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Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvβ3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 μg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvβ3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kairbaan Hodivala-Dilke reports a relationship with Ellipses Pharma that includes: consulting or advisory. Kairbaan Hodivala-Dilke reports a relationship with Vasodynamics that includes: consulting or advisory. Kairbaan Hodivala-Dilke reports a relationship with RGDscience Ltd that includes: consulting or advisory. Horst Kessler and Amnon Hoffman and Michael Weinmuller has patent #WO2019058374A1 pending to YISSUM RES DEV CO OF HEBREW UNIV JERUSALEM LTD [IL]; UNIV MUENCHEN TECH [DE]. Kairbaan Hodivala-Dilke has patent #WO2021032955A1 pending to UNIV LONDON QUEEN MARY [GB]. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1 CKO ) mouse model. PMCA1 CKO mice developed abnormal heart rhythms related to ventricular repolarisation dysfunction and displayed an increased susceptibility to ventricular arrhythmias. We further assessed the levels of cardiac ion channels using qPCR and found a downregulation of the voltage-dependent potassium channels, K v 4.2, with a corresponding reduction in the transient outward potassium current which underlies ventricular repolarisation in the murine heart. The changes in heart rhythm were found to occur in the absence of any structural cardiomyopathy. To further assess the molecular changes occurring in PMCA1 CKO hearts, we performed proteomic analysis. Functional characterisation of differentially expressed proteins suggested changes in pathways related to metabolism, protein-binding, and pathways associated cardiac function including β-adrenergic signalling. Together, these data suggest an important role for PMCA1 in basal cardiac function in relation to heart rhythm control, with reduced cardiac PMCA1 expression resulting in an increased risk of arrhythmia development., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Induction of endogenous regenerative capacity has emerged as one promising approach to repair damaged hearts following myocardial infarction (MI). Re-expression of factors that are exclusively expressed during embryonic development may reactivate the ability of adult cardiomyocytes to regenerate. Here, we identified miR-411 as a potent inducer of cardiomyocyte proliferation. Overexpression of miR-411 in the heart significantly increased cardiomyocyte proliferation and survival in a model MI. We found that miR-411 enhances the activity of YAP, the main downstream effector of the Hippo pathway, in cardiomyocytes. In conclusion, miR-411 induces cardiomyocyte regeneration and improves cardiac function post-MI likely by modulating the Hippo/YAP pathway., Competing Interests: This study was supported by British Heart Foundation Project and Programme grants [PG/17/78/33304 and RG/F/21/110055 to Dr Oceandy] and a Medical Research Council research grant [MR/P015816/1 to Dr Oceandy]. Dr Nugroho was supported by an Indonesian LPDP (Lembaga Pengelola Dana Pendidikan/Indonesia Endowment Funds for Education) PhD scholarship (S-476/LPDP.3/2016). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Mitochondrial dysfunction is a feature of type I and type II diabetes, but there is a lack of consistency between reports and links to disease development. We aimed to investigate if mitochondrial structure-function remodelling occurs in the early stages of diabetes by employing a mouse model (GENA348) of Maturity Onset Diabetes in the Young, exhibiting hyperglycemia, but not hyperinsulinemia, with mild left ventricular dysfunction. Employing 3-D electron microscopy (SBF-SEM) we determined that compared to wild-type, WT, the GENA348 subsarcolemma mitochondria (SSM) are ~ 2-fold larger, consistent with up-regulation of fusion proteins Mfn1, Mfn2 and Opa1. Further, in comparison, GENA348 mitochondria are more irregular in shape, have more tubular projections with SSM projections being longer and wider. Mitochondrial density is also increased in the GENA348 myocardium consistent with up-regulation of PGC1-α and stalled mitophagy (down-regulation of PINK1, Parkin and Miro1). GENA348 mitochondria have more irregular cristae arrangements but cristae dimensions and density are similar to WT. GENA348 Complex activity (I, II, IV, V) activity is decreased but the OCR is increased, potentially linked to a shift towards fatty acid oxidation due to impaired glycolysis. These novel data reveal that dysregulated mitochondrial morphology, dynamics and function develop in the early stages of diabetes., (© 2022. The Author(s).)

Ischaemic heart disease is the world's leading cause of mortality. Survival rates from acute myocardial infarction (MI) have improved in recent years; however, this has led to an increase in the prevalence of heart failure (HF) due to chronic remodelling of the infarcted myocardium, for which treatment options remain poor. We have previously shown that inhibition of isoform 4 of the plasma membrane calcium ATPase (PMCA4) prevents chronic remodelling and HF development during pressure overload, through fibroblast mediated Wnt signalling modulation. Given that Wnt signalling also plays a prominent role during remodelling of the infarcted heart, this study investigated the effect of genetic and functional loss of PMCA4 on cardiac outcomes following MI. Neither genetic deletion nor pharmacological inhibition of PMCA4 affected chronic remodelling of the post-MI myocardium. This was the case when PMCA4 was deleted globally, or specifically from cardiomyocytes or fibroblasts. PMCA4-ablated hearts were however less prone to acute arrhythmic events, which may offer a slight survival benefit. Overall, this study demonstrates that PMCA4 inhibition does not affect chronic outcomes following MI.

Inflammation plays a key role during cardiac hypertrophy and the development of heart failure. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine that is expressed in the heart and may play a crucial role in cardiac remodeling. Based on the evidence that IL-10 potentially reduces pathological hypertrophy, it was hypothesized that signaling via the IL-10 receptor (IL10R) in the heart produces a protective role in reducing cardiac hypertrophy. The aim of this study was to investigate the effects of the ablation of Il-10-r1 gene during pathological cardiac hypertrophy in mice. We found that IL-10R1 gene silencing in cultured cardiomyocytes diminished the anti-hypertrophic effect of Il-10 in TNF-α induced hypertrophy model. We then analyzed mice deficient in the Il-10-r1 gene (IL-10R1 -/- mice) and subjected them to transverse aortic constriction or isoproterenol infusion to induce pathological hypertrophy. In response to transverse aortic constriction for 2 weeks, IL-10R1 -/- mice displayed a significant increase in the hypertrophic response as indicated by heart weight/body weight ratio, which was accompanied by significant increases in cardiomyocyte surface area and interstitial fibrosis. In contrast, there was no difference in hypertrophic response to isoproterenol infusion (10 days) between the knockout and control groups. Analysis of cardiac function using echocardiography and invasive hemodynamic studies did not show any difference between the WT and IL-10R1 -/- groups, most likely due to the short term nature of the models. In conclusion, our data shows that signaling via the IL-10 receptor may produce protective effects against pressure overload-induced hypertrophy but not against β-adrenergic stimuli in the heart. Our data supports previous evidence that signaling modulated by IL-10 and its receptor may become a potential target to control pathological cardiac hypertrophy., (Copyright © 2020 Stafford, Assrafally, Prehar, Zi, De Morais, Maqsood, Cartwright, Muller and Oceandy.)

Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells. Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function, leading to severe cardiomyopathy and death. We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5'UTRs. Taken together we identify a critical role for BUD23 in bioenergetics gene expression, by promoting efficient translation of mRNA transcripts with low 5'UTR GC content. BUD23 emerges as essential to mouse development, and to postnatal cardiac function., Competing Interests: MB, MV, TP, GG, CP, LG, AK, KK, RM, MI, MZ, SP, EC, JG, LM, AA, NH, PR, MM, DB, AL, DR No competing interests declared, (© 2020, Baxter et al.)

Background: The mouse model of transverse aortic constriction (TAC) has been widely used as a cardiac stress in the investigation of the molecular mechanisms of cardiac hypertrophy. Recently, the International Knockout Mouse Consortium has selected the C57BL/6NTac (BL/6N) mouse strain to generate null alleles for all mouse genes; however, a range of genetic and cardiac phenotypic differences have been reported between this substrain and the commonly used C57BL/6J (BL/6J) substrain. It has been reported by Garcia-Menendez and colleagues that 12-week C57BL/6NTac mice are susceptible to heart failure but little is known about the cardiac remodeling in this substrain as cardiac function progresses from compensation to decompensation., Methods: BL/6J and BL/6N mice were subjected to pressure overload via TAC. The impact of both age and duration of cardiac pressure overload induced by TAC on cardiac remodelling were systematically assessed., Results: Our data showed that BL/6N mice developed eccentric hypertrophy with age- and time-dependent deterioration in cardiac function, accompanied by considerable interstitial fibrosis. In contrast, BL/6J mice were more resilient to TAC-induced cardiac stress and developed variable cardiac phenotypes independent of age and the duration of pressure overload. This was likely due to the greater variability in pre-TAC aortic arch dimension as measured by echocardiography. In addition to increased expression of brain natriuretic peptide and collagen gene type 1 and 3, BL/6N mice also had greater angiotensin II type 2 receptor (AT2R) gene expression than BL/6J counterparts at baseline and after 2-weeks TAC, which may contribute to the exacerbated interstitial fibrosis., Conclusions: BL/6N and BL/6J mice have very different responses to TAC stimulation and these differences should be taken into consideration when using the substrains to investigate the mechanisms of hypertrophy and heart failure., Competing Interests: Authors have no conflicts of interest to disclose., (© 2019 The Authors.)

Background and Purpose: The Hippo pathway has emerged as a potential therapeutic target to control pathological cardiac remodelling. The core components of the Hippo pathway, mammalian Ste-20 like kinase 1 (Mst1) and mammalian Ste-20 like kinase 2 (Mst2), modulate cardiac hypertrophy, apoptosis, and fibrosis. Here, we study the effects of pharmacological inhibition of Mst1/2 using a novel inhibitor XMU-MP-1 in controlling the adverse effects of pressure overload-induced hypertrophy., Experimental Approach: We used cultured neonatal rat cardiomyocytes (NRCM) and C57Bl/6 mice with transverse aortic constriction (TAC) as in vitro and in vivo models, respectively, to test the effects of XMU-MP-1 treatment. We used luciferase reporter assays, western blots and immunofluorescence assays in vitro, with echocardiography, qRT-PCR and immunohistochemical methods in vivo., Key Results: XMU-MP-1 treatment significantly increased activity of the Hippo pathway effector yes-associated protein and inhibited phenylephrine-induced hypertrophy in NRCM. XMU-MP-1 improved cardiomyocyte survival and reduced apoptosis following oxidative stress. In vivo, mice 3 weeks after TAC, were treated with XMU-MP-1 (1 mg·kg -1 ) every alternate day for 10 further days. XMU-MP-1-treated mice showed better cardiac contractility than vehicle-treated mice. Cardiomyocyte cross-sectional size and expression of the hypertrophic marker, brain natriuretic peptide, were reduced in XMU-MP-1-treated mice. Improved heart function in XMU-MP-1-treated mice with TAC, was accompanied by fewer TUNEL positive cardiomyocytes and lower levels of fibrosis, suggesting inhibition of cardiomyocyte apoptosis and decreased fibrosis., Conclusions and Implications: The Hippo pathway inhibitor, XMU-MP-1, reduced cellular hypertrophy and improved survival in cultured cardiomyocytes and, in vivo, preserved cardiac function following pressure overload., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor β (TGF-β) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-β signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-β signalling and likely represents a mechanism to limit fibrosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild-type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild-type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl-l-NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1 Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1 Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Background: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death., Methods: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell-derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell-derived cardiomyocytes., Results: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K + channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts., Conclusions: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported., (© 2016 American Heart Association, Inc.)

The G-protein-coupled receptors (GPCRs) family of proteins play essential roles in the heart, including in the regulation of cardiac hypertrophy. One member of this family, the oxoglutarate receptor 1 (OXGR1), may have a crucial role in the heart because it acts as a receptor for α-ketoglutarate, a metabolite that is elevated in heart failure patients. OXGR1 is expressed in the heart but its precise function during cardiac pathophysiological process is unknown. Here we used both in vivo and in vitro approaches to investigate the role of OXGR1 in cardiac hypertrophy. Genetic ablation of Oxgr1 in mice (OXGR1 -/- ) resulted in a significant increase in hypertrophy following transverse aortic constriction (TAC). This was accompanied by reduction in contractile function as indicated by cardiac fractional shortening and ejection fraction. Conversely, adenoviral mediated overexpression of OXGR1 in neonatal rat cardiomyocytes significantly reduced phenylephrine-induced cardiomyocyte hypertrophy, a result that was consistent with the in vivo data. Using a combination of yeast two hybrid screening and phospho-antibody array analysis we identified novel interacting partner and downstream signalling pathway that might be regulated by the OXGR1. First, we found that OXGR1 forms a molecular complex with the COP9 signalosome complex subunit 5 (CSN5). Secondly, we observed that the STAT3 signalling pathway was upregulated in OXGR1 -/- hearts. Since CSN5 interacts with TYK2, a major upstream regulator of STAT3, OXGR1 might regulate the pro-hypertrophic STAT3 pathway via interaction with the CSN5-TYK2 complex. In conclusion, our study has identified OXGR1 as a novel regulator of pathological hypertrophy via the regulation of the STAT3. Identification of molecules that can specifically activate or inhibit this receptor may be very useful in the development of novel therapeutic approach for pathological cardiac hypertrophy., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

Pathological cardiac hypertrophy is regarded as a critical intermediate step toward the development of heart failure. Many signal transduction cascades are demonstrated to dictate the induction and progression of pathological hypertrophy; however, our understanding in regulatory mechanisms responsible for the suppression of hypertrophy remains limited. In this study, we showed that exacerbated hypertrophy induced by pressure overload in cardiac-deleted Pak1 mice was attributable to a failure to upregulate the antihypertrophic E3 ligase, Fbxo32, responsible for targeting proteins for the ubiquitin-degradation pathway. Under pressure overload, cardiac overexpression of constitutively active Pak1 mice manifested strong resilience against pathological hypertrophic remodeling. Mechanistic studies demonstrated that subsequent to Pak1 activation, the binding of Smad3 on a critical singular AGAC(-286)-binding site on the FBXO32 promoter was crucial for its transcriptional regulation. Pharmacological upregulation of Fbxo32 by Berberine ameliorated hypertrophic remodeling and improved cardiac performance in cardiac-deficient Pak1 mice under pressure overload. Our findings discover Smad3 and Fbxo32 as novel downstream components of the Pak1-dependent signaling pathway for the suppression of hypertrophy. This discovery opens a new venue for opportunities to identify novel targets for the management of cardiac hypertrophy., (© 2015 American Heart Association, Inc.)

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2(-/-) mice showed no alteration in cardiomyocyte proliferation. However, Mst2(-/-) mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of cardiac hypertrophy and myocyte survival. Extracellular signal regulated protein kinase 1 and 2 (ERK1/2) are key components in the MAPK signaling pathways. Dysfunction of ERK1/2 in congenital heart diseases (Noonan syndrome and LEOPARD syndrome) leads to cardiac hypertrophy. ERK2 contributes 70% of protein content to total ERK1/2 content in myocardium; however, the specific role of ERK2 in regulating cardiac hypertrophy is yet to be further defined. To investigate the specific role of ERK2 played in the cardiomyocytes, we generated and examined mice with cardiomyocyte-specific deletion of the erk2 gene (ERK2(cko) mice). Following short-term pathological hypertrophic stresses, the mutant mice showed attenuated hypertrophic remodeling characterized by a blunted increase in the cross-sectional area of individual myocytes, downregulation of hypertrophic foetal gene markers (ANP and BNP), and less interstitial fibrosis. However, increased cardiomyocyte apoptosis was observed. Upon prolonged stimulation, ERK2(cko) mice developed deterioration in cardiac function. However, absence of ERK2 did not affect physiological hypertrophy induced by 4weeks of swimming exercise. These results revealed an essential role for ERK2 in cardiomyocytes in the development of pathological hypertrophic remodeling and resistance to cell death., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Aims: Tumour necrosis factor-α (TNF-α) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-α receptors (TNFRs), the mechanism of TNF-α signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-α signalling in cardiomyocytes., Methods and Results: We used RASSF1A knockout (RASSF1A(-/-)) mice and wild-type (WT) littermates in this study. Acute stimulation with a low dose of TNF-α (10 µg/kg iv) increased cardiac contractility and intracellular calcium transients' amplitude in WT mice. In contrast, RASSF1A(-/-) mice showed a blunted contractile response. Mechanistically, RASSF1A was essential in the formation of the TNFR complex (TNFRC), where it functions as an adaptor molecule to facilitate the recruitment of TNFR type 1-associated death domain protein and TNFR-associated factor 2 to form the TNF-α receptor complex. In the absence of RASSF1A, signal transmission from the TNF-α receptor complex to the downstream effectors, such as cytoplasmic phospholipase A2 and protein kinase A, was attenuated leading to the reduction in the activation of calcium handling molecules, such as L-type Ca(2+) channel and ryanodine receptors., Conclusion: Our data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNFRC and in signal transmission to the downstream targets., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Endurance athletes exhibit sinus bradycardia, that is a slow resting heart rate, associated with a higher incidence of sinus node (pacemaker) disease and electronic pacemaker implantation. Here we show that training-induced bradycardia is not a consequence of changes in the activity of the autonomic nervous system but is caused by intrinsic electrophysiological changes in the sinus node. We demonstrate that training-induced bradycardia persists after blockade of the autonomous nervous system in vivo in mice and in vitro in the denervated sinus node. We also show that a widespread remodelling of pacemaker ion channels, notably a downregulation of HCN4 and the corresponding ionic current, If. Block of If abolishes the difference in heart rate between trained and sedentary animals in vivo and in vitro. We further observe training-induced downregulation of Tbx3 and upregulation of NRSF and miR-1 (transcriptional regulators) that explains the downregulation of HCN4. Our findings provide a molecular explanation for the potentially pathological heart rate adaptation to exercise training.

Background: Hypertension or aortic stenosis causes pressure overload, which evokes hypertrophic myocardial growth. Sustained cardiac hypertrophy eventually progresses to heart failure. Growing evidence indicates that restraining hypertrophy could be beneficial; here, we discovered that FTY-720, an immunomodulator for treating multiple sclerosis, can reverse existing cardiac hypertrophy/fibrosis., Methods and Results: Male C57/Bl6 mice underwent transverse aortic constriction (TAC) for 1 week followed by FTY-720 treatment for 2 weeks under continuing TAC. Compared with vehicle-treated TAC hearts, FTY-720 significantly reduced ventricular mass, ameliorated fibrosis, and improved cardiac performance. Mechanistic studies led us to discover that FTY-720 appreciably inhibited nuclear factor of activated T-cells (NFAT) activity. Moreover, we found that in primary cardiomyocytes (rat and human) pertussis toxin (Gi-coupled receptor inhibitor) substantially blocked the antihypertrophic effect of FTY-720. This observation was confirmed in a mouse model of pressure overload. Interestingly, gene array analysis of TAC hearts revealed that FTY-720 profoundly decreased gene expression of a group of matricellular proteins, of which periostin was prominent. Analysis of periostin protein expression in TAC-myocardium, as well as in rat and human cardiac fibroblasts, confirmed the array data. Moreover, we found that FTY-720 treatment or knockdown of periostin protein was able to inhibit transforming growth factor-β responsiveness and decrease collagen expression., Conclusions: FTY-720 alleviates existing cardiac hypertrophy/fibrosis through mechanisms involving negative regulation of NFAT activity in cardiomyocytes and reduction of periostin expression allowing for a more homeostatic extracellular compartment milieu. Together, FTY-720 or its analogues could be a promising new approach for treating hypertrophic/fibrotic heart disease.

Background: Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy., Methods and Results: Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation., Conclusions: These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac β-adrenergic signaling.

There is little doubt that members of mitogen-activated protein kinase (MAPK) families play key roles in the transition from adaptive hypertrophic remodeling to heart failure. Mitogen-activated protein kinase kinase 7 (MKK7) is a critical component of stress-activated MAP kinase signaling pathway. The role of MKK7 plays in mediating cardiac remodeling in response to load stress has yet to be defined. Herein, we investigate the role of MKK7 in regulating cardiac remodeling in response to pressure overload. We generated and examined the phenotype of mice with cardiomyocyte-specific deletion of the mkk7 gene (MKK7(cko)). Following one week of pressure overload, MKK7(cko) mice exhibited characteristic phenotypes of heart failure evidenced by deterioration in ventricular function and pulmonary congestion. Cell death assays revealed an increased prevalence of cardiomyocyte apoptosis in the MKK7(cko) heart, in which elevated p53 levels and attenuated expression of manganese superoxide dismutase (MnSOD) were found. Moreover, extensive interstitial fibrosis was discovered in the knockout heart likely attributable to upregulation of transforming growth factor β (TGF-β) signaling. These results reveal an essential role of MKK7 in cardiomyocytes for protecting the heart from hypertrophic insults thereby preventing the transition to heart failure., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Rationale: Mitogen-activated protein kinase (MAPK) pathways provide a critical connection between extrinsic and intrinsic signals to cardiac hypertrophy. Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is activated in the heart by pressure overload. However, the role of ERK5 plays in regulating hypertrophic growth and hypertrophy-induced apoptosis is not completely understood., Objective: Herein, we investigate the in vivo role and signaling mechanism whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis., Methods and Results: We generated and examined the phenotypes of mice with cardiomyocyte-specific deletion of the erk5 gene (ERK5(cko)). In response to hypertrophic stress, ERK5(cko) mice developed less hypertrophic growth and fibrosis than controls. However, increased apoptosis together with upregulated expression levels of p53 and Bad were observed in the mutant hearts. Consistently, we found that silencing ERK5 expression or specific inhibition of its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs) reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs using a non-DNA binding mutant form of MEF2 was found to attenuate the ERK5-regulated hypertrophic response., Conclusions: These results reveal an important function of ERK5 in cardiac hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in hypertrophic remodeling is likely to be mediated via the regulation of MEF2 activity.

Background: Although, it is accepted that there is an excess of cardiovascular mortality in acromegaly, it is uncertain whether this is due to the direct effects of growth hormone-induced-cardiomyopathy or is a consequence of atherosclerosis secondary to the metabolic syndrome often observed in this condition. Direct comparison of a mouse model of acromegaly to a mouse model of Laron's syndrome allowed us to carry out detailed phenotyping and better understand the role GH plays in the circulatory system., Methods and Results: Transgenic mice that overexpress the growth hormone gene (GH) developed gigantism, including insulin resistance and higher blood pressures commensurate with increased body mass. In these giant mice, the hearts were hypertrophied but haemodynamic studies suggested contractile function was normal. Segments of small arteries mounted in a pressure myograph showed vascular wall hypertrophy but a preserved lumen diameter. Vascular contractile function was normal. Mice in which the GH receptor gene was disrupted or 'knocked out' were dwarf and had low blood pressure, small hearts and blood vessels but a normally functioning circulation. Correlations of body mass with cardiovascular parameters suggested that blood pressure and structural characteristics develop in line with body size., Conclusion: In this transgenic mouse model of acromegaly, there is cardiac and vascular hypertrophy commensurate with GH excess but normal function. Our findings support the contention that the excess mortality in this condition may be due to the development of hypertrophic cardiomyopathy rather than increased rates of atherosclerotic coronary artery disease.

Background: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy., Methods and Results: A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras., Conclusions: Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Mitogen-activated protein kinase kinase (MKK)4 is a critical member of the mitogen-activated protein kinase family. It is able to activate the c-Jun NH(2)-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase in response to environmental stresses. JNK and p38 are strongly implicated in pathological cardiac hypertrophy and heart failure; however, the regulatory mechanism whereby the upstream kinase MKK4 activates these signaling cascades in the heart is unknown. To elucidate the biological function of MKK4, we generated mice with a cardiac myocyte-specific deletion of mkk4 (MKK4(cko) mice). In response to pressure overload or chronic beta-adrenergic stimulation, upregulated NFAT (nuclear factor of activated T-cell) transcriptional activity associated with exacerbated cardiac hypertrophy and the appearance of apoptotic cardiomyocytes were observed in MKK4(cko) mice. However, when subjected to swimming exercise, MKK4(cko) mice displayed a similar level of physiological cardiac hypertrophy compared to controls (MKK4(f/f)). In addition, we also discovered that MKK4 expression was significantly reduced in heart failure patients. In conclusion, this study demonstrates for the first time that MKK4 is a key mediator which prevents the transition from an adaptive response to maladaptive cardiac hypertrophy likely involving the regulation of the NFAT signaling pathway.

Background: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo., Methods and Results: We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N omega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates., Conclusions: These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.

The cytokines IL-1 and IL-6 are able to induce prostaglandin (PG)-dependent activation of the hypothalamo-pituitary-adrenal axis (HPAA) and are thought to play key roles in immune-neuroendocrine interactions during inflammation. The present study shows that inflammation induced by im injection of turpentine (TPS) in the hind limb of mice causes an increase in the plasma concentration of IL-6, but not that of IL-1 alpha or IL-1 beta, together with a prolonged (>18-h) activation of the HPAA. IL-6 plays a causal role in the TPS-induced elevation in HPAA activity, because the sustained (8-18 h) increases in 1) plasma corticosterone, 2) plasma ACTH, and 3) induction of c-Fos in the hypothalamic paraventricular nucleus are all markedly blunted in IL-6-deficient (IL-6(-/-)) mice. Peripheral administration of a neutralizing IL-6 antiserum inhibited the plasma corticosterone response of normal (C57BL/6) mice to hind limb inflammation to an extent similar to that seen in IL-6(-/-) mice, suggesting that the IL-6 responsible for the increased HPAA activity is produced, or acts, on the blood side of the blood-brain barrier. We also show that IL-6 in the circulation is induced almost exclusively at the local inflammatory site, where IL-1 beta is produced. Induction of IL-6 and activation of the HPAA are dependent upon prior activation of an IL-1 type I receptor, as both are inhibited in type I IL-1 receptor-deficient mice. Furthermore, hind limb inflammation induced cyclooxygenase-2 protein expression around the cerebrovasculature of normal (IL-6(+/+)), but not IL-6(-/-), mice. Based on these data, we propose that IL-6 is produced at the local inflammatory site under the control of IL-1 beta and is the circulating afferent signal that is in part responsible for elevated HPAA activity, possibly acting via eicosanoid production within the cerebrovasculature.

In vitro receptor autoradiography has been used to study the distribution of [(125)I]endothelin binding sites in human coronary tissue from patients undergoing cardiac transplantation. Dense binding of [(125)I]endothelin was associated with the smooth muscle of epicardial coronary arteries as well as to perivascular regions of these vessels. Binding was also associated with the ventricular myocardium. There was an increased binding of [(125)I]endothelin to atheromatous tissue, both coronary arteries and vein graft. The [(125)I]endothelin binding sites identified using in vitro autoradiography are likely to be functionally relevant since endothelin causes a concentration-dependent contraction of segments of human epicardial coronary arteries in vitro and also has positive inotropic activity on isolated human cardiomyocytes. The presence of specific binding sites for [(125)I]endothelin on coronary tissue and the increased binding in atheromatous tissue suggest that endothelin is a peptide which may play a role in the maintenance of vascular tone and/or the pathogenesis of ischaemic heart disease.

The distribution of endothelin-1 (ET-1) receptors on human vascular tissue has been studied. High- and low-resolution autoradiography was used to determine the distribution of [125]ET binding sites in human blood vessels and ventricular myocardium. Dense, displaceable [125I]ET binding was associated with cardiac myocytes and the smooth muscle layer of all vessels were examined. There was also dense binding to vasa vasora. There was increased [125I]ET binding to atheromatous coronary arteries and vein graft, which was associated with the tunica media and vasa vasora or regions of neovascularization. Vasoconstrictor and positive inotropic activity of ET-1 has been established in vitro. The vasoconstrictor effect of ET-1 is likely to be mediated via the binding sites identified on vascular smooth muscle. The striking perivascular [125I]ET-1 binding suggests that ET-1 may also have constrictor activity on vasa vasora. There is experimental evidence that ET-1 has mitogenic activity on vascular smooth muscle cells in culture. The increased binding to both smooth muscle and regions of neovascularization in atheromatous vessels suggests that ET-1 may play a role in atherosclerosis.