Biology subjects

Alcaide, P.

Publications and source records attributed to Alcaide, P..

7 recordsLinked to original sources

bioRxiv2026

Gene therapy targeting of AKAP6β-CaMKII signalosomes improves myocardial inflammation and heart failure in a swine model of cardiometabolic syndrome

BackgroundCardiometabolic heart failure with preserved ejection fraction (HFpEF) is associated with systemic and cardiac inflammation and diastolic dysfunction. A-kinase anchoring protein 6{beta} (AKAP6{beta}) is a scaffold protein located at the cardiomyocyte outer nuclear membrane that promotes pathological cardiac remodeling via the recruitment of multiple regulatory proteins including protein kinases. In mice, adeno-associated virus (AAV) mediated expression of a peptide based upon a kinase binding domain (KBD) within AKAP6{beta} inhibited the development of heart failure due to chronic pressure overload. Whether KBD expression can also inhibit the development of cardiometabolic heart failure is unknown, and if so, the mechanism of KBD action in HFpEF has yet to be explored. MethodsThe efficacy of a cardiotropic self-complementary AAV gene therapy that expresses the AKAP6{beta} KBD peptide (AAV9sc.KBD) was tested in a female Ossabaw swine model of cardiometabolic syndrome and HFpEF. Single nucleus and bulk RNA sequencing of swine heart tissue and immunoprecipitation-mass spectrometry, live cell imaging, and biochemical assays using primary rat cardiomyocytes were employed to study KBD mechanism of action. ResultsAAV9sc.KBD inhibited the development of diastolic dysfunction and heart failure in the Ossabaw model, without negatively impacting systolic function. The improvement in cardiac phenotype was associated with decreased T-cell myocardial infiltrates and partial reversal of pathological gene expression. An unbiased interactome study revealed that the KBD peptide binds Ca2+/calmodulin-dependent protein kinase II (CaMKII), identifying CaMKII as a new AKAP6{beta} binding partner. Perinuclear CaMKII activity detected by live cell imaging required AKAP6{beta} expression and was inhibited by KBD expression. In addition, the CaMKII substrate Inhibitor of NF-{kappa}B Kinase {beta} (IKK{beta}) bound AKAP6{beta}. IKK phosphorylation in the Ossabaw model and in myocytes was inhibited by KBD expression, and NF-{kappa}B nuclear translocation in myocytes was dependent upon AKAP6{beta}-CaMKII protein complex formation. AAV9sc.KBD treatment inhibited cardiomyocyte NF-{kappa}B-dependent gene expression in the Ossabaw model. ConclusionsRegulated by perinuclear AKAP6{beta}-CaMKII signalosomes, NF-{kappa}B pro-inflammatory gene expression in cardiomyocytes participates in a positive feedback loop with cardiac inflammation promoting HFpEF. Proof-of-concept is provided in a large animal model that gene therapy-based cardiomyocyte expression of the KBD peptide will prevent cardiac dysfunction in cardiometabolic syndrome. Clinical PerspectiveO_ST_ABSWhat is newC_ST_ABSO_LIThe cardiomyocyte-selective gene therapy AAV9sc.KBD, which targets signalosomes organized by the scaffold protein AKAP6{beta}, is shown to inhibit myocardial T-cell infiltration and improve cardiac structure and function in a large animal model of cardiometabolic HFpEF. C_LIO_LIThe AKAP6{beta} KBD peptide is shown to bind and inhibit the function of CaMKII. C_LIO_LICaMKII and IKK{beta} are shown to participate in perinuclear AKAP6{beta} signalosomes, where they regulate activation of the NF-{kappa}B pro-inflammatory gene regulatory pathway. C_LI Clinical implicationsO_LIProof-of-concept for a novel strategy for the treatment of HFpEF is provided, intracellular expression by a cardiomyocyte-selective gene therapy vector of an inhibitory peptide, which will inhibit compartmentalized intracellular signal transduction. C_LIO_LIIn conjunction with previous studies in small rodents, the new data obtained in Ossabaw swine support clinical translation of the AAV9sc.KBD gene therapy. C_LI

physiology↗

bioRxiv2026

T cell-derived IFNγ instructs ECM crosslinking by cardiac fibroblasts through LOXL3 in experimental cardiometabolic HFpEF

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a major clinical challenge characterized by diastolic dysfunction. Left ventricular stiffening and inflammation are hallmarks of HFpEF, yet the contribution of extracellular matrix (ECM) stiffness and the immune-stromal mechanisms driving ECM stiffening in cardiometabolic HFpEF remain poorly understood. MethodsWe used the murine "2-hit model" of cardiometabolic HFpEF, in which the combination of high fat diet and hypertension induced by L-NAME causes diastolic dysfunction. We evaluated diastolic function by echocardiography and ECM mechanics by uniaxial tensile testing of decellularized cardiac tissue. Functional in vivo studies included genetic depletion of T cells, interferon-{gamma} (IFN{gamma}) knockout mice, and pharmacological lysyl oxidase inhibition. We combined co-cultures of CD4+ T cells and cardiac fibroblasts (CFB) with mechanical testing of cardiac ECM and molecular biology to elucidate cellular and molecular mechanisms. ResultsLeft ventricular ECM stiffness strongly correlated with impaired diastolic function in experimental cardiometabolic HFpEF. Cardiac CD4 T cell infiltration was required for ECM stiffening and upregulation of lysyl oxidase enzymes in CFB. CD4+ T cell-derived IFN{gamma} was both necessary and sufficient to induce LOXL3 in CFB, which increased ECM stiffness in vitro. Mechanistically, IFN{gamma} signaling activated hypoxia-inducible factor-1 (HIF1) in CFB, driving LOXL3 expression and subsequent collagen crosslinking. Genetic or pharmacologic disruption of this IFN{gamma}-HIF1-LOXL3 axis in vivo attenuated adverse ECM remodeling and improved diastolic function. ConclusionsCD4 T cells promote pathological ECM stiffening in cardiometabolic HFpEF through IFN{gamma}-mediated, LOXL3-dependent ECM crosslinking by CFB. Targeting this immune-stromal pathway may offer a novel therapeutic strategy for HFpEF.

pathology↗

bioRxiv2025

Targeting of CIP4-Calcineurin Signalosomes Improves Cardiac Structure and Function After Myocardial Infarction

BackgroundCalcineurin in a pleiotropic signaling enzyme that promotes pathological cardiac remodeling but also cardioprotection in ischemia-reperfusion injury. In addition, calcineurin inhibitors are immunosuppressants. This pleiotropy has precluded the use of calcineurin inhibitors as treatments for heart failure. Cdc42-interacting protein 4 (CIP4/TRIP10) is an endosomal scaffold protein that organizes a calcium and calcineurin A{beta}2 (CaNA{beta}2) signaling compartment activated by G-protein coupled receptors independently of contractile calcium. CIP4 binds CaNA{beta}2 via the CaNA{beta}-specific N-terminal polyproline (PP) domain. We previously showed that targeting of CIP4-CaNA{beta}2 signalosomes inhibited pathological cardiac hypertrophy and the development of heart failure induced by chronic pressure overload in mice. It is unknown whether CIP4-CaNA{beta}2 signalosomes contribute to cardioprotection and/or cardiac remodeling in ischemic heart disease. MethodsCIP4 conditional knock-out (CKO) mice were studied by echocardiography with strain analysis and histology following ischemia-reperfusion (I/R) injury and permanent left coronary artery (LCA) ligation to induce myocardial infarction. Wildtype C57BL/6NJ mice were transduced with adeno-associated virus (AAV) engineered for cardiomyocyte-specific expression of either a CaNA{beta}2 shRNA to inhibit CaNA{beta}2 expression, a VIVIT peptide to inhibit CaN-NFAT signaling, or a CaNA{beta}2 PP peptide to block CIP4-CaNA{beta}2 binding. AAV-transduced mice were studied by I/R injury. Additional mice were subjected to permanent LCA ligation and subsequently treated with AAV to test the effects of CaN inhibition in chronic ischemic cardiomyopathy. The effects of CaNA{beta}2 PP-GFP expression on primary T-cell activation were studied in vitro. ResultsCIP4 CKO mice and mice expressing the PP anchoring disruptor peptide exhibited preserved cardiac function after I/R injury and decreased infarct size and preserved cardiac function 8 weeks after myocardial infarction by permanent LCA ligation. In contrast, cardiomyocyte-specific depletion of CaNA{beta}2 and VIVIT peptide expression worsened outcome after I/R injury and in chronic ischemic cardiomyopathy. In addition, in contrast to cardiomyocytes, PP-mediated CaNA{beta} anchoring inhibition had no effect on T-cell activation and cytokine expression in vitro. ConclusionsCIP4-CaNA{beta}2 signalosomes promote adverse cardiac remodeling and are not cardioprotective. Proof-of-concept is provided for the treatment of ischemic cardiomyopathy by a PP anchoring disruptor gene therapy. Targeting these complexes may be beneficial in cardiovascular diseases, including ischemic cardiomyopathy and acute myocardial infarction. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LITargeting CIP4, which is a scaffold protein for the phosphatase calcineurin, improves cardiac function in mice after acute myocardial infarction due to ischemia-reperfusion injury and in chronic ischemic cardiomyopathy. C_LIO_LIGene therapy-based expression of a calcineurin A{beta}-derived polyproline peptide, which can compete CIP4-calcineurin binding, is beneficial in acute and chronic myocardial infarction. C_LI What Are the Clinical Implications?O_LIThis study establishes CIP4 signalosomes as a new drug target for the treatment of ischemia-reperfusion injury and chronic pathological cardiac remodeling. C_LIO_LIThis study provides proof-of-concept for a new gene therapy approach to treating acute myocardial infarction and chronic ischemic cardiomyopathy. C_LI

physiology↗

bioRxiv2024

STING promotes homeostatic maintenance of tissues and confers longevity with aging

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING- sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING- signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging. HIGHLIGHTSO_LIAging tissues are associated with tissue-autonomous immune fingerprints, primarily driven by interactions of tissue stromal and myeloid populations. C_LIO_LISTING shapes these immune fingerprints of aging tissues in unexpected ways. C_LIO_LILoss of STING alters the location, numbers, and viability of tissue resident macrophages. C_LIO_LISTING signaling is critical for longer lifespans and maintenance of tissue architecture. C_LI

immunology↗

bioRxiv2024

A Novel Therapeutic Approach using CXCR3 Blockade to Treat Immune Checkpoint Inhibitor-mediated Myocarditis

BackgroundImmune checkpoint inhibitors (ICIs) are successful in treating many cancers but may cause immune-related adverse events. ICI-mediated myocarditis has a high fatality rate of up to 40%, with severe cardiovascular consequences. Targeted therapies for ICI myocarditis are currently lacking. MethodsWe used a genetic mouse model of PD-1 deletion (MRL/Pdcd1-/-) along with a novel drug-treated ICI myocarditis mouse model to recapitulate the disease phenotype. We performed single-cell RNA-sequencing (scRNAseq), single-cell T-cell receptor sequencing (scTCR-seq), and cellular indexing of transcriptomes and epitopes (CITE-seq) on immune cells isolated from MRL and MRL/Pdcd1-/- mice at serial timepoints. We assessed the impact of macrophage deletion in MRL/Pdcd1-/- mice, then inhibited CXC chemokine receptor 3 (CXCR3) in ICI-treated mice to assess therapeutic effect on myocarditis phenotype. Furthermore, we delineated functional effects of CXCR3 blockade on T-cell and macrophage interactions in a transwell assay. We then correlated the results in human single-cell multi-omics data from blood and heart biopsy data from patients with ICI myocarditis. ResultsSingle-cell multi-omics demonstrated expansion of CXCL9/10+CCR2+ macrophages and CXCR3hi CD8+ effector T-lymphocytes in the hearts of MRL/Pdcd1-/- mice correlating with onset of myocarditis development. Both depletion of CXCL9/10+CCR2+ macrophages and CXCR3 blockade respectively led to decreased CXCR3hiCD8+ T-cell infiltration into the heart and significantly improved survival. A transwell assay showed that selective blockade of CXCR3 and its ligand, CXCL10 decreased CD8+ T-cell migration towards macrophages, implicating this interaction in T-cell cardiotropism towards cardiac macrophages. Cardiac biopsies from patients with confirmed ICI myocarditis demonstrated infiltrating CXCR3+ lymphocytes and CXCL9+/CXCL10+ macrophages. Both mouse cardiac immune cells and patient peripheral blood immune cells revealed expanded TCRs correlating with CXCR3hi CD8+ T-cells in ICI myocarditis samples. ConclusionsThese findings bring forth the CXCR3-CXCL9/10 axis as an attractive therapeutic target for ICI myocarditis treatment, and more broadly, as a druggable pathway in cardiac inflammation.

cell biology↗

bioRxiv2021

AKT1 is Required for a Complete Palbociclib-induced Senescence Phenotype in BRAF-V600E-Driven Human Melanoma

Cellular senescence is a carefully regulated process of proliferative arrest accompanied by numerous functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation, however induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of the senescence response may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like, non-proliferative state in tumor cells. The AKT kinase family plays an important role in cellular growth and division, and is commonly hyperactive in many cancers including melanoma. AKT activity has also been implicated in regulation of senescence. The three AKT isoforms play both redundant and unique roles in tumorigenesis and cancer progression. To interrogate the role of AKT isoforms in the induction of cellular senescence by Cdk4/6i, we generated isoform specific AKT knockout human BRAF-V600E mutated melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. As a potential mechanism, we evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence. While we showed cGAS-STING function to be dependent on AKT1, pharmacologic inhibition of either cGAS or STING had little effect on senescence. However, we found SASP factors to require NF-kB function, in part dependent on a stimulatory phosphorylation of IKK by AKT1 previously reported in other models. In summary, we provide the first evidence of a novel, isoform specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-kB but independent of cGAS-STING.

cancer biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗