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Myocardial edema is a consequence of many cardiovascular stressors, including myocardial infarction, cardiac bypass surgery, and hypertension. The aim of this study was to establish a murine model of myocardial edema and elucidate the response of cardiac lymphatics and the myocardium. Myocardial edema without infarction was induced in mice by cauterizing the coronary sinus, increasing pressure in the coronary venous system, and inducing myocardial edema. In male mice, there was rapid development of edema 3 h following coronary sinus cauterization (CSC), with associated dilation of cardiac lymphatics. By 24 h, males displayed significant cardiovascular contractile dysfunction. In contrast, female mice exhibited a temporal delay in the formation of myocardial edema, with onset of cardiovascular dysfunction by 24 h. Furthermore, myocardial edema induced a ring of fibrosis around the epicardial surface of the left ventricle in both sexes that included fibroblasts, immune cells, and increased lymphatics. Interestingly, the pattern of fibrosis and the cells that make up the fibrotic epicardial ring differ between sexes. We conclude that a novel surgical model of myocardial edema without infarct was established in mice. Cardiac lymphatics compensated by exhibiting both an acute dilatory and chronic growth response. Transient myocardial edema was sufficient to induce a robust epicardial fibrotic and inflammatory response, with distinct sex differences, which underscores the sex-dependent differences that exist in cardiac vascular physiology. NEW & NOTEWORTHY Myocardial edema is a consequence of many cardiovascular stressors, including myocardial infarction, cardiac bypass surgery, and high blood pressure. Cardiac lymphatics regulate interstitial fluid balance and, in a myocardial infarction model, have been shown to be therapeutically targetable by increasing heart function. Cardiac lymphatics have only rarely been studied in a noninfarct setting in the heart, and so we characterized the first murine model of increased coronary sinus pressure to induce myocardial edema, demonstrating distinct sex differences in the response to myocardial edema. The temporal pattern of myocardial edema induction and resolution is different between males and females, underscoring sex-dependent differences in the response to myocardial edema. This model provides an important platform for future research in cardiovascular and lymphatic fields with the potential to develop therapeutic interventions for many common cardiovascular diseases.

The complex and inaccessible space radiation environment poses an unresolved risk to astronaut cardiovascular health during long-term space exploration missions. To model this risk, healthy male c57BL/6 mice aged six months (corresponding to an astronaut of 34 years) were exposed to simplified galactic cosmic ray (GCR

This paper analyses the unusual mal-operation of the differential protection for transformer current differential protection during the external fault. The mal-operation reason is researched in detail, based on the mathematical analysis, the simulation, and the field recorded fault data analysis. It is disclosed that the CT saturation leads to the maloperation. Based on this, this paper analyses the improvement for the CT saturation identification method, and discusses the CT selection principles. This paper is aimed at attracting the attention on the problem of the CT saturation and proposing the reference of the analysis method the countermeasure for this problem.

Background and purpose Late cardiac toxicity is a major side effect of radiation therapy (RT) for breast cancer. We developed and characterized a mouse model of radiation-induced heart disease that mimics the dose, fractionation, and beam arrangement of left breast and chest wall RT. Material and methods Female wild-type (C57BL6/J) and atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice (on a C57BL/6J background) on regular chow were treated with 2 Gy × 25 fractions of partial-heart irradiation via opposed tangential beams to the left chest wall. The changes in myocardial perfusion and cardiac function of C57BL/6J mice were examined by single-photon emission computed tomography (SPECT) and echocardiography, respectively. In addition to SPECT and echocardiography, the formation of calcified plaques and changes in cardiac function of ApoE−/− mice were examined by dual-energy microCT (DE-CT) and pressure–volume (PV) loop analysis, respectively. The development of myocardial fibrosis was examined by histopathology. Results Compared to unirradiated controls, irradiated C57BL/6J mice showed no significant changes by SPECT or echocardiography up to 18 months after 2 Gy × 25 partial-heart irradiation even though irradiated mice exhibited a modest increase in myocardial fibrosis. For ApoE−/− mice, 2 Gy × 25 partial-heart irradiation did not cause significant changes by SPECT, DE-CT, or echocardiography. However, PV loop analysis revealed a significant decrease in load-dependent systolic and diastolic function measures including cardiac output, dV/dtmax and dV/dt min 12 months after RT. Conclusions Following clinically relevant doses of partial-heart irradiation in C57BL/6J and ApoE−/− mice, assessment with noninvasive imaging modalities such as echocardiography, SPECT, and DE-CT yielded no evidence of decreased myocardial perfusion and cardiac dysfunction related to RT. However, invasive hemodynamic assessment with PV loop analysis indicated subtle, but significant, changes in cardiac function of irradiated ApoE−/− mice. PV loop analysis may be useful for future preclinical studies of radiation-induced heart disease, especially if subtle changes in cardiac function are expected.

Reversible ubiquitination of G protein–coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial β1-adrenergic receptors (β1ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the β1AR. β1AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of β1AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the β1AR and failed to deubiquitinate the β1AR. USP20–KO mice showed normal baseline systolic function but impaired β1AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20–KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on β1AR ubiquitination, but USP33 was up-regulated in USP20–KO hearts suggesting compensatory regulation. Myocardial β1AR expression in USP20–KO was drastically reduced, whereas β2AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in β1AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates β1AR signaling in vitro and in vivo. Additionally, β1AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize β1AR expression and signaling during pathological insults to the myocardium.

A decrease in skeletal muscle strength and functional exercise capacity due to aging, frailty, and muscle wasting poses major unmet clinical needs. These conditions are associated with numerous adverse clinical outcomes including falls, fractures, and increased hospitalization. Clenbuterol, a β(2)-adrenergic receptor (β(2)AR) agonist enhances skeletal muscle strength and hypertrophy; however, its clinical utility is limited by side effects such as cardiac arrhythmias mediated by G protein signaling. We recently reported that clenbuterol-induced increases in contractility and skeletal muscle hypertrophy were lost in β-arrestin 1 knockout mice, implying that arrestins, multifunctional adapter and signaling proteins, play a vital role in mediating the skeletal muscle effects of β(2)AR agonists. Carvedilol, classically defined as a βAR antagonist, is widely used for the treatment of chronic systolic heart failure and hypertension, and has been demonstrated to function as a β-arrestin-biased ligand for the β(2)AR, stimulating β-arrestin-dependent but not G protein-dependent signaling. In this study, we investigated whether treatment with carvedilol could enhance skeletal muscle strength via β-arrestin-dependent pathways. In a murine model, we demonstrate chronic treatment with carvedilol, but not other β-blockers, indeed enhances contractile force in skeletal muscle and this is mediated by β-arrestin 1. Interestingly, carvedilol enhanced skeletal muscle contractility despite a lack of effect on skeletal muscle hypertrophy. Our findings suggest a potential unique clinical role of carvedilol to stimulate skeletal muscle contractility while avoiding the adverse effects with βAR agonists. This distinctive signaling profile could present an innovative approach to treating sarcopenia, frailty, and secondary muscle wasting.

Progesterone is primarily a pregnancy-related hormone, produced in substantial quantities after ovulation and during gestation. Traditionally known to function via nuclear receptors for transcriptional regulation, there is also evidence of nonnuclear action. A previously identified mitochondrial progesterone receptor (PR-M) increases cellular respiration in cell models. In these studies, we demonstrated that expression of PR-M in rat H9c2 cardiomyocytes resulted in a ligand-dependent increase in oxidative cellular respiration and beta-oxidation. Cardiac expression in a TET-On transgenic mouse resulted in gene expression of myofibril proteins for remodeling and proteins involved in oxidative phosphorylation and fatty acid metabolism. In a model of increased afterload from constant transverse aortic constriction, mice expressing PR-M showed a ligand-dependent preservation of cardiac function. From these observations, we propose that PR-M is responsible for progesterone-induced increases in cellular energy production and cardiac remodeling to meet the physiological demands of pregnancy.

Cardiac two-pore domain potassium channels (K2P) exist in organisms from Drosophila to humans; however, their role in cardiac function is not known. We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK-related potassium channel (TREK-1, also known Kcnk2), exhibit exaggerated pressure overload-induced concentric hypertrophy and alterations in fetal gene expression, yet retain preserved systolic and diastolic cardiac function. While cardiomyocyte-specific deletion of TREK-1 in response to in vivo pressure overload resulted in cardiac dysfunction, TREK-1 deletion in fibroblasts prevented deterioration in cardiac function. The absence of pressure overload-induced dysfunction in TREK-1-KO mice was associated with diminished cardiac fibrosis and reduced activation of JNK in cardiomyocytes and fibroblasts. These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload-induced cardiac dysfunction and serve as a conceptual basis for its inhibition as a potential therapy.

An increasing body of evidence suggests that bone marrow–derived myeloid cells play a critical role in the pathophysiology of pulmonary hypertension (PH). However, the true requirement for myeloid cells in PH development has not been demonstrated, and a specific disease-promoting myeloid cell population has not been identified. Using bone marrow chimeras, lineage labeling, and proliferation studies, we determined that, in murine hypoxia-induced PH, Ly6Clo nonclassical monocytes are recruited to small pulmonary arteries and differentiate into pulmonary interstitial macrophages. Accumulation of these nonclassical monocyte–derived pulmonary interstitial macrophages around pulmonary vasculature is associated with increased muscularization of small pulmonary arteries and disease severity. To determine if the sensing of hypoxia by nonclassical monocytes contributes to the development of PH, mice lacking expression of hypoxia-inducible factor-1α in the Ly6Clo monocyte lineage were exposed to hypoxia. In these mice, vascular remodeling and PH severity were significantly reduced. Transcriptome analyses suggest that the Ly6Clo monocyte lineage regulates PH through complement, phagocytosis, Ag presentation, and chemokine/cytokine pathways. Consistent with these murine findings, relative to controls, lungs from pulmonary arterial hypertension patients displayed a significant increase in the frequency of nonclassical monocytes. Taken together, these findings show that, in response to hypoxia, nonclassical monocytes in the lung sense hypoxia, infiltrate small pulmonary arteries, and promote vascular remodeling and development of PH. Our results demonstrate that myeloid cells, specifically cells of the nonclassical monocyte lineage, play a direct role in the pathogenesis of PH.

In response to heart injury, inflammation, or mechanical overload, quiescent cardiac fibroblasts (CFs) can become activated myofibroblasts leading to pathological matrix remodeling and decline in cardiac function. Specific targeting of fibroblasts may thus enable new therapeutic strategies to delay or reverse the progression of heart failure and cardiac fibrosis. However, it remains unknown if all CFs are equally responsive to specific pathological insults and if there exist sub-populations of resident fibroblasts in the heart that have distinctive pathogenic phenotypes. Here, we show that in response to transverse aortic constriction (TAC)-induced heart failure, previously uncharacterized Thy1neg (Thy1-/MEFSK4+/CD45-/CD31-) fraction of mouse ventricular fibroblasts became more abundant and attained a more activated, pro-fibrotic myofibroblast phenotype compared to Thy1Pos fraction. In a tissue-engineered 3D co-culture model of healthy cardiomyocytes and freshly isolated CFs, Thy1neg CFs from TAC hearts significantly decreased cardiomyocyte contractile function and calcium transient amplitude, and increased extracellular collagen deposition yielding a profibrotic heart tissue phenotype. In vivo, mice with global knockout of Thy1 developed more severe cardiac dysfunction and fibrosis in response to TAC-induced heart failure than wild-type mice. Taken together, our studies identify cardiac myofibroblasts lacking Thy1 as a pathogenic CF fraction in cardiac fibrosis and suggest important roles of Thy1 in pathophysiology of heart failure.

Objective: To explore the differences in the effect of peroral traction-assisted endoscopic submucosal dissection (ESD) and traditional ESD for the treatment of early gastric cancer and precancerous lesions. Methods: 112 patients with early gastric cancer and precancerous lesions treated in our hospital between May 2013 and May 2016 were collected and divided into control group and observation group according to the random number table (n=56). Observation group of patients received peroral traction-assisted ESD, and the control group of patients only received traditional ESD. The intraoperative dissected lesion diameter, mean operation time and intraoperative blood loss of two groups of patients were recorded, enzyme-linked immunosorbent assay was used to detect serum inflammatory factor levels, and RIA was used to detect serum stress hormone levels. Results: Dissected lesion diameter of observation group was greater than that of control group (P

The Frank-Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank-Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca2+ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca2+ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein β-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank-Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking β-arrestin 1, β-arrestin 2, or AT1R were unable to generate a Frank-Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a β-arrestin-biased AT1R ligand to selectively activate β-arrestin signaling preserved the Frank-Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca2+ sensitivity in β-arrestin 1, β-arrestin 2, and AT1R knockout mice. Our data reveal β-arrestin 1, β-arrestin 2, and AT1R as key regulatory molecules in the Frank-Starling mechanism, which potentially can be targeted therapeutically with β-arrestin-biased AT1R ligands.

Background β2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth. Methods The contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak’s or Tukey’s multiple comparison test and the Student’s t test. Results Chronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1. Conclusions We have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting. Electronic supplementary material The online version of this article (10.1186/s13395-018-0184-8) contains supplementary material, which is available to authorized users.

Reversible ubiquitination of G protein–coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial β(1)-adrenergic receptors (β(1)ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the β(1)AR. β(1)AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of β(1)AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the β(1)AR and failed to deubiquitinate the β(1)AR. USP20–KO mice showed normal baseline systolic function but impaired β(1)AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20–KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on β(1)AR ubiquitination, but USP33 was up-regulated in USP20–KO hearts suggesting compensatory regulation. Myocardial β(1)AR expression in USP20–KO was drastically reduced, whereas β(2)AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in β(1)AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates β(1)AR signaling in vitro and in vivo. Additionally, β(1)AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize β(1)AR expression and signaling during pathological insults to the myocardium.

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.

In mitochondria, the sirtuin SIRT5 is an NAD+-dependent protein deacylase that controls several metabolic pathways. Although a wide range of SIRT5 targets have been identified, the overall function of SIRT5 in organismal metabolic homeostasis remains unclear. Given that SIRT5 expression is highest in the heart and that sirtuins are commonly stress-response proteins, we used an established model of pressure overload-induced heart muscle hypertrophy caused by transverse aortic constriction (TAC) to determine SIRT5's role in cardiac stress responses. Remarkably, SIRT5KO mice had reduced survival upon TAC compared with wild-type mice but exhibited no mortality when undergoing a sham control operation. The increased mortality with TAC was associated with increased pathological hypertrophy and with key abnormalities in both cardiac performance and ventricular compliance. By combining high-resolution MS-based metabolomic and proteomic analyses of cardiac tissues from wild-type and SIRT5KO mice, we found several biochemical abnormalities exacerbated in the SIRT5KO mice, including apparent decreases in fatty acid oxidation and glucose oxidation as well as an overall decrease in mitochondrial NAD+/NADH. Together, these abnormalities suggest that SIRT5 deacylates protein substrates involved in cellular oxidative metabolism to maintain mitochondrial energy production. Overall, the functional and metabolic results presented here suggest an accelerated development of cardiac dysfunction in SIRT5KO mice in response to TAC, explaining increased mortality upon cardiac stress. Our findings reveal a key role for SIRT5 in maintaining cardiac oxidative metabolism under pressure overload to ensure survival.

Increasing NAD+ levels by supplementing with the precursor nicotinamide mononucleotide (NMN) improves cardiac function in multiple mouse models of disease. While NMN influences several aspects of mitochondrial metabolism, the molecular mechanisms by which increased NAD+ enhances cardiac function are poorly understood. A putative mechanism of NAD+ therapeutic action exists via activation of the mitochondrial NAD+-dependent protein deacetylase sirtuin 3 (SIRT3). We assessed the therapeutic efficacy of NMN and the role of SIRT3 in the Friedreich's ataxia cardiomyopathy mouse model (FXN-KO). At baseline, the FXN-KO heart has mitochondrial protein hyperacetylation, reduced Sirt3 mRNA expression, and evidence of increased NAD+ salvage. Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels. To determine whether SIRT3 is required for NMN therapeutic efficacy, we generated SIRT3-KO and SIRT3-KO/FXN-KO (double KO [dKO]) models. The improvement in cardiac function upon NMN treatment in the FXN-KO is lost in the dKO model, demonstrating that the effects of NMN are dependent upon cardiac SIRT3. Coupled with cardio-protection, SIRT3 mediates NMN-induced improvements in both cardiac and extracardiac metabolic function and energy metabolism. Taken together, these results serve as important preclinical data for NMN supplementation or SIRT3 activator therapy in Friedreich's ataxia patients.

In response to chronic pressure overload, the heart undergoes pathological hypertrophy associated with adverse remodeling, fibrosis, and cardiac dysfunction. Caveolae, membrane invaginations that organize protective signaling pathways and provide a reservoir to buffer membrane tension, can ameliorate maladaptive hypertrophy. However, the factors that govern caveolae assembly in the heart are not fully understood. Here, we report that the fibroblast growth factor (FGF) homologous factor FGF13 is a negative regulator of caveolae density in cardiomyocytes. Adult mice lacking FGF13 in the heart have increased caveolae abundance in cardiomyocytes and are thereby protected from the pathological effects of chronic pressure overload. Our findings, identifying an unexpected role for FGF13, provide insight into the mechanisms underlying caveolae-mediated adaptation to cardiac stress.

The fibroblast growth factor (FGF) homologous factor FGF13, a noncanonical FGF, has been best characterized as a voltage-gated Na+ channel auxiliary subunit. Other cellular functions have been suggested, but not explored. In inducible, cardiac-specific Fgf13 knockout mice, we found-even in the context of the expected reduction in Na+ channel current-an unanticipated protection from the maladaptive hypertrophic response to pressure overload. To uncover the underlying mechanisms, we searched for components of the FGF13 interactome in cardiomyocytes and discovered the complete set of the cavin family of caveolar coat proteins. Detailed biochemical investigations showed that FGF13 acts as a negative regulator of caveolae abundance in cardiomyocytes by controlling the relative distribution of cavin 1 between the sarcolemma and cytosol. In cardiac-specific Fgf13 knockout mice, cavin 1 redistribution to the sarcolemma stabilized the caveolar structural protein caveolin 3. The consequent increase in caveolae density afforded protection against pressure overload-induced cardiac dysfunction by two mechanisms: (i) enhancing cardioprotective signaling pathways enriched in caveolae, and (ii) increasing the caveolar membrane reserve available to buffer membrane tension. Thus, our results uncover unexpected roles for a FGF homologous factor and establish FGF13 as a regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling.

The graphene oxide (GO) nanocoating surface was fabricated by GO nanoparticles self-assembly on plain copper surface under nucleate pool boiling. Saturated and subcooled pool boiling experiments on GO nanocoating surface with distilled water at atmospheric pressure were conducted. Saturated pool boiling performance indicated enhancements of 78% in critical heat flux (CHF) and 41% in maximum heat transfer coefficient (HTC) when compared with the plain copper surface. It was found that the enhancement was mainly attributed to the improved wettability and high thermal conductivity of GO nanocoating, as well as the increased surface roughness. Subcooled pool boiling results showed that the liquid subcooling had significant effect on heat transfer performance of GO nanocoating surface. The CHF increased linearly with the increased subcooling degree, and the maximum HTC increased almost linearly with the increased subcooling degree. The CHF reached 274 W/cm2 at subcooling degree of 19 K, which translated an enhancement of 128% when compared with the plain copper surface. This high CHF can address the heat dissipation bottleneck in ultra high-power density electronic devices. To better understand the subcooling effect on nucleate boiling, visualization studies on bubble growth characteristics at subcooled pool boiling were investigated with a high-speed digital camera. It was shown that bubbles were smaller and grew more slowly with the increase of liquid subcooling, and microbubble jets were observed at moderate heat flux regime.

Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

Noninvasive small animal imaging techniques are essential for evaluation of cardiac disease and potential therapeutics. A novel preclinical iodinated contrast agent called eXIA 160 has recently been developed, which has been evaluated for micro-CT cardiac imaging. eXIA 160 creates strong contrast between blood and tissue immediately after its injection and is subsequently taken up by the myocardium and other metabolically active tissues over time. We focus on these properties of eXIA and show its use in imaging myocardial infarction in mice. Five C57BL/6 mice were imaged ~2 weeks after left anterior descending coronary artery ligation. Six C57BL/6 mice were used as controls. Immediately after injection of eXIA 160, an enhancement difference between blood and myocardium of ~340 HU enabled cardiac function estimation via 4D micro-CT scanning with retrospective gating. Four hours post-injection, the healthy perfused myocardium had a contrast difference of ~140 HU relative to blood while the infarcted myocardium showed no enhancement. These differences allowed quantification of infarct size via dual-energy micro-CT. In vivo micro-SPECT imaging and ex vivo triphenyl tetrazolium chloride (TTC) staining provided validation for the micro-CT findings. Root mean squared error of infarct measurements was 2.7% between micro-CT and SPECT, and 4.7% between micro-CT and TTC. Thus, micro-CT with eXIA 160 can be used to provide both morphological and functional data for preclinical studies evaluating myocardial infarction and potential therapies. Further studies are warranted to study the potential use of eXIA 160 as a CT molecular imaging tool for other metabolically active tissues in the mouse. Copyright © 2014 John Wiley & Sons, Ltd.

In pulmonary arterial hypertension (PAH), there is overexpression of the chemokine, C-C chemokine ligand type 2 (CCL2), and infiltration of myeloid cells into the pulmonary vasculature. Inhibition of CCL2 in animals decreases PAH, suggesting that the CCL2 receptor (CCR2) plays a role in PAH development. To test this hypothesis, we exposed wild-type (WT) and CCR2-deficient (Ccr2(-/-)) mice to chronic hypobaric hypoxia to induce PAH. After hypoxic stress, Ccr2(-/-) mice displayed a more severe PAH phenotype, as demonstrated by increased right ventricular (RV) systolic pressures, RV hypertrophy, and tachycardia relative to WT mice. However, these mice also exhibited increased RV systolic pressures and increased pulmonary artery muscularization under normoxic conditions. Moreover, Ccr2(-/-) mice displayed decreased pulmonary vascular branching at 3 weeks of age and increased vascular muscularization at birth, suggesting that an abnormality in pulmonary vascular development leads to spontaneous PAH in these animals. No significant differences in cytokine responses were observed between WT and Ccr2(-/-) mice during either normoxia or hypoxia. However, Ccr2(-/-) mice displayed increased Notch-3 signaling and dysregulated Notch ligand expression, suggesting a possible cause for their abnormal pulmonary vascular development. Our findings imply that CCR2 does not directly contribute to the development of PAH, but does play a previously unrecognized role in pulmonary vasculature development and remodeling wherein the absence of CCR2 results in spontaneous PAH, most likely via dysregulation of Notch signaling. Our results demonstrate that CCR2 has impacts beyond leukocyte recruitment, and is required for the proper expression of Notch signaling molecules.

Cardiac troponin I-interacting kinase (TNNI3K) is a cardiac-specific kinase whose biological function remains largely unknown. We have recently shown that TNNI3K expression greatly accelerates cardiac dysfunction in mouse models of cardiomyopathy, indicating an important role in modulating disease progression. To further investigate TNNI3K kinase activity in vivo, we have generated transgenic mice expressing both wild-type and kinase-dead versions of the human TNNI3K protein. Importantly, we show that the increased TNNI3K kinase activity induces mouse cardiac remodeling, and its kinase activity promotes accelerated disease progression in a left-ventricular pressure overload model of mouse cardiomyopathy. Using an in vitro kinase assay and proteomics analysis, we show that TNNI3K is a dual-function kinase with Tyr and Ser/Thr kinase activity. TNNI3K expression induces a series of cellular and molecular changes, including a reduction of sarcomere length and changes in titin isoform composition, which are indicative of cardiac remodeling. Using antisera to TNNI3K, we show that TNNI3K protein is located at the sarcomere Z disc. These combined data suggest that TNNI3K mediates cell signaling to modulate cardiac response to stress.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pharmacological blockade of the ANG II type 1 receptor (AT1R) is a common therapy for treatment of congestive heart failure and hypertension. Increasing evidence suggests that selective engagement of β-arrestin-mediated AT1R signaling, referred to as biased signaling, promotes cardioprotective signaling. Here, we tested the hypothesis that a β-arrestin-biased AT1R ligand TRV120023 would confer cardioprotection in response to acute cardiac injury compared with the traditional AT1R blocker (ARB), losartan. TRV120023 promotes cardiac contractility, assessed by pressure-volume loop analyses, while blocking the effects of endogenous ANG II. Compared with losartan, TRV120023 significantly activates MAPK and Akt signaling pathways. These hemodynamic and biochemical effects were lost in β-arrestin-2 knockout (KO) mice. In response to cardiac injury induced by ischemia reperfusion injury or mechanical stretch, pretreatment with TRV120023 significantly diminishes cell death compared with losartan, which did not appear to be cardioprotective. This cytoprotective effect was lost in β-arrestin-2 KO mice. The β-arrestin-biased AT1R ligand, TRV120023, has cardioprotective and functional properties in vivo, which are distinct from losartan. Our data suggest that this novel class of drugs may provide an advantage over conventional ARBs by supporting cardiac function and reducing cellular injury during acute cardiac injury.

2-Mesitylindenyl phosphine ligand (1) and [(2-mesitylindenyl)dicyclohexyl-phosphine]PdCl2 (2) have been synthesized and fully characterized by NMR and elemental analysis, as well as by X-ray crystallography for 2. A Highly active catalyst system derived from a palladium precatalyst and bulky 2-mesitylindenyl phosphine ligand (1) for the Buchwald–Hartwig amination reaction of aryl halides with primary and secondary amines has been developed. This method allows for the preparation of a wide variety of amines in moderate to excellent yields and displays a high level of activity for the coupling of aryl chlorides as well as hindered aryl bromides.

Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector.

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.

Secreted frizzled related protein 2 (Sfrp2) is known as an inhibitor for the Wnt signaling. In recent studies, Sfrp2 has been reported to inhibit the activity of Xenopus homolog of mammalian Tolloid-like 1 metalloproteinase. Bone morphogenic protein 1 (Bmp1)/Tolloid-like metalloproteinase plays a key role in the regulation of collagen biosynthesis and maturation after tissue injury. Here, we showed both endogenous Sfrp2 and Bmp1 protein expressions were up-regulated in rat heart after myocardial infarction (MI). We hypothesize that Sfrp2 could inhibit mammalian Bmp1 activity and, hence, the exogenous administration of Sfrp2 after MI would inhibit the deposition of mature collagen and improve heart function. Using recombinant proteins, we demonstrated that Sfrp2, but not Sfrp1 or Sfrp3, inhibited Bmp1 activity in vitro as measured by a fluorogenic peptide based procollagen C-proteinase activity assay. We also demonstrated that Sfrp2 at high concentration inhibited human and rat type I procollagen processing by Bmp1 in vitro. We further showed that exogenously added Sfrp2 inhibited type I procollagen maturation in primary cardiac fibroblasts. Two days after direct injection into the rat infarcted myocardium, Sfrp2 inhibited MI-induced type I collagen deposition. As early as 2 wk after injection, Sfrp2 significantly reduced left ventricular (LV) fibrosis as shown by trichrome staining. Four weeks after injection, Sfrp2 prevented the anterior wall thinning and significantly improved cardiac function as revealed by histological analysis and echocardiographic measurement. Our study demonstrates Sfrp2 at therapeutic doses can inhibit fibrosis and improve LV function at a later stage after MI.

Rationale: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein–coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. Objective: Transient receptor potential canonical (TRPC) channels are G protein–coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1 − / − mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1 − / − mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1 − / − mice. Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

Reduced sinusoidal endothelial nitric oxide (NO) production contributes to increased intrahepatic resistance and portal hypertension after liver injury. We hypothesized that V-PYRRO/NO, an NO donor prodrug metabolized “specifically” in the liver, would reduce portal venous pressure (PVP) without affecting the systemic vasculature. Liver injury was induced in male BALB/c mice by weekly CCl4gavage. PVP and mean arterial pressure were recorded during intravenous administration of V-PYRRO/NO. In vivo microscopy was used to monitor sinusoidal diameter and flow during drug administration. Mean PVP was increased in CCl4-treated mice compared with sham-treated mice. In dose-response experiments, the minimum dose of PYRRO/NO required to acutely lower PVP by 20%, the amount believed to yield a clinically meaningful outcome, was 200 nmol/kg. This dose decreased portal pressure in cirrhotic (23.4 ± 2.0%, P < 0.001 vs. vehicle) and sham-treated (19.5 ± 2.3%, P < 0.001 vs. vehicle) animals by a similar magnitude. This concentration also led to dilation of hepatic sinusoids and an increase in sinusoidal volumetric flow, consistent with a reduction of intrahepatic resistance. The effect of V-PYRRO/NO on mean arterial pressure was significant at all concentrations tested, including the lowest, 30 nmol/kg ( P < 0.001 vs. vehicle for all doses). We conclude that V-PYRRO/NO had widespread vascular effects and, as such, is unlikely to be suitable for treatment of portal hypertension. As the potential of this or other similar compounds for treatment of portal hypertension is evaluated, effects on the systemic vasculature will also need to be considered.

In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gsand Gi, the functional role of Gois still poorly defined. To investigate the role of Gαoin the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of Gαo1* (Gαo*), the predominant Gαoisoform in the heart. Gαoexpression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in Gαo* transgenic mice in vivo, along with increased L-type Ca2+channel current density, calcium transients, and cell shortening in ventricular Gαo*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in Gαo* ventricles. Density and affinity of β-adrenoceptors, cAMP levels, and PKA activity were comparable in Gαo* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon Gαo* expression, particularly in the vicinity of the ryanodine receptor. We conclude that Gαo* exerts a positive effect on Ca2+cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+handling proteins in hearts with constitutive Gαoactivation.

The “brain-gut” peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. Additionally, the GHSR1a signals through βarrestin-2 to regulate actin/stress fiber rearrangement, suggesting βarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific βarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The βarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for βarrestin-2 and show that pharmacological inhibition of βarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of βarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate βarrestin-2 activity differentially from G protein activity may be required.

Oxytocin is a potent uterotonic agent and is used clinically for induction and augmentation of labor, as well as for prevention and treatment of postpartum hemorrhage. Oxytocin increases uterine contractility by activating the oxytocin receptor (OXTR), a member of the G protein-coupled receptor family, which is prone to molecular desensitization. After oxytocin binding, the OXTR is phosphorylated by a member of the G protein-coupled receptor kinase (GRK) family, which allows for recruitment of β-arrestin, receptor internalization, and desensitization. According to previous in vitro analyses, desensitization of calcium signaling by the OXTR is mediated by GRK6. The objective of this study was to determine the role of GRK6 in mediating uterine contractility. Here, we demonstrate that uterine GRK6 levels increase in pregnancy and using a telemetry device to measure changes in uterine contractility in live mice during labor, show that mice lacking GRK6 produce a phenotype of enhanced uterine contractility during both spontaneous and oxytocin-induced labor compared with wild-type or GRK5 knockout mice. In addition, the observed enhanced contractility was associated with high rates of term stillbirth. Lastly, using a heterologous in vitro model, we show that β-arrestin recruitment to the OXTR, which is necessary for homologous OXTR desensitization, is dependent on GRK6. Our findings suggest that GRK6-mediated OXTR desensitization in labor is necessary for normal uterine contractile patterns and optimal fetal outcome.

Introduction: Resident c-Kit + cardiac progenitor cells (CPCs) differentiation into cardiomyocytes in vivo is controversial. Using lineage tracing and gene ablation techniques we documented that the Wnt signaling modulator Sfrp2 substantially increased differentiation of c-Kit + CPCs into cardiomyocytes in post-MI heart and restored cardiac function. Methods: c-Kit-CreERT2/mTeG mice were used where the c-Kit promoter drives expression of a tamoxifen (TX)-inducible Cre. Upon TX treatment Cre-mediated recombination drives expression of eGFP exclusively in c-Kit cells. Mice were crossed with a Diphtheria toxin receptor (DT) strain to selectively ablate heart c-Kit cells. Mice were subjected to myocardial infarction. 2 days later, mice were injected with Sfrp2 (0.5 μg) or saline at the infarct border zone. Heart tissue was analyzed 2 months post-MI for eGFP expression and various markers by microscopy. Results: c-Kit-CreERT2/mTeG model was tested. eGFP expression had no effect on in vitro c-Kit CPC differentiation or proliferation. In TX treated mice, >90% of c-Kit+ cells were eGFP+. No eGFP+ c-Kit+ cells were seen in vehicle-only mice (N=6, p95% N=4, P=0.01). Sfrp2 induction of c-Kit differentiation to cardiomyocytes and functional improvements were completely ablated by DT. Conclusion: Using c-Kit lineage-tracing and genetic ablation we have identified that these cells are important in cardiomyocyte generation in vivo in response to Sfrp2 and improves cardiac function following MI.

SummaryBoth iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

Cardiac pressure-volume loop analysis is the “gold-standard” in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.