Extracellular Vesicles Derived from Human Liver Stem Cells Counteract Chronic Kidney Disease Development and Cardiac Dysfunction in Remnant Kidney Murine Model: The Possible Involvement of Proteases.

Fibrosis is a marker of chronic kidney disease (CKD) and consists of the accumulation of the extracellular matrix (ECM) components, causing the progressive deterioration of kidney function. Human liver stem cells (HLSCs) have anti-fibrotic activity, and HLSC-derived extracellular vesicles (EVs) mediate this effect. Herein, we evaluated the ability of HLSC-EVs to reverse renal and cardiac alterations in a murine model of partial nephrectomy (PNx) that mimics human CKD development. Furthermore, we investigated the contribution of extracellular matrix remodeling-related proteases to the anti-fibrotic effect of HLSC-EVs. PNx was performed by ligation of both poles of the left kidney, followed one week later by the removal of the right kidney. EV treatment started 4 weeks after the nephrectomy, when renal and cardiac alternations were already established, and mice were sacrificed at week eight. HLSC-EV treatment improved renal function and morphology, significantly decreasing interstitial fibrosis, glomerular sclerosis, and capillary rarefaction. This improvement was confirmed by the decreased expression of pro-fibrotic genes. Moreover, EV treatment improved cardiac function and reduced cardiac fibrosis. HLSC-EVs shuttled different proteases with ECM remodeling activity, and matrix metalloproteinase 1 (MMP-1) was involved in their anti-fibrotic effect on renal tissue. HLSC-EV treatment interferes with CKD development and ameliorates cardiomyopathy in PNx mice.

Unravelling the metabolic rewiring in the context of doxorubicin-induced cardiotoxicity: Fuel preference changes from fatty acids to glucose oxidation.

Doxorubicin (DOX) is a highly effective chemotherapeutic agent whose clinical use is hindered by the onset of cardiotoxic effects, resulting in reduced ejection fraction within the first year from treatment initiation. Recently it has been demonstrated that DOX accumulates within mitochondria, leading to disruption of metabolic processes and energetic imbalance. We previously described that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, causing autophagy inhibition and accumulation of damaged mitochondria. Here we intend to describe the maladaptive metabolic rewiring occurring in DOX-treated hearts and the contribution of PI3Kγ signalling to this process. Metabolomic analysis of DOX-treated WT hearts revealed an accumulation of TCA cycle metabolites due to a cycle slowdown, with reduced levels of pyruvate, unchanged abundance of lactate and increased Acetyl-CoA production. Moreover, the activity of glycolytic enzymes was upregulated, and fatty acid oxidation downregulated, after DOX, indicative of increased glucose oxidation. In agreement, oxygen consumption was increased in after pyruvate supplementation, with the formation of cytotoxic ROS rather than energy production. These metabolic changes were fully prevented in KD hearts. Interestingly, they failed to increase glucose oxidation in response to DOX even with autophagy inhibition, indicating that PI3Kγ likely controls the fuel preference after DOX through an autophagy-independent mechanism. In vitro experiments showed that inhibition of PI3Kγ inhibits pyruvate dehydrogenase (PDH), the key enzyme of Randle cycle regulating the switch from fatty acids to glucose usage, while decreasing DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane and limiting the ensuing glucose uptake. These results demonstrate that PI3Kγ promotes a maladaptive metabolic rewiring in DOX-treated hearts, through a two-pronged mechanism controlling PDH activation and GLUT-4-mediated glucose uptake.

Gene therapy with phosphodiesterases 2A and 4B ameliorates heart failure and arrhythmias by improving subcellular cAMP compartmentation.

AIMS: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. METHODS AND RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. CONCLUSION: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.

Emerging Roles of Phospholipase C Beta Isozymes as Potential Biomarkers in Cardiac Disorders.

Phospholipase C (PLC) enzymes represent crucial participants in the plasma membrane of mammalian cells, including the cardiac sarcolemmal (SL) membrane of cardiomyocytes. They are responsible for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into 1,2-diacylglycerol (DAG) and inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), both essential lipid mediators. These second messengers regulate the intracellular calcium (Ca2+) concentration, which activates signal transduction cascades involved in the regulation of cardiomyocyte activity. Of note, emerging evidence suggests that changes in cardiomyocytes' phospholipid profiles are associated with an increased occurrence of cardiovascular diseases, but the underlying mechanisms are still poorly understood. This review aims to provide a comprehensive overview of the significant impact of PLC on the cardiovascular system, encompassing both physiological and pathological conditions. Specifically, it focuses on the relevance of PLCß isoforms as potential cardiac biomarkers, due to their implications for pathological disorders, such as cardiac hypertrophy, diabetic cardiomyopathy, and myocardial ischemia/reperfusion injury. Gaining a deeper understanding of the mechanisms underlying PLCß activation and regulation is crucial for unraveling the complex signaling networks involved in healthy and diseased myocardium. Ultimately, this knowledge holds significant promise for advancing the development of potential therapeutic strategies that can effectively target and address cardiac disorders by focusing on the PLCß subfamily.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

It Takes Two to Tango! Protein-Protein Interactions behind cAMP-Mediated CFTR Regulation.

Over the last fifteen years, with the approval of the first molecular treatments, a breakthrough era has begun for patients with cystic fibrosis (CF), the rare genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). These molecules, known as CFTR modulators, have led to unprecedented improvements in the lung function and quality of life of most CF patients. However, the efficacy of these drugs is still suboptimal, and the clinical response is highly variable even among individuals bearing the same mutation. Furthermore, not all patients carrying rare CFTR mutations are eligible for CFTR modulator therapies, indicating the need for alternative and/or add-on therapeutic approaches. Because the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) represents the primary trigger for CFTR activation and a major regulator of different steps of the life cycle of the channel, there is growing interest in devising ways to fine-tune the cAMP signaling pathway for therapeutic purposes. This review article summarizes current knowledge regarding the role of cAMP signalosomes, i.e., multiprotein complexes bringing together key enzymes of the cAMP pathway, in the regulation of CFTR function, and discusses how modulating this signaling cascade could be leveraged for therapeutic intervention in CF.

In Humanized Sickle Cell Mice, Imatinib Protects Against Sickle Cell-Related Injury.

Drug repurposing is a valuable strategy for rare diseases. Sickle cell disease (SCD) is a rare hereditary hemolytic anemia accompanied by acute and chronic painful episodes, most often in the context of vaso-occlusive crisis (VOC). Although progress in the knowledge of pathophysiology of SCD have allowed the development of new therapeutic options, a large fraction of patients still exhibits unmet therapeutic needs, with persistence of VOCs and chronic disease progression. Here, we show that imatinib, an oral tyrosine kinase inhibitor developed for the treatment of chronic myelogenous leukemia, acts as multimodal therapy targeting signal transduction pathways involved in the pathogenesis of both anemia and inflammatory vasculopathy of humanized murine model for SCD. In addition, imatinib inhibits the platelet-derived growth factor-B-dependent pathway, interfering with the profibrotic response to hypoxia/reperfusion injury, used to mimic acute VOCs. Our data indicate that imatinib might be considered as possible new therapeutic tool for chronic treatment of SCD.

Duality of Nrf2 in iron-overload cardiomyopathy.

Cardiomyopathy deeply affects quality of life and mortality of patients with b-thalassemia or with transfusion-dependent myelodysplastic syndromes. Recently, a link between Nrf2 activity and iron metabolism has been reported in liver ironoverload murine models. Here, we studied C57B6 mice as healthy control and nuclear erythroid factor-2 knockout (Nrf2-/-) male mice aged 4 and 12 months. Eleven-month-old wild-type and Nrf2-/- mice were fed with either standard diet or a diet containing 2.5% carbonyl-iron (iron overload [IO]) for 4 weeks. We show that Nrf2-/- mice develop an age-dependent cardiomyopathy, characterized by severe oxidation, degradation of SERCA2A and iron accumulation. This was associated with local hepcidin expression and increased serum non-transferrin-bound iron, which promotes maladaptive cardiac remodeling and interstitial fibrosis related to overactivation of the TGF-b pathway. When mice were exposed to IO diet, the absence of Nrf2 was paradoxically protective against further heart iron accumulation. Indeed, the combination of prolonged oxidation and the burst induced by IO diet resulted in activation of the unfolded protein response (UPR) system, which in turn promotes hepcidin expression independently from heart iron accumulation. In the heart of Hbbth3/+ mice, a model of b-thalassemia intermedia, despite the activation of Nrf2 pathway, we found severe protein oxidation, activation of UPR system and cardiac fibrosis independently from heart iron content. We describe the dual role of Nrf2 when aging is combined with IO and its novel interrelation with UPR system to ensure cell survival. We open a new perspective for early and intense treatment of cardiomyopathy in patients with b-thalassemia before the appearance of heart iron accumulation.

Cell signaling and regulation of CFTR expression in cystic fibrosis cells in the era of high efficiency modulator therapy.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and protein kinase A (PKA)-regulated channel, expressed on the luminal surface of secretory and absorptive epithelial cells. CFTR has a complex, cell-specific regulatory network playing a major role in cAMP- and Ca2+-activated secretion of electrolytes. It secretes intracellular Cl- and bicarbonate and regulates absorption of electrolytes by differentially controlling the activity of the epithelial Na+ channel (ENaC) in colon, airways, and sweat ducts. The CFTR gene expression is regulated by cell-specific, time-dependent mechanisms reviewed elsewhere [1]. This review will focus on the transcriptional, post-transcriptional, and translational regulation of CFTR by cAMP-PKA, non-coding (nc)RNAs, and TGF-ß signaling pathways in cystic fibrosis (CF) cells.

Priorities in Cardio-Oncology Basic and Translational Science: GCOS 2023 Symposium Proceedings: JACC: CardioOncology State-of-the-Art Review.

Despite improvements in cancer survival, cancer therapy-related cardiovascular toxicity has risen to become a prominent clinical challenge. This has led to the growth of the burgeoning field of cardio-oncology, which aims to advance the cardiovascular health of cancer patients and survivors, through actionable and translatable science. In these Global Cardio-Oncology Symposium 2023 scientific symposium proceedings, we present a focused review on the mechanisms that contribute to common cardiovascular toxicities discussed at this meeting, the ongoing international collaborative efforts to improve patient outcomes, and the bidirectional challenges of translating basic research to clinical care. We acknowledge that there are many additional therapies that are of significance but were not topics of discussion at this symposium. We hope that through this symposium-based review we can highlight the knowledge gaps and clinical priorities to inform the design of future studies that aim to prevent and mitigate cardiovascular disease in cancer patients and survivors.

Circulating hemopexin modulates anthracycline cardiac toxicity in patients and in mice.

Anthracyclines such as doxorubicin (Dox) are effective chemotherapies, but their use is limited by cardiac toxicity. We hypothesized that plasma proteomics in women with breast cancer could identify new mechanisms of anthracycline cardiac toxicity. We measured changes in 1317 proteins in anthracycline-treated patients (n = 30) and replicated key findings in a second cohort (n = 31). An increase in the heme-binding protein hemopexin (Hpx) 3 months after anthracycline initiation was associated with cardiac toxicity by echocardiography. To assess the functional role of Hpx, we administered Hpx to wild-type (WT) mice treated with Dox and observed improved cardiac function. Conversely, Hpx-/- mice demonstrated increased Dox cardiac toxicity compared to WT mice. Initial mechanistic studies indicate that Hpx is likely transported to the heart by circulating monocytes/macrophages and that Hpx may mitigate Dox-induced ferroptosis to confer cardioprotection. Together, these observations suggest that Hpx induction represents a compensatory response during Dox treatment.

Neurotoxic Effect of Doxorubicin Treatment on Cardiac Sympathetic Neurons.

Doxorubicin (DOXO) remains amongst the most commonly used anti-cancer agents for the treatment of solid tumors, lymphomas, and leukemias. However, its clinical use is hampered by cardiotoxicity, characterized by heart failure and arrhythmias, which may require chemotherapy interruption, with devastating consequences on patient survival and quality of life. Although the adverse cardiac effects of DOXO are consolidated, the underlying mechanisms are still incompletely understood. It was previously shown that DOXO leads to proteotoxic cardiomyocyte (CM) death and myocardial fibrosis, both mechanisms leading to mechanical and electrical dysfunction. While several works focused on CMs as the culprits of DOXO-induced arrhythmias and heart failure, recent studies suggest that DOXO may also affect cardiac sympathetic neurons (cSNs), which would thus represent additional cells targeted in DOXO-cardiotoxicity. Confocal immunofluorescence and morphometric analyses revealed alterations in SN innervation density and topology in hearts from DOXO-treated mice, which was consistent with the reduced cardiotropic effect of adrenergic neurons in vivo. Ex vivo analyses suggested that DOXO-induced denervation may be linked to reduced neurotrophic input, which we have shown to rely on nerve growth factor, released from innervated CMs. Notably, similar alterations were observed in explanted hearts from DOXO-treated patients. Our data demonstrate that chemotherapy cardiotoxicity includes alterations in cardiac innervation, unveiling a previously unrecognized effect of DOXO on cardiac autonomic regulation, which is involved in both cardiac physiology and pathology, including heart failure and arrhythmias.

An integrated approach to cardioprotection in lymphomas.

In potentially curable cancers, long-term survival depends not only on the successful treatment of the malignancy but also on the risks associated with treatment-related toxicity, especially cardiotoxicity. Malignant lymphomas affect patients at any age, with acute and late toxicity risks that could have a severe effect on morbidity, mortality, and quality of life. Although our understanding of chemotherapy-associated and radiotherapy-associated cardiovascular disease has advanced considerably, new drugs with potential cardiotoxicity have been introduced for the treatment of lymphomas. In this Review, we summarise the mechanisms of treatment-related cardiac injury, available clinical data, and protocols for optimising cardioprotection in lymphomas. We discuss ongoing research strategies to advance our knowledge of the molecular basis of drug-induced and radiation-induced toxicity. Additionally, we emphasise the potential for personalised follow-up and early detection, including the role of biomarkers and novel diagnostic tests, highlighting the role of the cardio-oncology team.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

PI(3,4)P2-mediated cytokinetic abscission prevents early senescence and cataract formation.

Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)­dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2­binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell type­specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.

PI3K as Mediator of Apoptosis and Contractile Dysfunction in TGFß1-Stimulated Cardiomyocytes.

BACKGROUND: TGFß1 is a growth factor that plays a major role in the remodeling process of the heart by inducing cardiomyocyte dysfunction and apoptosis, as well as fibrosis thereby restricting heart function. TGFß1 mediates its effect via the TGFß receptor I (ALK5) and the activation of SMAD transcription factors, but TGFß1 is also known as activator of phosphoinositide-3-kinase (PI3K) via the non-SMAD signaling pathway. The aim of this study was to investigate whether PI3K is also involved in TGFß1-induced cardiomyocytes apoptosis and contractile dysfunction. METHODS AND RESULTS: Incubation of isolated ventricular cardiomyocytes with TGFß1 resulted in impaired contractile function. Pre-incubation of cells with the PI3K inhibitor Ly294002 or the ALK5 inhibitor SB431542 attenuated the decreased cell shortening in TGFß1-stimulated cells. Additionally, TGFß-induced apoptosis was significantly reduced by the PI3K inhibitor Ly294002. Administration of a PI3Kγ-specific inhibitor AS605240 abolished the TGFß effect on apoptosis and cell shortening. This was also confirmed in cardiomyocytes from PI3Kγ KO mice. Induction of SMAD binding activity and the TGFß target gene collagen 1 could be blocked by the PI3K inhibitor Ly294002, but not by the specific PI3Kγ inhibitor AS605240. CONCLUSIONS: TGFß1-induced SMAD activation, cardiomyocyte apoptosis, and impaired cell shortening are mediated via both, the ALK5 receptor and PI3K, in adult cardiomyocytes. PI3Kγ specifically contributes to apoptosis induction and impairment of contractile function independent of SMAD signaling.