(24)

Department of Pediatrics (Cardiology), Stanford University School of Medicine, Palo Alto, CA 94304;

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.
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http://dx.doi.org/10.1073/pnas.2025030118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214707PMC
June 2021

Improving Right Ventricular Function by Increasing BMP Signaling with FK506.

Am J Respir Cell Mol Biol 2021 09;65(3):272-287

Division of Pulmonary and Critical Care Medicine, Department of Medicine.

Right ventricular (RV) function is the predominant determinant of survival in patients with pulmonary arterial hypertension (PAH). In preclinical models, pharmacological activation of BMP (bone morphogenetic protein) signaling with FK506 (tacrolimus) improved RV function by decreasing RV afterload. FK506 therapy further stabilized three patients with end-stage PAH. Whether FK506 has direct effects on the pressure-overloaded right ventricle is yet unknown. We hypothesized that increasing cardiac BMP signaling with FK506 improves RV structure and function in a model of fixed RV afterload after pulmonary artery banding (PAB). Direct cardiac effects of FK506 on the microvasculature and RV fibrosis were studied after surgical PAB in wild-type and heterozygous mutant mice. RV function and strain were assessed longitudinally via cardiac magnetic resonance imaging during continuous FK506 infusion. Genetic lineage tracing of endothelial cells (ECs) was performed to assess the contribution of ECs to fibrosis. Molecular mechanistic studies were performed in human cardiac fibroblasts and ECs. In mice, low BMP signaling in the right ventricle exaggerated PAB-induced RV fibrosis. FK506 therapy restored cardiac BMP signaling, reduced RV fibrosis in a BMP-dependent manner independent from its immunosuppressive effect, preserved RV capillarization, and improved RV function and strain over the time course of disease. Endothelial mesenchymal transition was a rare event and did not significantly contribute to cardiac fibrosis after PAB. Mechanistically, FK506 required ALK1 in human cardiac fibroblasts as a BMPR2 co-receptor to reduce TGFβ1-induced proliferation and collagen production. Our study demonstrates that increasing cardiac BMP signaling with FK506 improves RV structure and function independent from its previously described beneficial effects on pulmonary vascular remodeling.
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http://dx.doi.org/10.1165/rcmb.2020-0528OCDOI Listing
September 2021

Transcriptomic and Functional Analyses of Mitochondrial Dysfunction in Pressure Overload-Induced Right Ventricular Failure.

J Am Heart Assoc 2021 02 30;10(4):e017835. Epub 2021 Jan 30.

Department of Pediatrics (Cardiology) Stanford University Palo Alto CA.

Background In complex congenital heart disease patients such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload, leading to RV hypertrophy and eventually RV failure. The mechanisms that promote the transition from stable RV hypertrophy to RV failure are unknown. We evaluated the role of mitochondrial bioenergetics in the development of RV failure. Methods and Results We created a murine model of RV pressure overload by pulmonary artery banding and compared with sham-operated controls. Gene expression by RNA-sequencing, oxidative stress, mitochondrial respiration, dynamics, and structure were assessed in pressure overload-induced RV failure. RV failure was characterized by decreased expression of electron transport chain genes and mitochondrial antioxidant genes (aldehyde dehydrogenase 2 and superoxide dismutase 2) and increased expression of oxidant stress markers (heme oxygenase, 4-hydroxynonenal). The activities of all electron transport chain complexes decreased with RV hypertrophy and further with RV failure (oxidative phosphorylation: sham 552.3±43.07 versus RV hypertrophy 334.3±30.65 versus RV failure 165.4±36.72 pmol/(s×mL), <0.0001). Mitochondrial fission protein DRP1 (dynamin 1-like) trended toward an increase, while MFF (mitochondrial fission factor) decreased and fusion protein OPA1 (mitochondrial dynamin like GTPase) decreased. In contrast, transcription of electron transport chain genes increased in the left ventricle of RV failure. Conclusions Pressure overload-induced RV failure is characterized by decreased transcription and activity of electron transport chain complexes and increased oxidative stress which are associated with decreased energy generation. An improved understanding of the complex processes of energy generation could aid in developing novel therapies to mitigate mitochondrial dysfunction and delay the onset of RV failure.
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http://dx.doi.org/10.1161/JAHA.120.017835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955345PMC
February 2021

β-Adrenergic Signaling Modulates Mitochondrial Function and Morphology in Skeletal Muscle in Response to Aerobic Exercise.

Cells 2021 01 13;10(1). Epub 2021 Jan 13.

Department of Pediatrics, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.

The molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by aerobic exercise (AE) are not fully understood. We have previously shown that AE induces mitochondrial adaptations in cardiac muscle, mediated by sympathetic stimulation. Since direct sympathetic innervation of neuromuscular junctions influences skeletal muscle homeostasis, we tested the hypothesis that β-adrenergic receptor (β-AR)-mediated sympathetic activation induces mitochondrial adaptations to AE in skeletal muscle. Male FVB mice were subjected to a single bout of AE on a treadmill (80% Vmax, 60 min) under β-AR blockade with ICI 118,551 (ICI) or vehicle, and parameters of mitochondrial function and morphology/dynamics were evaluated. An acute bout of AE significantly increased maximal mitochondrial respiration in tibialis anterior (TA) isolated fiber bundles, which was prevented by β-AR blockade. This increased mitochondrial function after AE was accompanied by a change in mitochondrial morphology towards fusion, associated with increased Mfn1 protein expression and activity. β-AR blockade fully prevented the increase in Mfn1 activity and reduced mitochondrial elongation. To determine the mechanisms involved in mitochondrial modulation by β-AR activation in skeletal muscle during AE, we used C2C12 myotubes, treated with the non-selective β-AR agonist isoproterenol (ISO) in the presence of the specific β-AR antagonist ICI or during protein kinase A (PKA) and Gα protein blockade. Our in vitro data show that β-AR activation significantly increases mitochondrial respiration in myotubes, and this response was dependent on β-AR activation through a Gα-PKA signaling cascade. In conclusion, we provide evidence for AE-induced β-AR activation as a major mechanism leading to alterations in mitochondria function and morphology/dynamics. β-AR signaling is thus a key-signaling pathway that contributes to skeletal muscle plasticity in response to exercise.
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http://dx.doi.org/10.3390/cells10010146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828343PMC
January 2021

4HNE Impairs Myocardial Bioenergetics in Congenital Heart Disease-Induced Right Ventricular Failure.

Circulation 2020 10 18;142(17):1667-1683. Epub 2020 Aug 18.

Department of Pediatrics (Cardiology) (HT.V.H., N.S., S.L.P., S. Ranjbarvairi, D-Q.H., M.Z., G.F., D.B., S. Reddy), Stanford University, Palo Alto, CA.

Background: In patients with complex congenital heart disease, such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload stress, leading to RV hypertrophy and eventually RV failure. The role of lipid peroxidation, a potent form of oxidative stress, in mediating RV hypertrophy and failure in congenital heart disease is unknown.

Methods: Lipid peroxidation and mitochondrial function and structure were assessed in right ventricle (RV) myocardium collected from patients with RV hypertrophy with normal RV systolic function (RV fractional area change, 47.3±3.8%) and in patients with RV failure showing decreased RV systolic function (RV fractional area change, 26.6±3.1%). The mechanism of the effect of lipid peroxidation, mediated by 4-hydroxynonenal ([4HNE] a byproduct of lipid peroxidation) on mitochondrial function and structure was assessed in HL1 murine cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes.

Results: RV failure was characterized by an increase in 4HNE adduction of metabolic and mitochondrial proteins (16 of 27 identified proteins), in particular electron transport chain proteins. Sarcomeric (myosin) and cytoskeletal proteins (desmin, tubulin) also underwent 4HNE adduction. RV failure showed lower oxidative phosphorylation (moderate RV hypertrophy, 287.6±19.75 versus RV failure, 137.8±11.57 pmol/[sec×mL]; =0.0004), and mitochondrial structural damage. Using a cell model, we show that 4HNE decreases cell number and oxidative phosphorylation (control, 388.1±23.54 versus 4HNE, 143.7±11.64 pmol/[sec×mL]; <0.0001). Carvedilol, a known antioxidant did not decrease 4HNE adduction of metabolic and mitochondrial proteins and did not improve oxidative phosphorylation.

Conclusions: Metabolic, mitochondrial, sarcomeric, and cytoskeletal proteins are susceptible to 4HNE-adduction in patients with RV failure. 4HNE decreases mitochondrial oxygen consumption by inhibiting electron transport chain complexes. Carvedilol did not improve the 4HNE-mediated decrease in oxygen consumption. Strategies to decrease lipid peroxidation could improve mitochondrial energy generation and cardiomyocyte survival and improve RV failure in patients with congenital heart disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.045470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606813PMC
October 2020

Physiological Mitochondrial Fragmentation Is a Normal Cardiac Adaptation to Increased Energy Demand.

Circ Res 2018 01 12;122(2):282-295. Epub 2017 Dec 12.

From the Department of Pediatrics (Cardiology) (M.C., G.F., K.N., M.Z., K.K., G.J., D.-Q.H., S.R., E.S., D.B.) and Cardiovascular Research Institute (M.C., G.F., M.Z., K.K., G.J., D.-Q.H., S.R., E.S., D.B.), Stanford University, CA; and Molecular Cardiology Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (A.S., R.A.G.).

Rationale: Mitochondria play a dual role in the heart, responsible for meeting energetic demands and regulating cell death. Paradigms have held that mitochondrial fission and fragmentation are the result of pathological stresses, such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in decreased mitochondrial function and cardiac impairment, suggesting that fission is important for maintaining cardiac and mitochondrial bioenergetic homeostasis.

Objective: The purpose of this study is to determine whether mitochondrial fission and fragmentation can be an adaptive mechanism used by the heart to augment mitochondrial and cardiac function during a normal physiological stress, such as exercise.

Methods And Results: We demonstrate a novel role for cardiac mitochondrial fission as a normal adaptation to increased energetic demand. During submaximal exercise, physiological mitochondrial fragmentation results in enhanced, rather than impaired, mitochondrial function and is mediated, in part, by β1-adrenergic receptor signaling. Similar to pathological fragmentation, physiological fragmentation is induced by activation of dynamin-related protein 1; however, unlike pathological fragmentation, membrane potential is maintained and regulators of mitophagy are downregulated. Inhibition of fission with P110, Mdivi-1 (mitochondrial division inhibitor), or in mice with cardiac-specific dynamin-related protein 1 ablation significantly decreases exercise capacity.

Conclusions: These findings demonstrate the requirement for physiological mitochondrial fragmentation to meet the energetic demands of exercise, as well as providing additional support for the evolving conceptual framework, where mitochondrial fission and fragmentation play a role in the balance between mitochondrial maintenance of normal physiology and response to disease.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.310725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775047PMC
January 2018

Load-dependent effects of apelin on murine cardiomyocytes.

Prog Biophys Mol Biol 2017 11 18;130(Pt B):333-343. Epub 2017 Sep 18.

Institute for Experimental Cardiovascular Medicine, University Heart Centre Freiburg · Bad Krozingen, Medical School of the University of Freiburg, Germany; Imperial College London, NHLI, Heart Science Centre, UK.

The apelin peptide is described as one of the most potent inotropic agents, produced endogenously in a wide range of cells, including cardiomyocytes. Despite positive effects on cardiac contractility in multicellular preparations, as well as indications of cardio-protective actions in several diseases, its effects and mechanisms of action at the cellular level are incompletely understood. Here, we report apelin effects on dynamic mechanical characteristics of single ventricular cardiomyocytes, isolated from mouse models (control, apelin-deficient [Apelin-KO], apelin-receptor KO mouse [APJ-KO]), and rat. Dynamic changes in maximal velocity of cell shortening and relaxation were monitored. In addition, more traditional indicators of inotropic effects, such as maximum shortening (in mechanically unloaded cells) or peak force development (in auxotonic contracting cells, preloaded using the carbon fibre technique) were studied. The key finding is that, using Apelin-KO cardiomyocytes exposed to different preloads with the 2-carbon fibre technique, we observe a lowering of the slope of the end-diastolic stress-length relation in response to 10 nM apelin, an effect that is preload-dependent. This suggests a positive lusitropic effect of apelin, which could explain earlier counter-intuitive findings on an apelin-induced increase in contractility occurring without matching rise in oxygen consumption.
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http://dx.doi.org/10.1016/j.pbiomolbio.2017.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726609PMC
November 2017

miR-21 is associated with fibrosis and right ventricular failure.

JCI Insight 2017 May 4;2(9). Epub 2017 May 4.

Department of Pediatrics, Division of Cardiology, Stanford University, Stanford, California, USA.

Combined pulmonary insufficiency (PI) and stenosis (PS) is a common long-term sequela after repair of many forms of congenital heart disease, causing progressive right ventricular (RV) dilation and failure. Little is known of the mechanisms underlying this combination of preload and afterload stressors. We developed a murine model of PI and PS (PI+PS) to identify clinically relevant pathways and biomarkers of disease progression. Diastolic dysfunction was induced (restrictive RV filling, elevated RV end-diastolic pressures) at 1 month after generation of PI+PS and progressed to systolic dysfunction (decreased RV shortening) by 3 months. RV fibrosis progressed from 1 month (4.4% ± 0.4%) to 3 months (9.2% ± 1%), along with TGF-β signaling and tissue expression of profibrotic miR-21. Although plasma miR-21 was upregulated with diastolic dysfunction, it was downregulated with the onset of systolic dysfunction), correlating with RV fibrosis. Plasma miR-21 in children with PI+PS followed a similar pattern. A model of combined RV volume and pressure overload recapitulates the evolution of RV failure unique to patients with prior RV outflow tract surgery. This progression was characterized by enhanced TGF-β and miR-21 signaling. miR-21 may serve as a plasma biomarker of RV failure, with decreased expression heralding the need for valve replacement.
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http://dx.doi.org/10.1172/jci.insight.91625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414555PMC
May 2017

Nat1 Deficiency Is Associated with Mitochondrial Dysfunction and Exercise Intolerance in Mice.

Cell Rep 2016 10;17(2):527-540

Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

We recently identified human N-acetyltransferase 2 (NAT2) as an insulin resistance (IR) gene. Here, we examine the cellular mechanism linking NAT2 to IR and find that Nat1 (mouse ortholog of NAT2) is co-regulated with key mitochondrial genes. RNAi-mediated silencing of Nat1 led to mitochondrial dysfunction characterized by increased intracellular reactive oxygen species and mitochondrial fragmentation as well as decreased mitochondrial membrane potential, biogenesis, mass, cellular respiration, and ATP generation. These effects were consistent in 3T3-L1 adipocytes, C2C12 myoblasts, and in tissues from Nat1-deficient mice, including white adipose tissue, heart, and skeletal muscle. Nat1-deficient mice had changes in plasma metabolites and lipids consistent with a decreased ability to utilize fats for energy and a decrease in basal metabolic rate and exercise capacity without altered thermogenesis. Collectively, our results suggest that Nat1 deficiency results in mitochondrial dysfunction, which may constitute a mechanistic link between this gene and IR.
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http://dx.doi.org/10.1016/j.celrep.2016.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097870PMC
October 2016

Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

FASEB J 2016 Apr 16;30(4):1464-79. Epub 2015 Dec 16.

*Division of Cardiology, Department of Pediatrics, Stanford Cardiovascular Institute, Division of Cardiovascular Medicine, Department of Medicine, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA; Department of Mechanical Engineering, Stanford University School of Engineering, Stanford, California, USA; and Departments of Pharmacology and Medicine, University of California, San Diego, San Diego, California, USA

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.
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http://dx.doi.org/10.1096/fj.15-280982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799510PMC
April 2016

Epicardial FSTL1 reconstitution regenerates the adult mammalian heart.

Nature 2015 Sep 16;525(7570):479-85. Epub 2015 Sep 16.

Stanford Cardiovascular Institute and Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305, USA.

The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.
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http://dx.doi.org/10.1038/nature15372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762253PMC
September 2015

Deletion of the β2-adrenergic receptor prevents the development of cardiomyopathy in mice.

J Mol Cell Cardiol 2013 Oct 3;63:155-64. Epub 2013 Aug 3.

Department of Pediatrics, Stanford University, Stanford, CA, USA.

Beta adrenergic receptor (β-AR) subtypes act through diverse signaling cascades to modulate cardiac function and remodeling. Previous in vitro studies suggest that β1-AR signaling is cardiotoxic whereas β2-AR signaling is cardioprotective, and may be the case during ischemia/reperfusion in vivo. The objective of this study was to assess whether β2-ARs also play a cardioprotective role in the pathogenesis of non-ischemic forms of cardiomyopathy. To dissect the role of β1 vs β2-ARs in modulating MLP (Muscle LIM Protein) cardiomyopathy, we crossbred MLP-/- with β1-/- or β2-/- mice. Deletion of the β2-AR improved survival, cardiac function, exercise capacity and myocyte shortening; by contrast haploinsufficency of the β1-AR reduced survival. Pathologic changes in Ca(2+) handling were reversed in the absence of β2-ARs: peak Ca(2+) and SR Ca(2+) were decreased in MLP-/- and β1+/-/MLP-/- but restored in β2-/-MLP-/-. These changes were associated with reversal of alterations in troponin I and phospholamban phosphorylation. Gi inhibition increased peak and baseline Ca(2+), recapitulating changes observed in the β2-/-/MLP-/-. The L-type Ca(2+) blocker verapamil significantly decreased cardiac function in β2-/-MLP-/- vs WT. We next tested if the protective effects of β2-AR ablation were unique to the MLP model using TAC-induced heart failure. Similar to MLP, β2-/- mice demonstrated delayed progression of heart failure with restoration of myocyte shortening and peak Ca(2+) and Ca(2+) release. Deletion of β2-ARs prevents the development of MLP-/- cardiomyopathy via positive modulation of Ca(2+) due to removal of inhibitory Gi signaling and increased phosphorylation of troponin I and phospholamban. Similar effects were seen after TAC. Unlike previous models where β2-ARs were found to be cardioprotective, in these two models, β2-AR signaling appears to be deleterious, potentially through negative regulation of Ca(2+) dynamics.
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http://dx.doi.org/10.1016/j.yjmcc.2013.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791213PMC
October 2013

Altered ubiquitin-proteasome signaling in right ventricular hypertrophy and failure.

Am J Physiol Heart Circ Physiol 2013 Aug 31;305(4):H551-62. Epub 2013 May 31.

Department of Pediatrics (Cardiology Stanford University School of Medicine, Stanford, California;

Alterations in the ubiquitin-proteasome system (UPS) have been described in left ventricular hypertrophy and failure, although results have been inconsistent. The role of the UPS in right ventricular (RV) hypertrophy (RVH) and RV failure (RVF) is unknown. Given the greater percent increase in RV mass associated with RV afterload stress, as present in many congenital heart lesions, we hypothesized that alterations in the UPS could play an important role in RVH/RVF. UPS expression and activity were measured in the RV from mice with RVH/RVF secondary to pulmonary artery constriction (PAC). Epoxomicin and MG132 were used to inhibit the proteasome, and overexpression of the 11S PA28α subunit was used to activate the proteasome. PAC mice developed RVH (109.3% increase in RV weight to body weight), RV dilation with septal shift, RV dysfunction, and clinical RVF. Proteasomal function (26S β₅ chymotrypsin-like activity) was decreased 26% (P < 0.05). Protein expression of 19S subunit Rpt5 (P < 0.05), UCHL1 deubiquitinase (P < 0.0001), and Smurf1 E3 ubiquitin ligase (P < 0.01) were increased, as were polyubiquitinated proteins (P < 0.05) and free-ubiquitins (P = 0.05). Pro-apoptotic Bax was increased (P < 0.0001), whereas anti-apoptotic Bcl-2 decreased (P < 0.05), resulting in a sixfold increase in the Bax/Bcl-2 ratio. Proteasomal inhibition did not accelerate RVF. However, proteasome enhancement by cardiac-specific proteasome overexpression partially improved survival. Proteasome activity is decreased in RVH/RVF, associated with upregulation of key UPS regulators and pro-apoptotic signaling. Enhancement of proteasome function partially attenuates RVF, suggesting that UPS dysfunction contributes to RVF.
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http://dx.doi.org/10.1152/ajpheart.00771.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891246PMC
August 2013

Physiologic and molecular characterization of a murine model of right ventricular volume overload.

Am J Physiol Heart Circ Physiol 2013 May 15;304(10):H1314-27. Epub 2013 Mar 15.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

Pulmonary insufficiency (PI) is a common long-term sequel after repair of tetralogy of Fallot, causing progressive right ventricular (RV) dilation and failure. We describe the physiologic and molecular characteristics of the first murine model of RV volume overload. PI was created by entrapping the pulmonary valve leaflets with sutures. Imaging, catheterization, and exercise testing were performed at 1, 3, and 6 mo and compared with sham controls. RNA from the RV free wall was hybridized to Agilent whole genome oligonucleotide microarrays. Volume overload resulted in RV enlargement, decreased RV outflow tract shortening fraction at 1 mo followed by normalization at 3 and 6 mo (39 ± 2, 44 ± 2, and 41 ± 2 vs. 46 ± 3% in sham), early reversal of early and late diastolic filling velocities (E/A ratio) followed by pseudonormalization (0.87 ± 0.08, 0.82 ± 0.08, and 0.96 ± 0.08 vs. 1.04 ± 0.03; P < 0.05), elevated end-diastolic pressure (7.6 ± 0.7, 6.9 ± 0.8, and 7 ± 0.5 vs. 2.7 ± 0.2 mmHg; P < 0.05), and decreased exercise duration (26 ± 0.4, 26 ± 1, and 22 ± 1.3 vs. 30 ± 1.1 min; P < 0.05). Subendocardial RV fibrosis was evident by 1 mo. At 1 mo, 372 genes were significantly downregulated. Mitochondrial pathways and G protein-coupled receptor signaling were the most represented categories. At 3 mo, 434 genes were upregulated and 307 downregulated. While many of the same pathways continued to be downregulated, TNF-α, transforming growth factor-β(1) (TGF-β(1)), p53-signaling, and extracellular matrix (ECM) remodeling transitioned from down- to upregulated. We describe a novel murine model of chronic RV volume overload recapitulating aspects of the clinical disease with gene expression changes suggesting early mitochondrial bioenergetic dysfunction, enhanced TGF-β signaling, ECM remodeling, and apoptosis.
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http://dx.doi.org/10.1152/ajpheart.00776.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652064PMC
May 2013

Sacrificial layer technique for axial force post assay of immature cardiomyocytes.

Biomed Microdevices 2013 Feb;15(1):171-81

Department of Mechanical Engineering and Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA.

Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease, and offer potential for patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function, and to gain insight into cardiac growth and disease, tissue engineers must quantify the contractile forces of these single cells. Currently, axial contractile forces of isolated adult heart cells can only be measured by two-point methods such as carbon fiber techniques, which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificial support layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput, is functionally analogous to current gold-standard axial force assays for adult heart cells, and prescribes elongated cell shapes without protein patterning. Finally, we calibrate these force posts with piezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far better than that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culture platforms will enable improved cell placement and the complex suspension of cells across 3D constructs.
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http://dx.doi.org/10.1007/s10544-012-9710-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545035PMC
February 2013

Dynamic microRNA expression during the transition from right ventricular hypertrophy to failure.

Physiol Genomics 2012 May 27;44(10):562-75. Epub 2012 Mar 27.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA.

MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there are no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC vs. control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: four miRs (34a, 28, 148a, and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, extracellular matrix turnover, and impaired proteosomal function. The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload-stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.
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http://dx.doi.org/10.1152/physiolgenomics.00163.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426410PMC
May 2012

In vivo functional and transcriptional profiling of bone marrow stem cells after transplantation into ischemic myocardium.

Arterioscler Thromb Vasc Biol 2012 Jan 27;32(1):92-102. Epub 2011 Oct 27.

Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA, USA.

Objective: Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model.

Methods And Results: We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis.

Conclusions: Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.
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http://dx.doi.org/10.1161/ATVBAHA.111.238618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241895PMC
January 2012

Pharmacological inhibition of βIIPKC is cardioprotective in late-stage hypertrophy.

J Mol Cell Cardiol 2011 Dec 2;51(6):980-7. Epub 2011 Sep 2.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305-5174, USA.

We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, βIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a βIIPKC-specific inhibitor, βIIV5-3, prolonged their survival by at least 6weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca(2+)-handling proteins, SERCA2 and the Na(+)/Ca(2+) exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the βΙΙPKC-treated rats, suggesting that βΙΙPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of βIPKC, an alternative spliced form of βIIPKC, had no beneficial effects in these rats. We also found that βIIV5-3, but not βIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that βIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of βIIPKC may be a potential treatment for this disease.
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http://dx.doi.org/10.1016/j.yjmcc.2011.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418885PMC
December 2011

THE ROLE OF β-ADRENERGIC RECEPTORS IN HEART FAILURE: DIFFERENTIAL REGULATION OF CARDIOTOXICITY AND CARDIOPROTECTION.

Prog Pediatr Cardiol 2011 Jan;31(1):35-38

Division of Pediatric Cardiology, Department of Pediatrics, Stanford University.

β-adrenergic receptor blockers have demonstrated significant survival benefit and have become standard therapy for adults with dilated cardiomyopathy, although their efficacy in pediatric patients is still unproven. Recent data suggests that the two major cardiac β-adrenergic receptor subtypes (β1 and β2) couple differentially to intracellular signaling pathways regulating contractility and remodeling. This has led some to suggest that the β1 receptor is the "cardiotoxic subtype" whereas the β2 receptor is "cardioprotective." Given this paradigm, there could be situations where subtype selective β-blockade or even subtype selective β-stimulation might be beneficial. However, since most of these studies have been performed in isolated cardiomyocytes, their application to clinical practice is unclear. To better understand the roles of β1- vs. β2-receptors in the pathogenesis of clinical cardiomyopathy, we and others have taken advantage of several well-characterized murine models of cardiovascular disease. These studies demonstrate that β-receptor regulation of the balance between cardioprotection and cardiotoxicity is even more complex than previously appreciated: the role of each β-receptor subtype may vary depending on the specific cardiac stressor involved (e.g. ischemia, pressure overload, genetic mutation, cardiotoxin). Furthermore, the remodeling effects of β-receptor signaling have a temporal component, depending on whether a cardiac stress is acute vs. chronic.
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http://dx.doi.org/10.1016/j.ppedcard.2010.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135901PMC
January 2011

β2-adrenergic receptors mediate cardioprotection through crosstalk with mitochondrial cell death pathways.

J Mol Cell Cardiol 2011 Nov 2;51(5):781-9. Epub 2011 Jul 2.

Department of Pediatrics (Cardiology), Stanford University, Stanford, CA, USA.

β-adrenergic receptors (β-ARs) modulate cardiotoxicity/cardioprotection through crosstalk with multiple signaling pathways. We have previously shown that β2-ARs are cardioprotective during exposure to oxidative stress induced by doxorubicin (DOX). DOX cardiotoxicity is mediated in part through a Ca(2+)-dependent opening of the mitochondrial permeability transition (MPT), however the signals linking a cell surface receptor like the β2-AR to regulators of mitochondrial function are not clear. The objective of this study was to assess mechanisms of crosstalk between β2-ARs and mitochondrial cell death pathways. DOX administered to WT mice resulted in no acute mortality, however 85% of β2-/- mice died within 30 min. Several pro- and anti-survival pathways were altered. The pro-survival kinase, εPKC, was decreased by 64% in β2-/- after DOX vs WT (p<0.01); the εPKC activator ψεRACK partially rescued these mice (47% reduction in mortality). Activity of the pro-survival kinase Akt decreased by 76% in β2-/- after DOX vs WT (p<0.01). The α1-antagonist prazosin restored Akt activity to normal and also partially reversed the mortality (45%). Deletion of the β2-AR increased rate of Ca(2+) release by 75% and peak [Ca(2+)](i) by 20% respectively in isolated cardiomyocytes; the Ca(2+) channel blocker verapamil also partially rescued the β2-/- (26%). Mitochondrial architecture was disrupted and complex I and II activities decreased by 40.9% and 34.6% respectively after DOX only in β2-/-. The MPT blocker cyclosporine reduced DOX mortality by 41% and prazosin plus cyclosporine acted synergistically to decrease mortality by 85%. β2-ARs activate pro-survival kinases and attenuate mitochondrial dysfunction during oxidative stress; absence of β2-ARs enhances cardiotoxicity via negative regulation of survival kinases and enhancement of intracellular Ca(2+), thus predisposing the mitochondria to opening of the MPT.
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http://dx.doi.org/10.1016/j.yjmcc.2011.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184305PMC
November 2011

Cardiac pressure overload hypertrophy is differentially regulated by β-adrenergic receptor subtypes.

Am J Physiol Heart Circ Physiol 2011 Oct 24;301(4):H1461-70. Epub 2011 Jun 24.

Department of Pediatrics, Stanford University, Stanford, California 94304, USA.

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.
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http://dx.doi.org/10.1152/ajpheart.00453.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197363PMC
October 2011

Endogenous regulation of cardiovascular function by apelin-APJ.

Am J Physiol Heart Circ Physiol 2009 Nov 18;297(5):H1904-13. Epub 2009 Sep 18.

Department of Medicine (Cardiovascular Medicine), Stanford University, Stanford, CA 94305, USA.

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.
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http://dx.doi.org/10.1152/ajpheart.00686.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781363PMC
November 2009

Molecular and physiological characterization of RV remodeling in a murine model of pulmonary stenosis.

Am J Physiol Heart Circ Physiol 2008 Sep 27;295(3):H1351-H1368. Epub 2008 Jun 27.

Department of Pediatrics (Cardiology), Stanford University, Stanford, California, USA.

Right ventricular (RV) dysfunction is a common long-term complication in patients after the repair of congenital heart disease. Previous investigators have examined the cellular and molecular mechanisms of left ventricular (LV) remodeling, but little is known about the stressed RV. Our purpose was to provide a detailed physiological characterization of a model of RV hypertrophy and failure, including RV-LV interaction, and to compare gene alterations between afterloaded RV versus LV. Pulmonary artery constriction was performed in 86 mice. Mice with mild and moderate pulmonary stenosis (PS) developed stable hypertrophy without decompensation. Mice with severe PS developed edema, decreased RV function, and high mortality. Tissue Doppler imaging demonstrated septal dyssynchrony and deleterious RV-LV interaction in the severe PS group. Microarray analysis showed 196 genes with increased expression and 1,114 with decreased expression. Several transcripts were differentially increased in the afterloaded RV but not in the afterloaded LV, including clusterin, neuroblastoma suppression of tumorigenicity 1, Dkk3, Sfrp2, formin binding protein, annexin A7, and lysyl oxidase. We have characterized a murine model of RV hypertrophy and failure, providing a platform for studying the physiological and molecular events of RV remodeling. Although the molecular responses of the RV and LV to afterload stress are mostly concordant, there are several key differences, which may represent targets for RV failure-specific therapy.
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http://dx.doi.org/10.1152/ajpheart.91526.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2544484PMC
September 2008

Differential cardiotoxic/cardioprotective effects of beta-adrenergic receptor subtypes in myocytes and fibroblasts in doxorubicin cardiomyopathy.

J Mol Cell Cardiol 2006 Mar 3;40(3):375-83. Epub 2006 Feb 3.

Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.

beta-Adrenoceptor (beta-AR) subtypes act through different signaling pathways to regulate cardiac function and remodeling. Previous in vivo data show a markedly enhanced cardiotoxic response to doxorubicin in beta2-/- mice, which is rescued by the additional deletion of the beta1-AR. We determined whether this differential response was myocyte specific by examining the effects of doxorubicin in myocytes and fibroblasts from WT and beta1, beta2 and beta1/beta2-/- mice. Cells were exposed to doxorubicin at 1-50 microM and viability and apoptosis assessed at 6, 24 and 48 h. WT myocytes showed a time and dose-dependent decrease in viability (42% decrease at 1 microM after 24 h). beta2-/- Myocytes showed a greater decrease in viability vs. WT (20.8% less at 6 h; 14% less at 24 h, P<0.05); beta1-/- and beta1/beta2-/- myocytes showed enhanced survival (beta1-/- 11%; beta1/beta2-/- 18% greater than WT, P<0.05). TUNEL staining demonstrated a similar differential susceptibility (WT 26% apoptotic nuclei, beta2-/- 45.9%, beta1/beta2-/- 16.8%, P<0.05). beta2-/- Fibroblasts also showed enhanced toxicity. Pertussis toxin pretreatment of WT cells decreased survival similar to the beta2-/-, suggesting a role for Gi signaling. JNK was differentially activated in beta2-/- myocytes after doxorubicin and its inhibition increased cardiotoxicity. In conclusion, the differential cardioprotective/cardiotoxic effects mediated by beta1 vs. beta2-AR subtypes in knockout mice are recapitulated in myocytes isolated from these mice. beta2-ARs appear to play a cardioprotective role, whereas beta1-ARs a cardiotoxic role.
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http://dx.doi.org/10.1016/j.yjmcc.2005.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3140223PMC
March 2006

Differential cardioprotective/cardiotoxic effects mediated by beta-adrenergic receptor subtypes.

Am J Physiol Heart Circ Physiol 2005 Dec 22;289(6):H2441-9. Epub 2005 Jul 22.

Dept. of Pediatrics, 750 Welch Rd., Suite 305, Palo Alto, CA 94304, USA.

Recent data suggest that beta-adrenergic receptor subtypes couple differentially to signaling pathways regulating cardiac function vs. cardiac remodeling. To dissect the roles of beta1- vs. beta2-receptors in the pathogenesis of cardiomyopathy, doxorubicin was administered to beta1, beta2, and beta1/beta2 knockout (-/-) and wild-type mice. Expression and activation of MAPKs were measured. Wild-type and beta1-/- mice showed no acute cardiovascular effects, whereas beta2-/- mice all died within 30 min. The additional deletion of the beta1-receptor (beta1/beta2-/-) totally rescued this toxicity. beta2-/- mice developed decreased contractile function, hypotension, QTc prolongation, and ST segment changes and a 20-fold increase in p38 MAPK activity not seen in the other genotypes. The MAPK inhibitor SB-203580 rescued beta2-/- mice from this acute toxicity. The enhanced toxicity in beta2-/- mice was also recapitulated in wild-type mice with the beta2-selective antagonist ICI-118,551, although the rescue effect of the beta1-deletion was not recapitulated using the beta1-selective antagonist metoprolol or the nonselective beta-antagonist propranolol. These data suggest that beta2-adrenergic receptors play a cardioprotective role in the pathogenesis of cardiomyopathy, whereas beta1-adrenergic receptors mediate at least some of the acute cardiotoxicity of anthracyclines. Differential activation of MAPK isoforms, previously shown in vitro to regulate beta-agonist as well as doxorubicin cardiotoxicity, appears to play a role in mediating the differential effects of these beta-adrenergic receptor subtypes in vivo.
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http://dx.doi.org/10.1152/ajpheart.00005.2005DOI Listing
December 2005

Microarray analysis of gene expression after transverse aortic constriction in mice.

Physiol Genomics 2004 Sep 3;19(1):93-105. Epub 2004 Aug 3.

Department of Pediatrics, Stanford University, Stanford, California 94304, USA.

Cardiac hypertrophy is a compensatory response initially beneficial to heart function but can ultimately lead to cardiac decompensation. It is an integrated process involving multiple cellular signaling pathways and their cross talk. Microarray GeneChip technology is a powerful new tool to identify gene expression profiles of cardiac hypertrophy. To identify well-characterized as well as novel adaptive mechanisms, we utilized a murine model of compensated pressure overload hypertrophy (transverse aortic constriction, TAC). At 48 h, 10 days, and 3 wk, hearts were harvested and total RNA hybridized to Affymetrix U74Av2 GeneChips, which contain a 12,488-gene/EST probe set. Verification of gene expression was performed by SYBR quantitative real-time RT-PCR (QRT-PCR) for selected genes. A rigorous evaluation of the adequacy of the control condition was also performed. For statistical analysis we generated a four-step filtering criteria. Our results show an upregulation of 38 genes (48 h), 269 genes (10 days), and 203 genes (3 wk) and downregulation of 15 genes (48 h), 160 genes (10 days), and 124 genes (3 wk). Transcripts differentially expressed after TAC were categorized into 12 functional groups and revealed the presence of several intriguing transcripts, e.g., cell proliferation-related Ki-67 and several apoptosis-related genes. Overall changes in QRT-PCR were in accordance with GeneChip data, with the highest correlation for genes with the largest up- or downregulation with TAC. Thus TAC results in altered expression of genes in several pathways regulating both cardiac structure and function. However, for in vivo gene microarray experiments, it is critical to define adequate controls, perform rigorous statistical analysis, and provide validation by alternative methods.
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http://dx.doi.org/10.1152/physiolgenomics.00040.2004DOI Listing
September 2004
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