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Background Platelets are key players in the pathophysiology of coronary artery disease (CAD) and platelet hyperreactivity leads to increased risk of developing adverse cardiovascular events. Further, significant changes in the platelet lipidome occur in patients with acute coronary syndrome (ACS) and critically regulated lipids lead to platelet hyperresponsiveness. Statin treatment is crucial in the treatment and prevention of patients with CAD by remodeling lipid metabolism. Objective In this study, we investigate the platelet lipidome of CAD patients by untargeted lipidomics, highlighting significant changes between statin-treated and naïve patients. Methods We characterized the platelet lipidome in a CAD cohort (n = 105) by an untargeted lipidomics approach using liquid chromatography coupled to mass spectrometry. Results Among the annotated lipids, 41 lipids were significantly upregulated in statin-treated patients, whereas 6 lipids were downregulated compared to naïve patients. The most prominent upregulated lipids in statin-treated patients belong to the class of triglycerides, cholesteryl esters, palmitic acid, and oxidized phospholipids, whereas mainly glycerophospholipids were downregulated compared to untreated patients. A more pronounced effect of statin treatment on the platelet lipidome was observed in ACS patients. We further highlight a dose-dependent influence on the platelet lipidome. Conclusion Our results reveal that the platelet lipidome is altered in CAD patients with statin treatment and upregulated lipids embody mainly characteristic triglycerides, whereas downregulated lipids mostly compromise glycerophospholipids, which may play a role in the pathophysiology of CAD. Results of this study may contribute to the understanding of statin treatment softening the lipid phenotype.

Patients with cardiovascular disease (CVD) and acute SARS-CoV-2 infection might show an altered immune response during COVID-19.Twenty-three patients with CVD and SARS-CoV-2 infection were prospectively enrolled and received a cardiological assessment at study entry and during follow-up visit. Inclusion criteria of our study were age older than 18 years, presence of CVD, and acute SARS-CoV-2 infection. The median age of the patient cohort was 69 (IQR 55-79) years. 12 (52.2%) patients were men. Peripheral monocytes and chemokine/cytokine profiles were analysed.Numbers of classical and non-classical monocytes were significantly decreased during acute SARS-CoV-2 infection compared to 3-month recovery. While classical monocytes reached the expected level in peripheral blood after 3 months, the number of non-classical monocytes remained significantly reduced.All three monocyte subsets exhibited changes of established adhesion and activation markers. Interestingly, they also expressed higher levels of pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF) at the time of recovery, although MIF was only slightly increased during the acute phase.Changes of monocyte phenotypes and increased MIF expression after 3-month recovery from acute SARS-CoV-2 infection may indicate persistent, possibly long-lasting, pro-inflammatory monocyte function in CVD patients.

Introduction Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. Methods We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. Results We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. Conclusion In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Aims Platelets play a key role in the pathophysiology of coronary artery disease (CAD) and patients with enhanced platelet activation are at increased risk to develop adverse cardiovascular events. Beyond reliable cardiovascular risk factors such as dyslipoproteinaemia, significant changes of platelet lipids occur in patients with CAD. In this study, we investigate the platelet lipidome by untargeted liquid chromatography–mass spectrometry, highlighting significant changes between acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) patients. Additionally, we classify the platelet lipidome, spotlighting specific glycerophospholipids as key players in ACS patients. Furthermore, we examine the impact of significantly altered lipids in ACS on platelet-dependent thrombus formation and aggregation. Methods and results In this consecutive study, we characterized the platelet lipidome in a CAD cohort (n = 139) and showed significant changes of lipids between patients with ACS and CCS. We found that among 928 lipids, 7 platelet glycerophospholipids were significantly up-regulated in ACS, whereas 25 lipids were down-regulated compared to CCS. The most prominent up-regulated lipid in ACS, PC18:0 (PC 10:0-8:0), promoted platelet activation and ex vivo platelet-dependent thrombus formation. Conclusions Our results reveal that the platelet lipidome is altered in ACS and up-regulated lipids embody primarily glycerophospholipids. Alterations of the platelet lipidome, especially of medium chain lipids, may play a role in the pathophysiology of ACS.

Aims Beyond classical roles in thrombosis and haemostasis, it becomes increasingly clear that platelets contribute as key players to inflammatory processes. The involvement of platelets in these processes is often mediated through a variety of platelet-derived chemokines which are released upon activation and act as paracrine and autocrine factors. In this study, we investigate CXCL14, a newly described platelet chemokine and its role in thrombus formation as well as monocyte and platelet migration. In addition, we examine the chemokine receptor CXCR4 as a possible receptor for CXCL14 on platelets. Furthermore, with the use of artificially generated platelets derived from induced pluripotent stem cells (iPSC), we investigate the importance of CXCR4 for CXCL14-mediated platelet functions. Methods and results In this study, we showed that CXCL14 deficient platelets reveal reduced thrombus formation under flow compared with wild-type platelets using a standardized flow chamber. Addition of recombinant CXCL14 normalized platelet-dependent thrombus formation on collagen. Furthermore, we found that CXCL14 is a chemoattractant for platelets and mediates migration via CXCR4. CXCL14 promotes platelet migration of platelets through the receptor CXCR4 as evidenced by murine CXCR4-deficient platelets and human iPSC-derived cultured platelets deficient in CXCR4. We found that CXCL14 directly interacts with the CXCR4 as verified by immunoprecipitation and confocal microscopy. Conclusions Our results reveal CXCL14 as a novel platelet-derived chemokine that is involved in thrombus formation and platelet migration. Furthermore, we identified CXCR4 as principal receptor for CXCL14, an interaction promoting platelet migration.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion. CA extern

Supplemental Digital Content is available in the text., Objective: Patients with coronary artery disease (CAD) are at increased risk for cardiac death and respiratory failure following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Platelets are crucially involved in pathogenesis of CAD and might also contribute to pathophysiology of SARS-CoV-2 infection. Approach and Results: We enrolled a cohort of 122 participants from February 2020 to July 2020 including 55 patients with preexisting CAD and acute SARS-CoV-2 infection (CAD-SARS-CoV-2positive), 28 patients with CAD and without SARS-CoV-2 (CAD-SARS-CoV-2negative), and 39 healthy controls. Clinical and cardiac examination of the CAD-SARS-CoV-2positive group included blood sampling, echocardiography, and electrocardiography within 24 hours after hospital admission. Phenotyping of platelets was performed by flow cytometry; plasma levels of chemokines were analyzed by ELISA. Respiratory failure of patients was stratified by the Horovitz index as moderately/severely impaired when Horovitz index

Aims To elucidate the prognostic role of monocytes in the immune response of patients with coronary artery disease (CAD) at risk for life-threatening heart and lung injury as major complications of SARS-CoV-2 infection. Methods and Results From February to April 2020, we prospectively studied a cohort of 96 participants comprising 47 consecutive patients with CAD and acute SARS-CoV-2 infection (CAD+SARS-CoV-2), 19 CAD patients without infections, and 30 healthy controls. Clinical assessment included blood sampling, echocardiography, and electrocardiography within 12 hours of admission. Respiratory failure was stratified by the Horovitz Index (HI) as moderately/severely impaired when HI ≤ 200 mmHg. The clinical endpoint (EP) was defined as HI ≤ 200 mmHg with subsequent mechanical ventilation within a follow-up of 30 days. The numbers of CD14dimCD16+ non-classical monocytes in peripheral blood were remarkably low in CAD+SARS-CoV-2 compared to CAD patients without infection and healthy controls (p, Graphical Abstract Graphical Abstract

The purpose of this study was to investigate the diagnostic value of simultaneous hybrid cardiac magnetic resonance (CMR) andLate gadolinium enhancement (LGE) CMR and FDG-PET are both established imaging techniques for the detection of CS. However, there are limited data regarding the value of a comprehensive simultaneous hybrid CMR/FDG-PET imaging approach that includes CMR mapping techniques.Forty-three patients with biopsy-proven extracardiac sarcoidosis (median age: 48 years, interquartile range: 37-57 years, 65% male) were prospectively enrolled for evaluation of suspected CS. After dietary preparation for suppression of myocardial glucose metabolism, patients were evaluated on a 3-T hybrid PET/MR scanner. The CMR protocol included T1 and T2 mapping, myocardial function, and LGE imaging. We assumed aCS if PET and CMR (ie, LGE or T1/T2 mapping) were both positive (PET+/CMR+), cCS if PET was negative but CMR was positive (PET-/CMR+), and no CS if patients were CMR negative regardless of PET findings.Among the 43 patients, myocardial glucose uptake was suppressed successfully in 36 (84%). Hybrid CMR/FDG-PET revealed aCS in 13 patients (36%), cCS in 5 (14%), and no CS in 18 (50%). LGE was present in 14 patients (39%); T1 mapping was abnormal in 10 (27%) and T2 mapping abnormal in 2 (6%). CS was diagnosed based on abnormal T1 mapping in 4 out of 18 CS patients (22%) who were LGE negative. PET FDG uptake was present in 17 (47%) patients.Comprehensive simultaneous hybrid CMR/FDG-PET imaging is useful for the detection of CS and provides additional value for identifying active disease. Our results may have implications for enhanced diagnosis as well as improved identification of patients with aCS in whom anti-inflammatory therapy may be most beneficial.

AMP-activated protein kinase (Ampk) regulates myocardial energy metabolism and plays a crucial role in the response to cell stress. In the failing heart, an isoform shift of the predominant Ampkα2 to the Ampkα1 was observed. The present study explored possible isoform specific effects of Ampkα1 in cardiomyocytes. To this end, experiments were performed in HL-1 cardiomyocytes, as well as in Ampkα1-deficient and corresponding wild-type mice and mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1. As a result, in HL-1 cardiomyocytes, overexpression of constitutively active Ampkα1 increased the phosphorylation of Pkcζ. Constitutively active Ampkα1 further increased AP-1-dependent transcriptional activity and mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1, effects blunted by Pkcζ silencing. In HL-1 cardiomyocytes, angiotensin-II activated AP-1, an effect blunted by silencing of Ampkα1 and Pkcζ, but not of Ampkα2. In wild-type mice, angiotensin-II infusion increased cardiac Ampkα1 and cardiac Pkcζ protein levels, as well as c-Fos, Il6 and Ncx1 mRNA expression, effects blunted in Ampkα1-deficient mice. Pressure overload by transverse aortic constriction (TAC) similarly increased cardiac Ampkα1 and Pkcζ abundance as well as c-Fos, Il6 and Ncx1 mRNA expression, effects again blunted in Ampkα1-deficient mice. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 increased Pkcζ protein abundance and the mRNA expression of c-Fos, Il6 and Ncx1 in cardiac tissue. In conclusion, Ampkα1 promotes myocardial AP-1 activation in a Pkcζ-dependent manner and thus contributes to cardiac stress signaling.

Aims Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification. Methods and results Experiments were performed in hyperphosphatemic klotho-hypomorphic mice ( kl/kl ), in subtotal nephrectomy and vitamin D3–overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 ( Pai1 ) and of osteogenic markers Msx2 , Cbfa1, and Alpl . Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1 , MSX2 , CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine. Conclusion Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.

Klotho, a protein expressed mainly in the kidney, is required for the inhibitory effect of FGF23 on renal 1,25(OH)2D3 formation. Klotho counteracts vascular calcification and diverse age-related disorders. Klotho-hypomorphic mice (kl/kl) suffer from severe vascular calcification and rapid aging. The calcification is at least in part caused by excessive 1,25(OH)2D3, Ca(2+), and phosphate concentrations in blood, which trigger osteogenic signaling including upregulation of alkaline phosphatase (Alpl). As precipitation of calcium and phosphate is fostered by alkaline pH, extracellular acidosis could counteract tissue calcification. In order to induce acidosis, acetazolamide was added to drinking water (0.8 g/l) of kl/kl and wild-type mice. As a result, acetazolamide treatment of kl/kl mice partially reversed the growth deficit, tripled the life span, almost completely reversed the calcifications in trachea, lung, kidney, stomach, intestine, and vascular tissues, the excessive aortic alkaline phosphatase mRNA levels and the plasma concentrations of osteoprotegerin, osteopontin as well as fetuin-A, without significantly decreasing FGF23, 1,25(OH)2D3, Ca(2+), and phosphate plasma concentrations. In primary human aortic smooth muscle cells, acidotic environment prevented phosphate-induced alkaline phosphatase mRNA expression. The present study reveals a completely novel effect of acetazolamide, i.e., interference with osteoinductive signaling and tissue calcification in kl/kl mice.Klotho deficient (kl/kl) mice suffer from hyperphosphatemia with dramatic tissue calcification. Acetazolamide (ACM) treatment partially reversed the growth deficit of kl/kl mice. In kl/kl mice, ACM reversed tissue calcification despite continued hyperphosphatemia. ACM tripled the life span of kl/kl mice. In human aortic smooth muscle cells, low extracellular pH prevented osteogenic signaling.

Background/Aims: Consequences of obstructive nephropathy include tissue fibrosis, a major pathophysiological mechanism contributing to development of end-stage renal disease. Transforming growth factor β 1 (Tgfβ1) is involved in the progression of renal fibrosis. According to recent observations, ammonium chloride (NH4Cl) prevented phosphate-induced vascular remodeling, effects involving decrease of Tgfβ1 expression and inhibition of Tgfβ1-dependent signaling. The present study, thus, explored whether NH4Cl influences renal Tgfβ1-induced pro-fibrotic signaling in obstructive nephropathy induced by unilateral ureteral obstruction (UUO). Methods: UUO was induced for seven days in C57Bl6 mice with or without additional treatment with NH4Cl (0.28 M in drinking water). Transcript levels were determined by RT-PCR as well as protein abundance by Western blotting, blood pH was determined utilizing a blood gas and chemistry analyser. Results: UUO increased renal mRNA expression of Tgfb1, Tgfβ-activated kinase 1 (Tak1) protein abundance and Smad2 phosphorylation in the nuclear fraction of the obstructed kidney tissues, effects blunted in NH4Cl treated mice as compared to control treated mice. The mRNA levels of the transcription factors nuclear factor of activated T cells 5 (Nfat5) and SRY (sex determining region Y)-box 9 (Sox9) as well as of tumor necrosis factor α (Tnfα), interleukin 6 (Il6), plasminogen activator inhibitor 1 (Pai1) and Snai1 were up-regulated in the obstructed kidney tissues following UUO, effects again significantly ameliorated following NH4Cl treatment. Furthermore, the increased protein and mRNA expression of α-smooth muscle actin (α-Sma), fibronectin and collagen type I in the obstructed kidney tissues following UUO were significantly attenuated following NH4Cl treatment. Conclusion: NH4Cl treatment ameliorates Tgfβ1-dependent pro-fibrotic signaling and renal tissue fibrosis markers following obstructive nephropathy.

Angiotensin-II is a key factor in renal fibrosis. Obstructive nephropathy induces an isoform shift from catalytic Ampkα2 towards Ampkα1 which contributes to signaling involved in renal tissue injury. The present study explored whether the Ampkα1 isoform contributes to the renal effects of angiotensin-II. To this end, angiotensin-II was infused by subcutaneous implantation of osmotic minipumps in gene-targeted mice lacking functional Ampkα1 (Ampkα1(-/-)) and corresponding wild-type mice (Ampkα1(+/+)). Western blotting and qRT-PCR were employed to determine protein abundance and mRNA levels, respectively, in renal tissue. In Ampkα1(+/+) mice, angiotensin-II increased renal Ampkα1 protein expression without significantly modifying renal Ampkα2 protein expression. The renal phosphorylated Ampkα (Thr(172)) protein abundance was not affected by angiotensin-II in neither genotypes, but was significantly lower in Ampkα1(-/-) mice than Ampkα1(+/+) mice. Angiotensin-II increased the phosphorylation of Tak1 (Ser(412)) in renal tissue of Ampkα1(+/+) mice, an effect virtually absent in the Ampkα1(-/-) mice. Furthermore, angiotensin-II treatment significantly increased renal protein and mRNA expression of α-smooth muscle actin (αSma) as well as Tak1-target gene expression: Cox2, Il6 and Pai1 in Ampkα1(+/+) mice, all effects significantly less pronounced in Ampkα1(-/-) mice. In conclusion, angiotensin-II up-regulates the Ampkα1 isoform in renal tissue. Ampkα1 participates in renal Tak1 activation and Tak1-dependent signaling induced by angiotensin-II.

Background: Chorein, a protein supporting activation of phosphoinositide 3 kinase (PI3K), participates in the regulation of actin polymerization and cell survival. A loss of function mutation of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) leads to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with simultaneous erythrocyte akanthocytosis. In blood platelets chorein deficiency has been shown to compromise expression of vesicle-associated membrane protein 8 (VAMP8) and thus degranulation. The present study explored whether chorein is similarly involved in VAMP8 expression and dopamine release of pheochromocytoma (PC12) cells. Methods: Chorein was down-regulated by silencing in PC12 cells. Transmission electron microscopy was employed to quantify the number of vesicles, RTPCR to determine transcript levels, Western blotting to quantify protein expression and ELISA to determine dopamine release. Results: Chorein silencing significantly reduced the number of vesicles, VAMP8 transcript levels and VAMP8 protein abundance. Increase of extracellular K + from 5 mM to 40 mM resulted in marked stimulation of dopamine release, an effect significantly blunted by chorein silencing. Conclusions: Chorein deficiency down-regulates VAMP8 expression, vesicle numbers and dopamine release in pheochromocytoma cells.

Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH4Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH4Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H+ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H+ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.

Background/Aims: The serum- and glucocorticoid-inducible kinase SGK1 participates in the orchestration of cardiac hypertrophy and remodeling. Signaling linking SGK1 activity to cardiac remodeling is, however, incompletely understood. SGK1 phosphorylation targets include cyclin-dependent kinase inhibitor 1B (p27), a protein which suppresses cardiac hypertrophy. The present study explored how effects of SGK1 on nuclear p27 localization might modulate the hypertrophic response in cardiomyocytes. Methods: Experiments were performed in HL-1 cardiomyocytes and in SGK1-deficient (sgk1-/-) and corresponding wild-type (sgk1+/+) mice following pressure overload by transverse aortic constriction (TAC). Transcript levels were quantified by RT-PCR, protein abundance by Western blotting and protein localization by confocal microscopy. Results: In HL-1 cardiomyocytes, overexpression of constitutively active SGK1 (SGK1S422D) but not of inactive SGK1 (SGK1K127N) increased significantly the cell size and transcript levels encoding Acta1, a molecular marker of hypertrophy. Those effects were paralleled by almost complete relocation of p27 in the cytoplasm. Treatment of HL-1 cardiomyocytes with isoproterenol was followed by up-regulation of SGK1 expression. Moreover, isoproterenol treatment stimulated the hypertrophic response and was followed by disappearance of p27 from the nuclei, effects prevented by the SGK1 inhibitor EMD638683. The effect of SGK1S422D overexpression on Acta1 mRNA levels was disrupted by overexpression of p27 and of the p27T197A mutant lacking the SGK1 phosphorylation site, but not of the phosphomimetic p27T197D mutant. In sgk1+/+ mice, TAC increased significantly SGK1 and Acta1 mRNA levels and decreased the nuclear to cytoplasmic protein ratio of p27 in cardiac tissue, effects blunted in the sgk1-/- mice. Conclusion: SGK1-induced hypertrophy of cardiomyocytes involves p27 phosphorylation at T197, which fosters cytoplasmic p27 localization.