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BACKGROUND – Sepsis is characterized by an overwhelming systemic inflammatory response to infection that may lead to multiple organ failure and death in over 20% of the estimated 49 million annual cases worldwide. Because sepsis-related inflammatory processes disturbing cardiac and endothelial cells homeostasis are still poorly understood, only few therapeutic strategies reached added-value efficacy in terms of mortality reduction in the critical care units. Using an innovative transgenic mouse line to detect the acute changes of newly synthetized proteins, we propose to assess the protein expression profile of cardiomyocytes and endothelial cells in a murine model of polymicrobial sepsis. METHODS – The Cre-recombinase mediated expression of mutant L274G-methionyl-tRNA synthetase (MetRS*) in transgenic mouse crosses enables the selective in vivo incorporation of azidonorleucine (ANL), a non-canonical methionine analog, in newly synthetized proteins. MetRS* mice expressing CDH5-creERT2 (CDH5, endothelial cells) or αMHC-MerCreMer (MCM, cardiomyocytes) underwent CLP (cecal ligation and puncture). The sepsis model was validated by echocardiography, inflammatory cytokine profiling and histology. Labeling efficiency of nascent proteins from MCM and CDH5 cells (heart and lung) were visualized by FUNCAT (Fluorescence non-canonical amino acid tagging) or purified by BONCAT (bioorthogonal non-canonical amino acid tagging) for mass spectrometry analysis. RESULTS – Significant elevation of circulating inflammatory cytokines proves the systemic inflammatory state of mice submitted to CLP compared to SHAM mice. Our experimental model also displays a significant impairment of the LV function at both systolic (reduced cardiac index) and diastolic levels (increased E/A and isovolumic relaxation time) confirming hemodynamics alteration related to sepsis. Metascape analysis showed expression changes for several proteins involved in the acute response to stress in both MCM and CDH5 samples. Overall, nascent protein synthesis is mainly reduced in MCM samples in sepsis (14 up/98 down), something not observed in CDH5 samples both in the heart (69 up/97 down) and in the lungs (137 up/43 down). Alterations in metabolic pathways such as pyruvate metabolism and glycolysis/gluconeogenesis were only observed in MCM samples while protein associated with focal adhesion, for example, was observed in heart and lungs CDH5 samples. CONCLUSIONS – The results of this study provide a better understanding of which proteins are altered in specific cell types in a sepsis model. We confirmed previously identified markers of inflammatory response in tissues, while identifying new protein candidates and signaling pathways that may contribute to key steps of sepsis detrimental effects and thus, offering potential new therapeutic avenues to curb the fatal outcomes of this disease. Supported by Canadian Institute of Health Research (CIHR) - Project Grants (399567) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUNDPeripheral artery disease (PAD) is a major risk factor for lower-extremity amputation in diabetic patients caused by an insufficient angiogenic response. Unfortunately, therapeutic angiogenesis using growth factors, such as the vascular endothelial growth factor (VEGF), are ineffective in diabetic conditions due to diabetes-induced growth factor resistance. The apelinergic system (APJ receptor/apelin) is highly upregulated under hypoxic condition and acts as an activator of angiogenesis. Apelin treatment has been shown to improve revascularization in nondiabetic models of ischemia, however, its role on angiogenesis in diabetic conditions remains poorly investigated. Thus, this study explored the impact of Pyr-apelin-13 in endothelial cell function and diabetic mouse model of hindlimb ischemia.METHODSNondiabetic and diabetic mice underwent femoral artery ligation to induce lower limb ischemia. A group of diabetic mice was implanted subcutaneously with osmotic pumps delivering Pyr-apelin-13 for 28 days. Blood flow reperfusion was measured for 4 weeks post-surgery and exercise willingness was assessed in individual cages with voluntary wheels.In vitro, BAECs were exposed to normal (NG) or high glucose (HG) levels and hypoxia. Cell migration, proliferation and tube formation assays were performed following either VEGF or Pyr-apelin-13 stimulation.RESULTSFollowing limb ischemia, blood flow reperfusion, functional recovery of the limb and vascular density were improved in diabetic mice receiving Pyr-apelin-13 compared to untreated diabetic mice. In cultured BAECs, exposure to HG concentrations and hypoxia reduced VEGF proangiogenic actions, whereas apelin proangiogenic effects remained unaltered. Pyr-apelin-13 induced its proangiogenic actions through Akt/AMPK/eNOS and RhoA/ROCK signaling pathways under both NG or HG concentrations and hypoxia exposure.CONCLUSIONSPyr-apelin-13 promoted endothelial cell function and angiogenesis in the ischemic limb despite diabetes and HG level exposure. Therefore, our results identified the apelinergic system as a potential therapeutic target for angiogenic therapy in diabetic patients with PAD.HighlightsSustained delivery of Pyr-apelin-13 improves blood flow reperfusion, functional recovery of the hindlimb and capillary density in diabetic mice following ischemia.Unlike VEGF, signaling pathways and proangiogenic actions induced by apelin/APJ stimulation are not impaired by high glucose exposure.Despite the presence of tissue ischemia, diabetes reduces the expression of apelin and APJ in the ischemic adductor muscle, an effect overcome by apelin administration.

Sepsis is a prevalent life-threatening condition related to a systemic infection, and with unresolved issues including refractory septic shock and organ failures. Endogenously released catecholamines are often inefficient to maintain blood pressure, and low reactivity to exogenous catecholamines with risk of sympathetic overstimulation is well documented in septic shock. In this context, apelinergics are efficient and safe inotrope and vasoregulator in rodents. However, their utility in a larger animal model as well as the limitations with regards to the enzymatic breakdown during sepsis, need to be investigated. The therapeutic potential and degradation of apelinergics in sepsis were tested experimentally and in a cohort of patients. (1) 36 sheep with or without fecal peritonitis-induced septic shock (a large animal experimental design aimed to mimic the human septic shock paradigm) were evaluated for hemodynamic and renal responsiveness to incremental doses of two dominant apelinergics: apelin-13 (APLN-13) or Elabela (ELA), and (2) 52 subjects (33 patients with sepsis/septic shock and 19 healthy volunteers) were investigated for early levels of endogenous apelinergics in the blood, the related enzymatic degradation profile, and data regarding sepsis outcome. APLN-13 was the only one apelinergic which efficiently improved hemodynamics in both healthy and septic sheep. Endogenous apelinergic levels early rose, and specific enzymatic breakdown activities potentially threatened endogenous apelin system reactivity and negatively impacted the outcome in human sepsis. Short-term exogenous APLN-13 infusion is helpful in stabilizing cardiorenal functions in ovine septic shock; however, this ability might be impaired by specific enzymatic systems triggered during the early time course of human sepsis. Strategies to improve resistance of APLN-13 to degradation and/or to overcome sepsis-induced enzymatic breakdown environment should guide future works.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here we describe that defects in the endothelial cell (EC) compartment of the perivascular stem cell niche in three different types of MD are associated with inefficient mobilization of MuSCs following tissue damage. Using chemoinformatic analysis, we identified the 13 amino acid form of the peptidic hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. In dystrophic mice, administration of AP-13 generates a pro-myogenic EC-rich niche that supports MuSC function and markedly improves tissue regeneration, muscle strength, and physical performance. Moreover, we demonstrate that EC specific knockout of the AP-13 receptor leads to regenerative defects that phenocopy major pathological features of MD. Altogether, we provide in vivo proof-of-concept that enhancing endogenous repair by targeting the perivascular niche is a viable therapeutic avenue for MD and characterize AP-13 as a novel drug candidate for systemic treatment of stem cell dysfunction.

Side-chain-constrained amino acids are useful tools to modulate the biological properties of peptides. In this study, we applied side-chain constraints to apelin-13 (Ape13) by substituting the Pro12 and Phe13 positions, affecting the binding affinity and signaling profile on the apelin receptor (APJ). The residues 1Nal, Trp, and Aia were found to be beneficial substitutions for Pro12, and the resulting analogues displayed high affinity for APJ (Ki 0.08-0.18 nM vs Ape13 Ki 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Dbzg; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, Ki 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Dbzg, Ki 0.08 nM) are the most potent Ape13 analogues activating the Gα12 pathways (53, EC50 Gα12 2.8 nM vs Ape13, EC50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro (t1/2 5.8-7.3 h in rat plasma) and in vivo.

Introduction Radiofrequency (RF) is the preferred thermal energy used in electrophysiology. RF catheter must deliver the energy close to arrhythmia foci. A new method to deliver RF to deeper locations using a pacemaker lead is explored. Methods A Medtronic 3830 lead screwed in chicken breasts delivered 50 watts RF energy in three methods: A) direct fashion (RF catheter touching the proximal end of the 3830 lead, acting as an extension of RF catheter), or B) 3830 lead as a return patch (RF delivered in the bath without contact), or C) 3830 lead as a return patch (RF delivered touching the breast surface close to the 3830 lead screwed deep in the flesh). Different power settings were also tested. Lesion surface area is reported in cm2. Results 76 measurements were available. Bigger lesions were obtained at 10W method A (0.78cm2), 50W method C (0.72cm2) and 5W method B (0.44cm2). High impedances were noted at 10W and 50W with tissue remaining attached to the lead when removed. Conclusion RF can be delivered to deeper foci through a 3830-pacemaker lead with maximum size lesion formation using proximal unipolar direct delivery and proximal close bipolar as the return patch. In humans, it opens a path to attain deep septal foci (LV summit) or epicardial structures (vein of Marshall, transmural ablation from RF endocardial to LV coronary sinus lead as return patch): using standard, 4F pacemaker leads, and 2F small EP catheters or even isolated guidewires.

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca2+ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca2+ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies.

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and β-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the β-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels.NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.

ELABELA (ELA) is the second endogenous ligand of the apelin receptor (APJ). Although apelin-13 and ELA both target APJ, there is limited information on structure-activity relationship (SAR) of ELA. In the present work, we identified the shortest bioactive C-terminal fragment ELA23-32, which possesses high affinity for APJ (Ki 4.6 nM) and produces cardiorenal effects in vivo similar to those of ELA. SAR studies on conserved residues (Leu25, His26, Val29, Pro30, Phe31, Pro32) show that ELA and apelin-13 may interact differently with APJ. His26 and Val29 emerge as important for ELA binding. Docking and binding experiments suggest that Phe31 of ELA may bind to a tight groove distinct from that of Phe13 of Ape13, while the Phe13 pocket may be occupied by Pro32 of ELA. Further characterization of signaling profiles on the Gαi1, Gα12, and β-arrestin2 pathways reveals the importance of aromatic residue at the Phe31 or Pro32 position for receptor activation.

Srsf3 was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodelling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibres from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating mitochondrial integrity.

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gαq led to a Gαq-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT1R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT1R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gαq-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

The ArfGAP with dual PH domains 1 (ADAP1) regulates the activation of the hypertrophic mitogen-activated protein kinase ERK1/2 pathway in non-cardiomyocytes. However, its role in cardiomyocytes is unknown. Our aim was to characterize the role of ADAP1 in the hypertrophic process of cardiomyocytes. We assessed the expression of ADAP1 in the hearts of adult and neonatal rats by RT-qPCR and Western blotting and showed that it is preferentially expressed in cardiomyocytes. Adenoviral-mediated ADAP1 overexpression in cultured rat neonatal ventricular cardiomyocytes limited their serum-induced hypertrophic response as measured by immunofluorescence microscopy. Furthermore, ADAP1 overexpression completely blocked phenylephrine- and Mek1 constitutively active (Mek1ca) mutant-induced hypertrophy in these cells. The anti-hypertrophic effect of ADAP1 was not caused by a reduction in protein synthesis, interference with the Erk1/2 pathway, or disruption of the fetal gene program activation, as assessed by nascent protein labeling, Western blotting, and RT-qPCR, respectively. An analysis of cultured cardiomyocytes by confocal microscopy revealed that ADAP1 partially re-organizes α-actinin into dense puncta, a phenomenon that is synergized by Mek1ca overexpression. Biotin labeling of cell surface proteins from cardiomyocytes overexpressing ADAP1 revealed that it reduces the surface expression of β1-integrin, an effect that is strongly potentiated by Mek1ca overexpression. Our findings provide insights into the anti-hypertrophic function of ADAP1 in cardiomyocytes.

The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue 15, Ki 0.15 nM and t1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues 13 and 15, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system.

The apelinergic pathway has been generating increasing interest in the past few years for its potential as a therapeutic target in several conditions associated with the cardiovascular and metabolic systems. Indeed, preclinical and, more recently, clinical evidence both point to this G protein-coupled receptor as a target of interest in the treatment of not only cardiovascular disorders such as heart failure, pulmonary arterial hypertension, atherosclerosis, or septic shock, but also of additional conditions such as water retention/hyponatremic disorders, type 2 diabetes, and preeclampsia. While it is a peculiar system with its two classes of endogenous ligand, the apelins and Elabela, its intricacies are a matter of continuing investigation to finely pinpoint its potential and how it enables crosstalk between the vasculature and organ systems of interest. In this perspective article, we first review the current knowledge on the role of the apelinergic pathway in the above systems, as well as the associated therapeutic indications and existing pharmacological tools. We also offer a perspective on the challenges and potential ahead to advance the apelinergic system as a target for therapeutic intervention in several key areas.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is essential for normal heart function. However, p38 also contributes to heart failure pathogenesis by affecting cardiomyocytes contractility and survival. To unravel part of the complex role of p38 in cardiac function, we performed an APEX2-based proximity assay in cultured neonatal rat ventricular myocytes and identified the protein interaction networks (interactomes) of two highly expressed p38 isoforms in the heart. We found that p38α and p38γ have distinct interactomes in cardiomyocytes under both basal and osmotic stress-activated states. Interestingly, the activated p38α interactome contains many RNA-binding proteins implicated in splicing, including the serine/arginine-rich splicing factor 3 (SRSF3). Its interaction with the activated p38α was validated by co-immunoprecipitation. The cytoplasmic abundance and alternative splicing function of SRSF3 are also both modulated by the p38 signaling pathway. Our findings reveal a new function for p38 as a specific regulator of SRSF3 in cardiomyocytes.

Diabetic nephropathy (DN) remains the leading cause of end-stage renal disease. Hyperglycemia-induced podocyte dysfunction is a major contributor of renal function impairment in DN. Previous studies showed that activation of mitogen-activated protein kinase (MAPK) in diabetes promotes podocyte dysfunction and cell death. Dual specificity phosphatases (DUSPs) are a family of phosphatases mainly responsible for MAPK inhibition. In this study, we demonstrated that diabetes and high glucose exposure decreased DUSP4 expression in cultured podocytes and glomeruli. Diabetes-induced DUSP4 reduction enhanced p38 and c-Jun N-terminal kinase (JNK) activity and podocyte dysfunction. The overexpression of DUSP4 prevented the activation of p38, JNK, caspase 3/7 activity, and NADPH oxidase 4 expression induced by high glucose level exposure. Deletion of DUSP4 exacerbated albuminuria and increased mesangial expansion and glomerular fibrosis in diabetic mice. These morphological changes were associated with profound podocyte foot process effacement, cell death, and sustained p38 and JNK activation. Moreover, inhibition of protein kinase C-δ prevented DUSP4 expression decline and p38/JNK activation in the podocytes and renal cortex of diabetic mice. Analysis of DUSP4 expression in the renal cortex of patients with diabetes revealed that decreased DUSP4 mRNA expression correlated with reduced estimated glomerular filtration rate (

ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELA's structure- activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the G alpha(i1) and beta-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases

Apelin is the endogenous ligand of the APJ receptor, a member of the G protein-coupled receptor family. This system plays an important role in the regulation of blood pressure and cardiovascular functions. To better understand the role of its C-terminal Phe(13) residue on ligand binding, receptor signaling, and hypotension, we report a series of modified analogues in which Phe(13) was substituted by unnatural amino acids. These modifications delivered new compounds exhibiting higher affinity and potency to inhibit cAMP accumulation compared to apelin-13. In particular, analogues Bpa(13) or (α-Me)Phe(13) were 30-fold more potent to inhibit cAMP accumulation than apelin-13. Tyr(OBn)(13) substitution led to a 60-fold improvement in binding affinity and induced stronger and more sustained drop in blood pressure compared to apelin-13. Our study identified new potent analogues of apelin-13, which represent valuable probes to better understand its structure-function relationship.

Objectives Apelin-13 was recently proposed as an alternative to the recommended β-adrenergic drugs for supporting endotoxin-induced myocardial dysfunction. Since Apelin-13 signals through its receptor (Apelin peptide jejunum) to exert singular inotropic/vasotropic actions and to optimize body fluid balance, this candidate pathway might benefit septic shock management. Whether the newly discovered ELABELA (ELA), a second endogenous ligand of the Apelin peptide jejunum receptor highly expressed in the kidney, further improves cardio-renal impairment remains unknown. Design, setting, and subjects Interventional study in a rat model of septic shock (128 adult males) to assess the effects of ELA and Apelin-13 on vascular and cardio-renal function. Experiments were performed in a tertiary care University-based research institute. Interventions Polymicrobial sepsis-induced cardiac dysfunction was produced by cecal ligation puncture to assess hemodynamic efficacy, cardioprotection, and biomechanics under acute or continuous infusions of the apelinergic agonists ELA or Apelin-13 (39 and 15 µg/kg/hr, respectively) versus normal saline. Measurements and main results Apelinergic agonists improved 72-hour survival after sepsis induction, with ELA providing the best clinical outcome after 24 hours. Apelinergic agonist infusion counteracted cecal ligation puncture-induced myocardial dysfunction by improving left ventricular pressure-volume relationship. ELA-treated cecal ligation puncture rats were the only group to 1) display a significant improvement in left ventricular filling as shown by increased E-wave velocity and left ventricular end-diastolic volume, 2) exhibit a higher plasma volume, and 3) limit kidney injury and free-water clearance. These beneficial renal effects were superior to Apelin-13, likely because full-length ELA enabled a distinctive regulation of pituitary vasopressin release. Conclusions Activation of the apelinergic system by exogenous ELA or Apelin-13 infusion improves cardiovascular function and survival after cecal ligation puncture-induced sepsis. However, ELA proved better than Apelin-13 by improving fluid homeostasis, cardiovascular hemodynamics recovery, and limiting kidney dysfunction in a vasopressinergic-dependent manner.

The Arf GTPase activating protein with dual PH domains 1 (Adap1), a putative regulator of the small G protein ADP-ribosylation factor 6 (Arf6), might be expressed to some extent in the heart. Although Arf6 roles in cardiomyocytes are not well defined, this small G protein was shown to alter surface expression of integrins in other cell types. Considering the importance of integrins on cellular mechanisms of adhesion and spreading, we hypothesized that Adap1 might impact cardiomyocyte hypertrophy by regulating integrin signaling . In order to determine the role of Adap1 in cardiomyocytes, we analyzed its cardiac expression in Sprague Dawley rats. Comparatively to its high mRNA expression in the brain, we showed that Adap1 transcript is expressed at relatively low levels in the heart of adult and neonatal rats. However, Western blot detection of Adap1 showed that it is well expressed in the whole heart of adult and neonatal rats at levels reaching 13% and 17% relative to the brain, respectively. Moreover, Adap1 was 3-fold more abundant in isolated rat neonatal ventricular cardiomyocyte (RNVC) comparatively to partially enriched non-cardiomyocyte cells. Adenoviral-mediated overexpression of ADAP1 partially prevented the increase in cell surface area of cultured RNVCs in response to increasing serum concentrations. Interestingly, ADAP1 overexpression completely blocked constitutively active mutant Mek1- and phenylephrine-induced hypertrophy of RNVCs. In these conditions, ADAP1 did not impair the Mek1ca-induced fetal gene program in RNVCs, suggesting that its anti-hypertrophic effect was not mediated by transcriptional regulation. In fact, cell surface biotin labeling indicated that ADAP1 significantly reduced the localization of integrin β1 at the membrane of RNVCs without changing its total expression. Strikingly, integrin β1 was almost completely absent from the surface of RNVCs overexpressing both ADAP1 and Mek1ca. These findings demonstrate for the first time that Adap1 expressed in cardiomyocytes interferes with the surface expression of integrin β1, thereby slowing the development of hypertrophy in vitro. Further in vivo studies are needed to characterize the anti-hypertrophic function of Adap1 in response to pathological stresses.

Apelin is the endogenous ligand for the G protein-coupled receptor APJ and exerts a key role in regulating cardiovascular functions. We report herein a novel series of macrocyclic analogues of apelin-13 in which the N- and C-terminal residues as well as the macrocycle composition were chemically modified to modulate structure–activity relationships on the APJ receptor. To this end, the binding affinity and the ability to engage G protein-dependent and G protein-independent signalling pathways of the resulting analogues were assessed. In this series, the position and the nature of the C-terminal aromatic residue is a determinant for APJ interaction and β-arrestin recruitment, as previously demonstrated for linear apelin-13 derivatives. We finally discovered compounds 1, 4, 11 and 15, four potent G protein-biased apelin receptor agonists exhibiting affinity in the nanomolar range for APJ. These macrocyclic compounds represent very useful pharmacological tools to explore the therapeutic potential of the apelinergic system.

Dobutamine is the currently recommended β-adrenergic inotropic drug for supporting sepsis-induced myocardial dysfunction when cardiac output index remains low after preload correction. Better and safer therapies are nonetheless mandatory because responsiveness to dobutamine is limited with numerous side effects. Apelin-13 is a powerful inotropic candidate that could be considered as an alternative noncatecholaminergic support in the setting of inflammatory cardiovascular dysfunction.Interventional controlled experimental animal study.Tertiary care university-based research institute.One hundred ninety-eight adult male rats.Using a rat model of "systemic inflammation-induced cardiac dysfunction" induced by intraperitoneal lipopolysaccharide injection (10 mg/kg), hemodynamic efficacy, cardioprotection, and biomechanics were assessed under IV osmotic pump infusions of apelin-13 (0.25 μg/kg/min) or dobutamine (7.5 μg/kg/min).In this model and in both in vivo and ex vivo studies, apelin-13 compared with dobutamine provoked distinctive effects on cardiac function: 1) optimized cardiac energy-dependent workload with improved cardiac index and lower vascular resistance, 2) upgraded hearts' apelinergic responsiveness, and 3) consecutive downstream advantages, including increased urine output, enhanced plasma volume, reduced weight loss, and substantially improved overall outcomes. In vitro studies confirmed that these apelin-13-driven processes encompassed a significant and rapid reduction in systemic cytokine release with dampening of myocardial inflammation, injury, and apoptosis and resolution of associated molecular pathways.In this inflammatory cardiovascular dysfunction, apelin-13 infusion delivers distinct and optimized hemodynamic support (including positive fluid balance), along with cardioprotective effects, modulation of circulatory inflammation and extended survival.

Introduction: p62 is a pleiotropic protein with defined roles in TNFα signaling, protein aggregate formation, and protein degradation processes. Current data suggest that p62 is a stress-response protein, with increased protein levels reported in TAC, MI, I/R, and protein aggregation models of cardiac disease. To date, there has been little study on the gain- or loss- of p62 function in cardiomyocyte/cardiac pathology. Our preliminary data found that adenoviral overexpression of p62 caused cardiomyocyte hypertrophy and cytotoxicity and that p62 was upregulated by pressure-overload stress. Hypothesis: Loss of p62 will be cardioprotective against pressure-overload pathology. Methods: Systemic p62 knockout mice underwent sham or transverse-aortic constriction surgery and were studied longitudinally to 8 weeks post-surgery by echocardiography. Results: Hearts from p62-null mice had significantly preserved cardiac function (%Fractional Shortening) over wild-type controls. p62-deficient mice had significantly less cardiac hypertrophy (heart weight/body weight ratios and myofiber cross-sectional areas) and showed no chamber dilation (LVED) in response to pressure-overload stress, unlike wild-types. Hearts from wild-type mice showed pronounced fibrotic remodeling and induction of apoptosis (TUNEL), while p62 knockouts had significantly less collagen staining and no evidence of apoptotic stimulation. Overexpression of p62 in rat neonatal cardiomyocytes significantly inhibited proteasomal catalytic activities (>50%) and showed increased indices of cardiomyocyte cell death. Conclusion: Our data show that induction of p62 is deleterious in vitro and that loss of p62 imparts cardioprotection against hemodynamic stress in vivo . The beneficial phenotype observed in hearts from p62-deficient mice may be due to p62-dependent mechanisms responsible for proteasomal dysfunction and apoptosis activation.

Rationale: Both nuclear factors of activated T cells (NFAT) and nuclear factor-κB (NFκB) are Rel homology domain (RHD)-containing transcription factors whose independent activities are critically involved in regulating cardiac hypertrophy and failure. Objective: To determine the potential functional interaction between NFAT and NFκB signaling pathways in cardiomyocytes and its role in cardiac hypertrophy and remodeling. Methods and Results: We identified a novel transcriptional regulatory mechanism whereby NFκB and NFAT directly interact and synergistically promote transcriptional activation in cardiomyocytes. We show that the p65 subunit of NFκB coimmunoprecipitates with NFAT in cardiomyocytes, and this interaction maps to the RHD within p65. Overexpression of the p65-RHD disrupts the association between endogenous p65 and NFATc1, leading to reduced transcriptional activity. Overexpression of IκB kinase β (IKKβ) or p65-RHD causes nuclear translocation of NFATc1, and expression of a constitutively nuclear NFATc1-SA mutant similarly facilitated p65 nuclear translocation. Combined overexpression of p65 and NFATc1 promotes synergistic activation of NFAT transcriptional activity in cardiomyocytes, whereas inhibition of NFκB with IκBαM or dominant negative IKKβ reduces NFAT activity. Importantly, agonist-induced NFAT activation is reduced in p65 null mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs. In vivo, cardiac-specific deletion of p65 using a Cre-loxP system causes a ≈50% reduction in NFAT activity in luciferase reporter mice. Moreover, ablation of p65 in the mouse heart decreases the hypertrophic response after pressure overload stimulation, reduces the degree of pathological remodeling, and preserves contractile function. Conclusions: Our results suggest a direct interaction between NFAT and NFκB that effectively integrates 2 disparate signaling pathways in promoting cardiac hypertrophy and ventricular remodeling.

Antagonists of L-type Ca²⁺ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²⁺ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-β (TGF-β) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future.

Hypertrophic heart disease is a leading health problem in Western countries. Here we identified the small EF hand domain-containing protein Ca(2+) and integrin-binding protein-1 (CIB1) in a screen for previously unknown regulators of cardiomyocyte hypertrophy. Yeast two-hybrid screening for CIB1-interacting partners identified a related EF hand domain-containing protein, calcineurin B, the regulatory subunit of the prohypertrophic protein phosphatase calcineurin. CIB1 localizes primarily to the sarcolemma in mouse and human myocardium, where it anchors calcineurin to control its activation in coordination with the L-type Ca(2+) channel. CIB1 protein amounts and membrane association were enhanced in cardiac pathological hypertrophy, but not in physiological hypertrophy. Consistent with these observations, Cib1-deleted mice showed a marked reduction in myocardial hypertrophy, fibrosis, cardiac dysfunction and calcineurin-nuclear factor of activated T cells (NFAT) activity after pressure overload, whereas the degree of physiologic hypertrophy after swimming exercise was not altered. Transgenic mice with inducible and cardiac-specific overexpression of CIB1 showed enhanced cardiac hypertrophy in response to pressure overload or calcineurin signaling. Moreover, mice lacking Ppp3cb (encoding calcineurin A, beta isozyme) showed no enhancement in cardiac hypertrophy associated with CIB1 overexpression. Thus, CIB1 functions as a previously undescribed regulator of cardiac hypertrophy through its ability to regulate the association of calcineurin with the sarcolemma and its activation.

Background— Cardiac cachexia is characterized by an exaggerated loss of skeletal muscle, weakness, and exercise intolerance, although the cause of these effects remains unknown. Here, we hypothesized that the heart functions as an endocrine organ in promoting systemic cachexia by secreting peptide factors such as myostatin. Myostatin is a cytokine of the transforming growth factor-β superfamily that is known to control muscle wasting. Methods and Results— We used a Cre/loxP system to ablate myostatin ( Mstn gene) expression in a cell type-specific manner. As expected, elimination of Mstn selectively in skeletal muscle with a myosin light chain 1f ( MLC1f )- cre allele induced robust hypertrophy in all skeletal muscle. However, heart-specific deletion of Mstn with an Nkx2.5-cre allele did not alter baseline heart size or secondarily affect skeletal muscle size, but the characteristic wasting and atrophy of skeletal muscle that typify heart failure were not observed in these heart-specific null mice, indicating that myocardial myostatin expression controls muscle atrophy in heart failure. Indeed, myostatin levels in the plasma were significantly increased in wild-type mice subjected to pressure overload-induced cardiac hypertrophy but not in Mstn heart-specific deleted mice. Moreover, cardiac-specific overexpression of myostatin, which increased circulating levels of myostatin by 3- to 4-fold, caused a reduction in weight of the quadriceps, gastrocnemius, soleus, and even the heart itself. Finally, to investigate myostatin as a potential therapeutic target for the treatment of muscle wasting in heart failure, we infused a myostatin blocking antibody (JA-16), which promoted greater maintenance of muscle mass in heart failure. Conclusions— Myostatin released from cardiomyocytes induces skeletal muscle wasting in heart failure. Targeted inhibition of myostatin in cardiac cachexia might be a therapeutic option in the future.

Nitric oxide (NO) is known to affect the properties of various proteins via the S-nitrosylation of cysteine residues. This study evaluated the direct effects of the NO donor sodium nitroprusside (SNP) on the pharmacological properties of the AT1 receptor for angiotensin II expressed in HEK-293 cells. SNP dose-dependently decreased the binding affinity of the AT1 receptor without affecting its total binding capacity. This modulatory effect was reversed within 5 min of removing SNP. The effect of SNP was not modified in the presence of the G protein uncoupling agent GTPγS or the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The binding properties of a mutant AT1 receptor in which all five cysteine residues within the transmembrane domains had been replaced by serine was not affected by SNP. Systematic analysis of mutant AT1 receptors revealed that cysteine 289 conferred the sensitivity to SNP. These results suggest that NO decreased the binding affinity of the AT1 receptor by S-nitrosylation of cysteine 289. This modulatory mechanism may be particularly relevant in pathophysiological situations where the beneficial effects of NO oppose the deleterious effects of angiotensin II. Keywords: AT1 receptor, S-nitrosylation, post-translational modification, redox-related regulation, sodium nitroprusside, reactive cysteine thiols, G-protein coupled receptor Introduction The octapeptide hormone angiotensin II (Ang II) produces a wide variety of physiological effects, including vascular contraction, aldosterone secretion, sodium and water retention, neuronal activation, and cardiovascular cell growth and proliferation (for review, see (de Gasparo et al., 2000)). Whereas Ang II can interact with two receptor subtypes (AT1 and AT2), the vast majority of its effects are produced through the activation of the AT1 receptor, which belongs to the G protein-coupled receptor (GPCR) superfamily (Murphy et al., 1991; Sasaki et al., 1991; Kambayashi et al., 1993; Mukoyama et al., 1993; Timmermans et al., 1993). The AT1 receptor functions primarily by coupling to the heterotrimeric G protein (Gq/11), which activates phospholipase C (PLC), which in turn generates the second messengers inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (Balla et al., 1989; Spat et al., 1991). InsP3 causes the release of Ca2+ from the endoplasmic reticulum while diacylglycerol recruits and activates protein kinase C at the plasma membrane. The functional properties of the AT1 receptor are regulated by different post-translational modifications such as N-glycosylation (Deslauriers et al., 1999; Jayadev et al., 1999; Lanctot et al., 1999) and phosphorylation (Smith et al., 1998a, 1998b; Thomas et al., 1998; Qian et al., 2001). N-glycosylation is important for the maturation of the AT1 receptor and for targeting it to the cell surface. Serine/threonine phosphorylation of the AT1 receptor participates in the recruitment of arrestin and the internalization of the receptor (Kule et al., 2004), whereas tyrosine phosphorylation of the AT1 receptor reportedly mediates the transactivation of the epidermal growth factor receptor (Seta & Sadoshima, 2003). The modification of the cysteine thiol by nitric oxide (NO) is another post-translational modification reported to regulate the activity of proteins (Stamler et al., 1992, 1997). This process, which is called S-nitrosylation, is a reversible redox-related post-translational modification that modulates protein functionality. NO is an unstable, gaseous, second messenger that can diffuse across membranes and that is involved in vascular tone, neurotransmission, and immune defense (Lowenstein et al., 1994). It is generated by nitric oxide synthase (NOS) from L-arginine and molecular oxygen (Alderton et al., 2001) and activates soluble guanylyl cyclase, which produces cGMP (Foster et al., 1999). Whereas most of the effects of NO have been attributed to cGMP production, recent evidence suggests that S-nitrosylation is involved in the regulation of several proteins. For example, S-nitrosylation of cysteine 118 of the GTPase p21ras, S-nitrosylation of cysteine 3635 of the ryanodine receptor , and denitrosylation of caspase-3 all lead to the activation of these proteins (Lander et al., 1997; Mannick et al., 1999; Eu et al., 2000; Sun et al., 2001). Among the 10 cysteines in the sequence of the AT1 receptor, four cysteines on the extracellular loops are involved in the formation of intramolecular disulfide bridges (Yamano et al., 1992; Ohyama et al., 1995) while only one cysteine is located in the cytoplasmic tail of the receptor. The other five cysteines are distributed within the seven transmembrane domains of the receptor at positions 76, 121, 149, 289, and 296. As NO distributes preferentially in membranes (Liu et al., 1998) and because hydrophobic environment promotes S-nitrosylation (Hess et al., 2005), we hypothesized that the five cysteines located in the transmembrane domains are potential targets for S-nitrosylation. To verify this, we treated HEK-293 cells that stably express the AT1 receptor with the NO donor sodium nitroprusside (SNP) and evaluated the pharmacological and functional properties of the AT1 receptor. We also analyzed the susceptibility to S-nitrosylation of a series of AT1 receptor mutants in which the cysteines had been replaced by serines. Our results suggest that S-nitrosylation of cysteine 289 decreases the binding affinity of the AT1 receptor.

The diverse cellular changes brought about by the expression of a constitutively active receptor are poorly understood. QBI-human embryonic kidney 293A cells stably expressing the constitutively active N111G-AT1 receptor (N111G cells) showed elevated levels of inositol phosphates and frequent spontaneous intracellular Ca2+ oscillations. Interestingly, Ca2+ transients triggered with maximal doses of angiotensin II were much weaker in N111G cells than in wild-type cells. These blunted responses were observed independently of the presence or absence of extracellular Ca2+ and were also obtained when endogenous muscarinic and purinergic receptors were activated, revealing a heterologous desensitization process. The desensitized component of the Ca2+ signaling cascade was neither the G protein Gq nor phospholipase C. The intracellular Ca2+ store of N111G cells and their mechanism of Ca2+ entry also appeared to be intact. The most striking adaptive response of N111G cells was a down-regulation of their inositol 1,4,5-trisphosphate receptor (IP3R) as revealed by reduced IP3-induced Ca2+ release, lowered [3H]IP3 binding capacity, diminished IP3R immunoreactivity, and accelerated IP3R degradation involving the lysosomal pathway. Treatment with the inverse agonist EXP3174 reversed the desensitized phenotype of N111G cells. Down-regulation of IP3R represents a reversible adaptive response to protect cells against the adverse effects of constitutively active Ca2+-mobilizing receptors.

The type 1 receptor for angiotensin II (AT(1)) is a member of the G protein-coupled receptor family. The presence of a caveolin-binding-like motif (phiXphiXXXXphiXXphi where phi is an aromatic residue) within the cytoplasmic tail of the AT(1) receptor suggests an implication for caveolae in the functionality of this receptor. We constructed a mutant AT(1) receptor where each of the aromatic residues in the caveolin-binding-like motif were replaced by alanine (AT(1)-YFFY/A). Mutation of this motif considerably reduced the plasma membrane expression of the receptor that accumulated in a perinuclear compartment. The agonist-induced internalization rate of the AT(1)-YFFY/A receptor was also significantly reduced. Finally, the AT(1)-YFFY/A receptor was poorly activated as indicated by a low agonist-induced production of inositol phosphates. Unexpectedly, the proportion of AT(1) receptor found in caveolae was minor under basal conditions and did not increase under stimulated conditions. Coexpression of the AT(1) receptor with dopamine receptor interacting protein of 78 kDa, a protein implicated in the cellular routing of the dopamine D1 receptor, increased plasma membrane expression of the AT(1) receptor. However, dopamine receptor interacting protein of 78 kDa had no effect on the expression of the AT(1)-YFFY/A receptor. Taken together, these results suggest that the caveolin-binding-like motif of the AT(1) receptor does not promote localization of the receptor to caveolae but rather may act as a docking site for regulatory proteins modulating the routing and the functionality of the receptor.

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.

Rationale: Mitogen-activated protein kinases (MAPKs) are activated in the heart by disease-inducing and stress-inducing stimuli, where they participate in hypertrophy, remodeling, contractility, and heart failure. A family of dual-specificity phosphatases (DUSPs) directly inactivates each of the MAPK terminal effectors, potentially serving a cardioprotective role. Objective: To determine the role of DUSP1 and DUSP4 in regulating p38 MAPK function in the heart and the effect on disease. Methods and Results: Here, we generated mice and mouse embryonic fibroblasts lacking both Dusp1 and Dusp4 genes. Although single nulls showed no molecular effects, combined disruption of Dusp1/4 promoted unrestrained p38 MAPK activity in both mouse embryonic fibroblasts and the heart, with no change in the phosphorylation of c-Jun N-terminal kinases or extracellular signal-regulated kinases at baseline or with stress stimulation. Single disruption of either Dusp1 or Dusp4 did not result in cardiac pathology, although Dusp1/4 double-null mice exhibited cardiomyopathy and increased mortality with aging. Pharmacological inhibition of p38 MAPK with SB731445 ameliorated cardiomyopathy in Dusp1/4 double-null mice, indicating that DUSP1/4 function primarily through p38 MAPK in affecting disease. At the cellular level, unrestrained p38 MAPK activity diminished cardiac contractility and Ca 2+ handling, which was acutely reversed with a p38 inhibitory compound. Poor function in Dusp1/4 double-null mice also was partially rescued by phospholamban deletion. Conclusions: Our data demonstrate that Dusp1 and Dusp4 are cardioprotective genes that play a critical role in the heart by dampening p38 MAPK signaling that would otherwise reduce contractility and induce cardiomyopathy.

Antagonists of L-type Ca2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C–/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C–/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C–/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C+/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca(2+) following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca(2+) handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca(2+)-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the alpha-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca(2+)-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.

Introduction: Skeletal muscle wasting during heart failure constitutes a major therapeutic challenge. The TGFβ superfamily member myostatin is a negative regulator of skeletal muscle growth. For example, elimination of myostatin (MSTN) in adult mice through an inducible Cre/lox recombination strategy has been shown to increase skeletal muscle mass by about 25%. Previous studies identified skeletal muscle and to a lesser extent cardiac and fat tissue as the source of MSTN production in the body. MSTN is produced as a precursor protein, which has been suggested to constitute the main reservoir of the protein in skeletal muscle. In mouse serum, however, MSTN is abundantly present in its mature form, which consists of the C-terminal fragment of the precursor protein. Results: We detected high levels of the mature MSTN protein (MM) in the mouse myocardium by western blotting. Interestingly, MM was significantly upregulated in the myocardium of mice subjected to long-term myocardial pressure overload (TAC, 12 weeks; protein levels: sham 100±22% vs. TAC 218±18%, p Conclusion: Myocardial MSTN contributes to the development of skeletal muscle wasting in heart failure, most likely through an endocrine mechanism involving its secretion into the circulatory system.

The expression of a constitutively active G protein-coupled receptor is expected to trigger diverse cellular changes ranging from normal to adaptive responses. We report that confluent HEK-293 cells stably expressing the constitutively active mutant N111G-AT1 receptor for angiotensin II spontaneously exhibited dramatic morphological changes and cytoskeletal reorganization. Phase-contrast microscopy revealed that these cells formed a dense monolayer, whereas cells expressing the WT-AT1 receptor displayed large intercellular spaces and numerous filopodia. Confocal microscopy revealed an elaborate web of polymerized actin at the apical and basolateral surfaces of cells expressing the N111G-AT1 receptor. Interestingly, these phenotypic changes were prevented by culturing the cells in the presence of the inverse agonist EXP3174. Similar morphologic rearrangements and de novo polymerized actin structures were found in Ang II-stimulated cells expressing the WT-AT1 receptor. We further showed that AT1 receptor-induced cell–cell contact formation did not require an increase in intracellular Ca2+ concentration or the activity of protein kinase C. However, pretreatment with Y-27632 revealed that Rho-kinase activity was required for cell–cell contact formation upon AT1 receptor activation. These observations demonstrate that the expression of the constitutively active mutant N111G-AT1 receptor had a significant impact on the morphology and cytoskeletal organization of HEK-293 cells, possibly via a mechanism involving the activity of Rho-kinase.

Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity (without agonist) for N111A-AT1 and N111G-AT1 to a significant loss of activity (upon maximal stimulation) for N111Q-AT1 and a major loss of activity for N111W-AT1. In Ca2+ mobilization studies, N111W-AT1 produced a weak Ca2+ transient and, unexpectedly, N111G-AT1 also produced a Ca2+ transient that was much weaker than that of the wild-type AT1. The agonist binding affinity of N111W-AT1 was not modified in the presence of GTPγ S, suggesting that this receptor is not basally coupled to a G protein. GTPγ S did not modify the high agonist-binding affinity of N111G-AT1 but abolished the coimmunoprecipitation of Gq/11α with this constitutively active mutant receptor. These results are a direct demonstration that the N111G-AT1 receptor maintains a high affinity conformation despite being uncoupled from the G protein Gq/11.

The human angiotensin II type 1 receptor (hAT 1 ) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, 1 2 5 I-[Sar 1 , Val 5 , p-Benzoyl-L-phenylalanine 8 ]AngII ( 1 2 5 I-[Sar 1 ,Bpa 8 ]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT 1 . Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe 2 9 3 and Asn 2 9 4 within the seventh transmembrane domain of the hAT 1 . Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT 1 produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT 1 receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.