Regulation Of Cardiac Function Research Articles

Calcium Signalling in Heart and Vessels: Role of Calmodulin and Downstream Calmodulin-Dependent Protein Kinases.

Cardiovascular disease is the major cause of death worldwide. The success of medication and other preventive measures introduced in the last century have not yet halted the epidemic of cardiovascular disease. Although the molecular mechanisms of the pathophysiology of the heart and vessels have been extensively studied, the burden of ischemic cardiovascular conditions has risen to become a top cause of morbidity and mortality. Calcium has important functions in the cardiovascular system. Calcium is involved in the mechanism of excitation-contraction coupling that regulates numerous events, ranging from the production of action potentials to the contraction of cardiomyocytes and vascular smooth muscle cells. Both in the heart and vessels, the rise of intracellular calcium is sensed by calmodulin, a protein that regulates and activates downstream kinases involved in regulating calcium signalling. Among them is the calcium calmodulin kinase family, which is involved in the regulation of cardiac functions. In this review, we present the current literature regarding the role of calcium/calmodulin pathways in the heart and vessels with the aim to summarize our mechanistic understanding of this process and to open novel avenues for research.

M6A-mRNA binding protein YTHDF1 is a novel regulator of cardiac function

m6A-mRNA binding protein YTHDF1 is a novel regulator of cardiac function

Abstract 12581: The N 6 -Methyladenosine Methylase Wtap is Essential for Cardiac Homeostasis

Introduction: N 6 -methyladenosine (m 6 A) methylation is one of the most common internal modifications in mammalian mRNA, and WTAP is a critical regulatory subunit of the RNA N 6 -methyladenosine methyltransferase complex. However, the roles of WTAP in cardiac structure and function are rarely known. Methods and Results: We found that the levels of m 6 A RNA methylation and the expression of WTAP were both decreased in myocardial tissue of the patients with hypertrophic cardiomyopathy, as well as in pressure overload-induced cardiac hypertrophy in mice. To determine the regulatory roles of WTAP, a conditional cardiac-specific knockout mice of WTAP was generated through Tamoxifen-inducible αMHC-Cre/loxP system. Depletion of WTPA resulted in heart failure in five days after Tamoxifen treatment, shown by enlarged heart and decreased left ventricular ejection fraction. Furthermore, we found WTAP knockout aggravated the progress of pathogenic cardiac remodeling induced by pressure overload, and abrogated the compensated response cardiac hypertrophy to hypertrophic stimulation. We found MYLK4 was a direct target of WTAP through a methylated RNA immunoprecipitation (MeRIP) sequencing, and it was validated by MeRIP-qPCR, RT-qPCR and RIP-qPCR. RNA stability analysis and dual-luciferase assays showed that depression of WTAP decreased m6A level of MYLK4 mRNA and resulted in a down-regulation of MYLK4 by reducing its RNA-stability. Forced overexpression of MYLK4 adeno-associated virus was able to partly alleviate the cardiac dysfunction induced by WTAP deletion. Finally, it was surprising that overexpression of WTAP also promotes pathogenic cardiomyocyte hypertrophy both in vitro and in vivo, as well as exacerbated cardiac remolding induced by pressure overload. Conclusions: In conclusion, our study demonstrates that WTAP is a critical regulator of cardiac function and indicates that appropriate m 6 A RNA methylation is required for cardiac homeostasis.

Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiac hypertrophy and heart failure.

Cyclic nucleotide phosphodiesterases (PDEs) modulate the neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple PDE isozymes with different enzymatic properties and subcellular localization regulate local pools of cyclic nucleotides and specific functions. This organization is heavily perturbed during cardiac hypertrophy and heart failure (HF), which can contribute to disease progression. Clinically, PDE inhibition has been considered a promising approach to compensate for the catecholamine desensitization that accompanies HF. Although PDE3 inhibitors, such as milrinone or enoximone, have been used clinically to improve systolic function and alleviate the symptoms of acute HF, their chronic use has proved to be detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as new potential targets to treat HF, each having a unique role in local cyclic nucleotide signalling pathways. In this Review, we describe cAMP and cGMP signalling in cardiomyocytes and present the various PDE families expressed in the heart as well as their modifications in pathological cardiac hypertrophy and HF. We also appraise the evidence from preclinical models as well as clinical data pointing to the use of inhibitors or activators of specific PDEs that could have therapeutic potential in HF.

Retinoic acid regulates pyruvate dehydrogenase kinase 4 (Pdk4) to modulate fuel utilization in the adult heart: Insights from wild-type and β-carotene 9',10' oxygenase knockout mice.

Regulation of the pyruvate dehydrogenase (PDH) complex by the pyruvate dehydrogenase kinase PDK4 enables the heart to respond to fluctuations in energy demands and substrate availability. Retinoic acid, the transcriptionally active form of vitamin A, is known to be involved in the regulation of cardiac function and growth during embryogenesis as well as under pathological conditions. Whether retinoic acid also maintains cardiac health under physiological conditions is unknown. However, vitamin A status and intake of its carotenoid precursor β‐carotene have been linked to the prevention of heart diseases. Here, we provide in vitro and in vivo evidence that retinoic acid regulates cardiac Pdk4 expression and thus PDH activity. Furthermore, we show that mice lacking β‐carotene 9′,10′‐oxygenase (BCO2), the only enzyme of the adult heart that cleaves β‐carotene to generate retinoids (vitamin A and its derivatives), displayed cardiac retinoic acid insufficiency and impaired metabolic flexibility linked to a compromised PDK4/PDH pathway. These findings provide novel insights into the functions of retinoic acid in regulating energy metabolism in adult tissues, especially the heart.

Abstract P3018: A Requirement For P21 Activated Kinase1 To Preserve Cardiac Function And Metabolic Homeostasis

Background/Hypothesis: Heart failure with preserved ejection fraction (HFpEF, EF>50%) continues to lack effective treatment. Obesity and diabetes are strong predictors of HF, with >80% of HFpEF patients being obese or overweight and ~40% suffering from diabetes. P21 activated kinase-1 (PAK1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Cdc42/Rac1. We tested the hypothesis that PAK1 is an important element in the regulation of cardiac function and metabolic homeostasis. Material and methods: 3-6 months male and female global PAK1 knock-out mice (PAK1 -/- ) and wild type (WT) controls were used to measure % of fat mass (by NMR), adipocyte size and glucose tolerance. We used echocardiography to assess cardiac dysfunction and western blots (WB) to examine cardiac protein alterations. Results (see Table): PAK1 -/- hearts exhibited preserved ejection fraction and diastolic dysfunction with slower e’, increased E/e’ ratio and prolonged IVRT. Additionally, PAK1 -/- hearts showed dilated left atrium and thickening of posterior wall. WB analyses showed decreased phosphorylation of cMyBP-C and dysregulation of calcium handling proteins. Moreover, PAK1 -/- mice exhibited increased % fat mass, adipocyte hypertrophy and impaired glucose tolerance test. Conclusion: Deletion of PAK1 resulted in obesity, impaired glucose homeostasis and HFpEF. Thus, PAK1 is required to support cardiometabolic health.

Abstract P2081: Beclin1 Ameliorates The HIV-1 Induced Cardiac Dysfunction In Mice Model Of HIV

Background: Despite antiretroviral drugs-mediated control of active HIV infection, cardiovascular disease (CVD) remains one of the leading causes of comorbidity in people living with HIV/AIDS (PLWHA). Recent studies suggest that activation of the immune system and chronic inflammation increases CVD risk in PLWHA. Dysregulation of the immune system can interact with several cellular functions, including autophagy and mitophagy. The autophagic factor Beclin1 is a crucial regulator of cellular autophagy and mitophagy. In this study, we tested the role of Beclin1 in cardiac function using a mice model of HIV. Methods and Results: This study was performed using transgenic mice of HIV-1 Tg26 mice, which was constructed before using HIV-1 DNA with deletion of 3-kb region of the gag/pol genes. Tg26 mice were crossed with Beclin1 transgenic mice to test the role of Beclin1. Cardiac function of Tg26 and Tg26 mice crossed with Beclin1 was evaluated by echocardiography. Cardiac autophagy was determined by western blots using autophagy regulatory markers. Our data suggest that Tg26 mice have significantly reduced cardiac function. Further, Tg26 mice show increased reactive Oxygen species (ROS) level, accumulation of LC3II, increased P62 level, and reduced autophagy flux. Additionally, western blots with cardiac tissue show upregulation of electron transport chain (ETC) complex proteins of mitochondria in the Tg26 mice that may be due to defective mitochondria accumulation in Tg26 mice heart. Interestingly, Beclin1 expression can improve the cardiac function of Tg26 mice heart. Mechanistically, we found that Beclin1 overexpression can restore the autophagy flux and reduce the cellular ROS level in the heart of Tg26 mice. Conclusion: Our study suggests that Beclin1 plays critical role in regulation of cardiac function during HIV induced cardiomyopathy and can be used as a potential therapeutic tool to control comorbidity in the PLWHA.

Molecular and functional characterization of the mouse intrinsic cardiac nervous system.

The intrinsic cardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate in the neuronal regulation of cardiac functions and that are involved in the initiation of cardiac arrhythmias. Therefore, deciphering its role in cardiac physiology and physiopathology is mandatory. The aim of this study was to provide a phenotypic, electrophysiological, and pharmacological characterization of the mouse ICNS, which is still poorly characterized. Global cardiac innervation and phenotypic diversity were investigated using immunohistochemistry on cleared murine hearts and on tissue sections. The patch clamp technique was used for the electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons. We have identified the expression of 7 distinct neuronal markers within the mouse ICNS, thus proving the neurochemical diversity of this network. Of note, it was the first time that the existenceof neurons expressing the calcium-binding protein calbindin, neuropeptide Y, and cocaine and amphetamine regulated transcript peptide was described in the mouse. Electrophysiology studies also revealed the existence of 4 different neuronal populations on the basis of their electrical behavior. Finally, we showed that these neurons can be modulated by several neuromodulators. This study showed that the mouse ICNS presents a molecular and functional complexity similar to other species and is therefore a suitable model to decipher the role of individual neuronal subtypes regarding the modulation of cardiac function and the initiation of cardiac arrhythmias.

The Acute Effects of Leptin on the Contractility of Isolated Rat Atrial and Ventricular Cardiomyocytes.

Leptin is a pleiotropic peptide playing an important role in the regulation of cardiac functions. It is not clear whether leptin directly modulates the mechanical function of atrial cardiomyocytes. We compared the acute effects of leptin on the characteristics of mechanically non-loaded sarcomere shortening and cytosolic Ca2+ concentration ([Ca2+]i) transients in single rat atrial and ventricular cardiomyocytes. We also studied the functional properties of myosin obtained from cardiomyocytes using an in vitro motility assay and assessed the sarcomeric protein phosphorylation. Single cardiomyocytes were exposed to 5, 20, and 60 nM leptin for 60 min. In ventricular cardiomyocytes, 60 nM leptin depressed sarcomere shortening amplitude and decreased the rates of shortening and relaxation. These effects were accompanied by a decrease in the phosphorylation of cMyBP-C, an increase in Tpm phosphorylation, and a slowdown of the sliding velocity of thin filaments over myosin in the in vitro motility assay. In contrast, in atrial cardiomyocytes, the phosphorylation of cMyBP-C and TnI increased, and the characteristics of sarcomere shortening did not change. Leptin had no effect on the characteristics of [Ca2+]i transients in ventricular cardiomyocytes, while 5 nM leptin prolonged [Ca2+]i transients in atrial cardiomyocytes. Thus, leptin-induced changes in contractility of ventricular cardiomyocytes may be attributed to the direct effects of leptin on cross-bridge kinetics and sarcomeric protein properties rather than changes in [Ca2+]i. We also suggest that the observed differences between atrial and ventricular cardiomyocytes may be associated with the peculiarities of the expression of leptin receptors, as well as signaling pathways in the atrial and ventricular myocardium.

Subcellular β-Adrenergic Receptor Signaling in Cardiac Physiology and Disease.

Adrenergic receptors are critical regulators of cardiac function with profound effects on cardiac output during sympathetic stimulation. Chronic stimulation of the adrenergic system of the heart under conditions of cardiac stress leads to cardiac dysfunction, hypertrophy, and ultimately failure. Emerging data have revealed that G protein-coupled receptors in intracellular compartments are functionally active and regulate distinct cellular processes from those at the cell surface. β2 adrenergic receptors internalize onto endosomes in various cell types where they have recently been shown to continue to stimulate cAMP production to selectively regulate gene expression. Other studies have identified β1 adrenergic receptors at the nuclear envelope and the Golgi apparatus. Here, we discuss data on signaling by β1 and β2 adrenergic receptors in the heart and the possible influence of their subcellular locations on their divergent physiological functions in cardiac myocytes and in cardiac pathology. Understanding the relative roles of these receptors at these locations could have a significant impact on pharmacological targeting of these receptors for the treatment of heart failure and cardiac diseases.

New Perspectives on Sex Steroid and Mineralocorticoid Receptor Signaling in Cardiac Ischemic Injury.

The global burden of ischemic heart disease is burgeoning for both men and women. Although advances have been made, the need for new sex-specific therapies targeting key differences in cardiovascular disease outcomes in men and women remains. Mineralocorticoid receptor directed treatments have been successfully used for blood pressure control and heart failure management and represent a potentially valuable therapeutic option for ischemic cardiac events. Clinical and experimental data indicate that mineralocorticoid excess or inappropriate mineralocorticoid receptor (MR) activation exacerbates ischemic damage, and many of the intracellular response pathways activated in ischemia and subsequent reperfusion are regulated by MR. In experimental contexts, where MR are abrogated genetically or mineralocorticoid signaling is suppressed pharmacologically, ischemic injury is alleviated, and reperfusion recovery is enhanced. In the chronic setting, mineralocorticoid signaling induces fibrosis, oxidative stress, and inflammation, which can predispose to ischemic events and exacerbate post-myocardial infarct pathologies. Whilst a range of cardiac cell types are involved in mineralocorticoid-mediated regulation of cardiac function, cardiomyocyte-specific MR signaling pathways are key. Selective inhibition of cardiomyocyte MR signaling improves electromechanical resilience during ischemia and enhances contractile recovery in reperfusion. Emerging evidence suggests that the MR also contribute to sex-specific aspects of ischemic vulnerability. Indeed, MR interactions with sex steroid receptors may differentially regulate myocardial nitric oxide bioavailability in males and females, potentially determining sex-specific post-ischemic outcomes. There is hence considerable impetus for exploration of MR directed, cell specific therapies for both women and men in order to improve ischemic heart disease outcomes.

Ultrastructural changes in the myocardium of animals under conditions of simulated hyperhomocysteinemia, hyper- and hypothyroidism and their combination

Thyroid hormones have a significant impact on heart function through both genomic and non-genomic effects. Deficiency or excess of thyroid hormones leads to profound changes in the regulation of cardiac function and cardiovascular hemodynamics. The heart is the main target organ for the action of thyroid hormones and in patients with hypo- or hyperthyroidism there are marked changes in the work of the heart. The aim of the work was to establish ultrastructural changes in myocardial components in experimental hyperhomocysteinemia (HHCy) against the background of hyper- and hypothyroidism. Thiolactone HHCy was modelized by administering to animals an exogenous HCy in the form of thiolactone at a dose of 100 mg/kg body weight once a day for 28 days. Hyperthyroidism was modelized by daily administration of L-thyroxine at a dose of 200 μg/kg for the 21 days, hypothyroidism – daily administration of thiamazole at a dose of 10 mg/kg for the 21 days. Individual groups of animals were administered L-thyroxine and thiamazole in parallel with HCy. High levels of HCy adversely affected the walls of myocardial blood vessels. The lumens of hemocapillaries were plethoric, filled with erythrocytes. Changes in endotheliocytes were revealed, and cardiomyocytes contained deformed nuclei. In laboratory animals with hyperthyroidism, an increase in ultrastructural changes in the walls of blood vessels (edema of the walls of hemocapillaries, damaged cristae in mitochondria) were established. In animals that were modeled for hyperthyroidism and HHCy, more significant changes in endotheliocytes were revealed, most of the mitochondria were destroyed. More pronounced alterative changes were revealed in cardiomyocytes. An electron microscopic examination of the myocardium of animals with hypothyroidism showed significant degenerative changes in the ultrastructure of the walls of blood vessels, and hypertrophied mitochondria were also found. The combined influence of hypothyroidism and HHCy caused the most profound disturbances in the ultrastructure of cardiomyocytes and hemocapillaries in comparison with other groups of animals. The integrity of intercellular contacts was impaired, most of the mitochondria of myocytes had destroyed cristae and the outer membrane.

Computer simulations demonstrate dual mechanisms through which cardiac myosin binding protein C regulates contraction

Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cardiac function. Mutations in cMyBP-C are a common cause of hypertrophic cardiomyopathy. Despite its significance in cardiac health, how cMyBP-C mediates its functional effects is not perfectly understood yet.

Evolution of the N-Terminal Regulation of Cardiac Troponin I for Heart Function of Tetrapods: Lungfish Presents an Example of the Emergence of Novel Submolecular Structure to Lead the Capacity of Adaptation.

Troponin-based Ca2+ regulation of striated muscle contraction emerged approximately 700 million years ago with largely conserved functions during evolution. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI is specifically expressed in the adult heart and has a unique N-terminal extension implicating a specific value during natural selection. The N-terminal extension of cardiac TnI in higher vertebrates contains β-adrenergic-regulated protein kinase A (PKA) phosphorylation sites as a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Phylogenic studies showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. This apparently rapid evolution of β-adrenergic regulation of cardiac function suggests a high selection value for the heart of vertebrate animals on land to work under higher metabolic demands. Sequencing and PKA phosphorylation data showed that lungfish cardiac TnI has evolved with an amphibian-like N-terminal extension with prototype PKA phosphorylation sites while its overall structure remained fish like. The data demonstrate that the submolecular structure of TnI may evolve ahead of the whole protein for cardiac muscle contractility to adapt to new environmental conditions. Understanding the evolution of the β-adrenergic regulation of TnI and cardiac adaptation to the increased energetic demands of life on land adds knowledge for the treatment of human heart diseases and failure.

Optogenetic Stimulation of Gi Signaling Enables Instantaneous Modulation of Cardiomyocyte Pacemaking.

G-protein signaling pathways are central in the regulation of cardiac function in physiological and pathophysiological conditions. Their functional analysis through optogenetic techniques with selective expression of opsin proteins and activation by specific wavelengths allows high spatial and temporal precision. Here, we present the application of long wavelength-sensitive cone opsin (LWO) in cardiomyocytes for activation of the Gi signaling pathway by red light. Murine embryonic stem (ES) cells expressing LWO were generated and differentiated into beating cardiomyocytes in embryoid bodies (EBs). Illumination with red light (625 nm) led to an instantaneous decrease up to complete inhibition (84–99% effectivity) of spontaneous beating, but had no effect on control EBs. By using increasing light intensities with 10 s pulses, we determined a half maximal effective light intensity of 2.4 μW/mm2 and a maximum effect at 100 μW/mm2. Pre-incubation of LWO EBs with pertussis toxin completely inhibited the light effect proving the specificity for Gi signaling. Frequency reduction was mainly due to the activation of GIRK channels because the specific channel blocker tertiapin reduced the light effect by ~80%. Compared with pharmacological stimulation of M2 receptors with carbachol with slow kinetics (>30 s), illumination of LWO had an identical efficacy, but much faster kinetics (<1 s) in the activation and deactivation demonstrating the temporal advantage of optogenetic stimulation. Thus, LWO is an effective optogenetic tool for selective stimulation of the Gi signaling cascade in cardiomyocytes with red light, providing high temporal precision.

Depletion of cardiac cardiolipin synthase alters systolic and diastolic function

SummaryCardiolipin (CL) is a major cardiac mitochondrial phospholipid maintaining regular mitochondrial morphology and function in cardiomyocytes. Cardiac CL production includes its biosynthesis and a CL remodeling process. Here we studied the impact of CL biosynthesis and the enzyme cardiolipin synthase (CLS) on cardiac function. CLS and cardiac CL species were significantly downregulated in cardiomyocytes following catecholamine-induced cardiac damage in mice, accompanied by increased oxygen consumption rates, signs of oxidative stress, and mitochondrial uncoupling. RNAi-mediated cardiomyocyte-specific knockdown of CLS in Drosophila melanogaster resulted in marked cardiac dilatation, severe impairment of systolic performance, and slower diastolic filling velocity assessed by fluorescence-based heart imaging. Finally, we showed that CL72:8 is significantly decreased in cardiac samples from patients with heart failure with reduced ejection fraction (HFrEF). In summary, we identified CLS as a regulator of cardiac function. Considering the cardiac depletion of CL species in HFrEF, pharmacological targeting of CLS may be a promising therapeutic approach.

Screening of β1- and β2-Adrenergic Receptor Modulators through Advanced Pharmacoinformatics and Machine Learning Approaches.

Cardiovascular diseases (CDs) are a major concern in the human race and one of the leading causes of death worldwide. β-Adrenergic receptors (β1-AR and β2-AR) play a crucial role in the overall regulation of cardiac function. In the present study, structure-based virtual screening, machine learning (ML), and a ligand-based similarity search were conducted for the PubChem database against both β1- and β2-AR. Initially, all docked molecules were screened using the threshold binding energy value. Molecules with a better binding affinity were further used for segregation as active and inactive through ML. The pharmacokinetic assessment was carried out on molecules retained in the above step. Further, similarity searching of the ChEMBL and DrugBank databases was performed. From detailed analysis of the above data, four compounds for each of β1- and β2-AR were found to be promising in nature. A number of critical ligand-binding amino acids formed potential hydrogen bonds and hydrophobic interactions. Finally, a molecular dynamics (MD) simulation study of each molecule bound with the respective target was performed. A number of parameters obtained from the MD simulation trajectories were calculated and substantiated the stability between the protein-ligand complex. Hence, it can be postulated that the final molecules might be crucial for CDs subjected to experimental validation.

Upregulation of miR-128 Mediates Heart Injury by Activating Wnt/β-catenin Signaling Pathway in Heart Failure Mice

ABSTRACT Cardiac hypertrophy contributes to heart failure and is pathogenically modulated by a network of signaling cascades including Wnt/β-catenin signaling pathway. miRNAs have been widely demonstrated to regulate gene expression in heart development. miR-128 was routinely found as a brain-enriched gene and has been functionally associated with regulation of cardiac function. However, its role and molecular mechanisms that regulate cardiac hypertrophy remain largely unclear. Adeno-associated virus serotype 9 (AAV9)-mediated constructs with miR-128 or anti-miR-128 were generated and delivered to overexpression or blockade of miR-128 in vivo followed by HF induction with isoproterenol (ISO) or transverse aortic constriction (TAC). Cardiac dysfunction and hypertrophy, coupled with involved gene and protein level, were then assessed. Our data found that miR-128, Wnt1, and β-catenin expressions were upregulated in both patients and mice model with HF. Interference with miR-128 reduces Wnt1/β-catenin expression in mouse failing hearts and ameliorates heart dysfunctional properties. We identified miR-128 directly targets to Axin1, an inhibitor of Wnt/β-catenin signaling, and suppresses its inhibition on Wnt1/β-catenin. Our study provides evidence indicating miR-128 as an inducer of HF and cardiac hypertrophy by enhancing Wnt1/β-catenin in an Axin1-dependent nature. We thus suggest miR-128 has potential value in the treatment of heart failure.

β-Arrestin-Biased Allosteric Modulator Potentiates Carvedilol-Stimulated β Adrenergic Receptor Cardioprotection.

β 1 adrenergic receptors (β 1ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein-coupled receptor family, β 1ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein β-arrestin. Carvedilol, a traditional β-blocker widely used in treating high blood pressure and heart failure by blocking β adrenergic receptor-mediated G protein activation, can selectively stimulate Gs-independent β-arrestin signaling of β adrenergic receptors, a process known as β-arrestin-biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied β 2 adrenergic receptors (β 2ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on β 2ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the β-arrestin-biased ligand carvedilol at β 2ARs. Here we describe the surprising finding that at β 1ARs unlike β 2ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for β 1ARs and potentiates carvedilol-stimulated, β-arrestin-dependent β 1AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on β-arrestins since it is lost in mice with myocyte-specific deletion of β-arrestins. Our findings demonstrate that Cmpd-6 is a selective β-arrestin-biased allosteric modulator of β 1ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT: This study demonstrates the positive cooperativity of Cmpd-6 on β1ARs as a β-arrestin-biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known β-arrestin-biased β-blocker for β1ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the β-arrestin-dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury-induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.

The evolutionary and physiological significance of the Hif pathway in teleost fishes.

The hypoxia-inducible factor (HIF) pathway is a key regulator of cellular O2 homeostasis and an important orchestrator of the physiological responses to hypoxia (low O2) in vertebrates. Fish can be exposed to significant and frequent changes in environmental O2, and increases in Hif-α (the hypoxia-sensitive subunit of the transcription factor Hif) have been documented in a number of species as a result of a decrease in O2. Here, we discuss the impact of the Hif pathway on the hypoxic response and the contribution to hypoxia tolerance, particularly in fishes of the cyprinid lineage, which includes the zebrafish (Danio rerio). The cyprinids are of specific interest because, unlike in most other fishes, duplicated paralogs of the Hif-α isoforms arising from a teleost-specific genome duplication event have been retained. Positive selection has acted on the duplicated paralogs of the Hif-α isoforms in some cyprinid sub-families, pointing to adaptive evolutionary change in the paralogs. Thus, cyprinids are valuable models for exploring the evolutionary significance and physiological impact of the Hif pathway on the hypoxic response. Knockout in zebrafish of either paralog of Hif-1α greatly reduces hypoxia tolerance, indicating the importance of both paralogs to the hypoxic response. Here, with an emphasis on the cardiorespiratory system, we focus on the role of Hif-1α in the hypoxic ventilatory response and the regulation of cardiac function. We explore the effects of the duration of the hypoxic exposure (acute, sustained or intermittent) on the impact of Hif-1α on cardiorespiratory function and compare relevant data with those from mammalian systems.