Dr. Kapiloff earned his Ph.D. in Biomedical Sciences in 1991 at the University of California, San Diego, in the laboratory of Dr. M. Geoffrey Rosenfeld, a HHMI investigator and member of the National Academy of Sciences. He graduated from UCSD with a Doctorate of Medicine in 1992 and completed a residency in General Pediatrics at the University of Utah in 1995. In 1997, Dr. Kapiloff became a Research Assistant Professor, performing research in the laboratory of HHMI Investigator Dr. John Scott at the Vollum Institute in Portland, OR. From 1999 to 2007, Dr. Kapiloff was an Assistant Professor of Pediatrics at the Oregon Health and Science University in Portland. From 2007 to 2017, he was Director of the Cardiac Signal Transduction and Cellular Biology Laboratory at the University of Miami Miller School of Medicine in Miami, Florida, where he was, as of 2013, a tenured Professor of Pediatrics and Medicine in the Division of Cardiology. Since 2011, Dr. Kapiloff has also been studying signal transduction pathways in the eye. Dr. Kapiloff was recruited to Stanford in July, 2017 in a joint effort by the Department of Ophthalmology and the Stanford Cardiovascular Institute in recognition of his work in both fields.
Honors & Awards
Beneficial-Hodson Scholar, The Johns Hopkins University (1981-1984)
Member, Phi Beta Kappa Society (1983)
Medical Scientist Training Program, University of California, San Diego (1984-1992)
Fellow, American Heart Association (2008)
Member, American Society for Clinical Investigation (2011)
Micah Batchelor Award For Excellence In Children's Health Research, University of Miami (2013)
Fellow, American Physiological Society, Cardiovascular Section (2014)
Reinhard Family Professorship, Stanford University (2021)
Boards, Advisory Committees, Professional Organizations
Guest Editor, "AKAPs - regulators of signaling in space and time”, Journal of Cardiovascular Pharmacology (2011 - 2011)
Editorial Board, American Journal of Physiology – Heart and Circulatory Physiology (2011 - 2020)
Member, Marcus Young Investigator Award in Cardiovascular Sciences Committee, American Heart Association (2012 - 2014)
Co-Chair, North American Section Meeting, International Society for Heart Research (2014 - 2014)
Chair, Molecular Signaling 1 Study Section, American Heart Association (2014 - 2015)
Faculty Member, Cardiovascular Pharmacology Section, Faculty of 1000 F1000 Prime (2014 - 2018)
Editorial Board, Journal of Molecular and Cellular Cardiology (2014 - 2019)
Member, Early Career Committee for Council on Basic Cardiovascular Sciences, American Heart Association (2015 - 2017)
Leadership Committee for Council on Basic Cardiovascular Sciences, American Heart Association (2016 - 2020)
Co-Chair, Specialty Conference of the Council on Basic Cardiovascular Sciences, American Heart Association (2017 - 2018)
Member, Cardiac Contractility, Hypertrophy, and Failure (CCHF) Study Section, National Institutes of Health (2017 - 2020)
BA, The Johns Hopkins University, Humanistic Studies (1984)
PhD, University of California, San Diego, Biomedical Sciences (1991)
MD, University of California, San Diego, Medicine (1992)
Residency, University of Utah and Primary Children's Medical Centers, General Pediatrics (1995)
Current Research and Scholarly Interests
Dr. Michael S. Kapiloff is a faculty member in the Departments of Ophthalmology and Medicine (Cardiovascular Medicine) and a member of the Stanford Cardiovascular Institute. Although Dr. Kapiloff was at one time a Board-Certified General Pediatrician, he is currently involved in full-time basic science and translational research. His laboratory studies the basic molecular mechanisms underlying the response of the retinal ganglion cell and cardiac myocyte to disease. The longstanding interest of his laboratory is the role in intracellular signal transduction of multimolecular complexes organized by scaffold proteins. Recently, his lab has also been involved in the translation of these concepts into new therapies, including the development of new AAV gene therapy biologics for the prevention and treatment of heart failure and for neuroprotection in the eye.
URL to NCBI listing of all published works:
For more information see Dr. Kapiloff's lab website: http://med.stanford.edu/kapilofflab.html
A molecular switch for neuroprotective astrocyte reactivity.
The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system (CNS) degeneration and repair remain poorly understood. Here, we show injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cAMP derived from soluble adenylyl cyclase (sAC) and show proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell (RGC) survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show elevating nuclear or depleting cytoplasmic cAMP in reactive astrocytes inhibits deleterious microglial/macrophage cell activation and promotes RGC survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cAMP in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand upon and define new reactive astrocyte subtypes and represent a novel step toward the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.
View details for DOI 10.1038/s41586-023-06935-3
View details for PubMedID 38086421
Differential expression of PIEZO1 and PIEZO2 mechanosensitive channels in ocular tissues implicates diverse functional roles.
Experimental eye research
PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.
View details for DOI 10.1016/j.exer.2023.109675
View details for PubMedID 37820892
Dock10 Regulates Cardiac Function under Neurohormonal Stress
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
2022; 23 (17)
Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the α-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte.
View details for DOI 10.3390/ijms23179616
View details for Web of Science ID 000851094300001
View details for PubMedID 36077014
View details for PubMedCentralID PMC9455810
A perinuclear calcium compartment regulates cardiac myocyte hypertrophy.
Journal of molecular and cellular cardiology
2022; 172: 26-40
The pleiotropic Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac myocyte hypertrophy. The selective activation of hypertrophic calcineurin signaling under stress conditions has been attributed to compartmentation of Ca2+ signaling in cardiac myocytes. Here, perinuclear signalosomes organized by the scaffold protein muscle A-Kinase Anchoring Protein beta (mAKAPbeta/AKAP6beta) are shown to orchestrate local Ca2+ transients, inducing calcineurin-dependent NFATc nuclear localization and myocyte hypertrophy in response to beta-adrenergic receptor activation. Fluorescent biosensors for Ca2+ and calcineurin and protein kinase A (PKA) activity, both diffusely expressed and localized by nesprin-1alpha to the nuclear envelope, are used to define an autonomous mAKAPbeta signaling compartment in adult and neonatal rat ventricular myocytes. Notably, beta-adrenergic-stimulated perinuclear Ca2+ and PKA and CaN activity transients depended upon mAKAPbeta expression, while Ca2+ elevation and PKA and CaN activity in the cytosol were mAKAPbeta independent. Buffering perinuclear cAMP and Ca2+ prevented calcineurin-dependent NFATc nuclear translocation and myocyte hypertrophy, without affecting cardiac myocyte contractility. Additional findings suggest that the perinuclear Ca2+ transients were mediated by signalosome-associated ryanodine receptors regulated by local PKA phosphorylation. These results demonstrate the existence of a functionally independent Ca2+ signaling compartment in the cardiac myocyte regulating hypertrophy and provide a premise for targeting mAKAPbeta signalosomes to prevent selectively cardiac hypertrophy in disease.
View details for DOI 10.1016/j.yjmcc.2022.07.007
View details for PubMedID 35952391
Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.
Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein beta (mAKAPbeta) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 x 105 vg/g gDNA for both infusion techniques at a dose ~1013 vg/kg body weight, demonstrating a delivery of ~1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable to levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.
View details for DOI 10.1152/physiolgenomics.00032.2022
View details for PubMedID 35648460
FGF21-FGFR4 signaling in cardiac myocytes promotes concentric cardiac hypertrophy in mouse models of diabetes.
2022; 12 (1): 7326
Fibroblast growth factor (FGF) 21, a hormone that increases insulin sensitivity, has shown promise as a therapeutic agent to improve metabolic dysregulation. Here we report that FGF21 directly targets cardiac myocytes by binding beta-klotho and FGF receptor (FGFR) 4. In combination with high glucose, FGF21 induces cardiac myocyte growth in width mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. While short-term FGF21 elevation can be cardio-protective, we find that in type 2 diabetes (T2D) in mice, where serum FGF21 levels are elevated, FGFR4 activation induces concentric cardiac hypertrophy. As T2D patients are at risk for heart failure with preserved ejection fraction (HFpEF), we propose that induction of concentric hypertrophy by elevated FGF21-FGFR4 signaling may constitute a novel mechanism promoting T2D-associated HFpEF such that FGFR4 blockade might serve as a cardio-protective therapy in T2D. In addition, potential adverse cardiac effects of FGF21 mimetics currently in clinical trials should be investigated.
View details for DOI 10.1038/s41598-022-11033-x
View details for PubMedID 35513431
Soluble alpha-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease.
Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble alpha-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions, and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4, and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.
View details for DOI 10.1016/j.kint.2022.03.028
View details for PubMedID 35513125
Targeting mAKAPbeta expression as a therapeutic approach for ischemic cardiomyopathy.
Ischemic cardiomyopathy is a leading cause of death and an unmet clinical need. Adeno-associated virus (AAV) gene-based therapies hold great promise for treating and preventing heart failure. Previously we showed that muscle A-kinase Anchoring Protein beta (mAKAPbeta, AKAP6beta), a scaffold protein that organizes perinuclear signalosomes in the cardiomyocyte, is a critical regulator of pathological cardiac hypertrophy. Here, we show that inhibition of mAKAPbeta expression in stressed adult cardiomyocytes in vitro was cardioprotective, while conditional cardiomyocyte-specific mAKAP gene deletion in mice prevented pathological cardiac remodeling due to myocardial infarction. We developed a new self-complementary serotype 9 AAV gene therapy vector expressing a short hairpin RNA for mAKAPbeta under the control of a cardiomyocyte-specific promoter (AAV9sc.shmAKAP). This vector efficiently downregulated mAKAPbeta expression in the mouse heart in vivo. Expression of the shRNA also inhibited mAKAPbeta expression in human induced cardiomyocytes in vitro. Following myocardial infarction, systemic administration of AAV9sc.shmAKAP prevented the development of pathological cardiac remodeling and heart failure, providing long-term restoration of left ventricular ejection fraction. Our findings provide proof-of-concept for mAKAPbeta as a therapeutic target for ischemic cardiomyopathy and support the development of a translational pipeline for AAV9sc.shmAKAP for the treatment of heart failure.
View details for DOI 10.1038/s41434-022-00321-w
View details for PubMedID 35102273
Mechanism-Driven Modeling to Aid Non-invasive Monitoring of Cardiac Function via Ballistocardiography.
Frontiers in medical technology
2022; 4: 788264
Left ventricular (LV) catheterization provides LV pressure-volume (P-V) loops and it represents the gold standard for cardiac function monitoring. This technique, however, is invasive and this limits its applicability in clinical and in-home settings. Ballistocardiography (BCG) is a good candidate for non-invasive cardiac monitoring, as it is based on capturing non-invasively the body motion that results from the blood flowing through the cardiovascular system. This work aims at building a mechanistic connection between changes in the BCG signal, changes in the P-V loops and changes in cardiac function. A mechanism-driven model based on cardiovascular physiology has been used as a virtual laboratory to predict how changes in cardiac function will manifest in the BCG waveform. Specifically, model simulations indicate that a decline in LV contractility results in an increase of the relative timing between the ECG and BCG signal and a decrease in BCG amplitude. The predicted changes have subsequently been observed in measurements on three swine serving as pre-clinical models for pre- and post-myocardial infarction conditions. The reproducibility of BCG measurements has been assessed on repeated, consecutive sessions of data acquisitions on three additional swine. Overall, this study provides experimental evidence supporting the utilization of mechanism-driven mathematical modeling as a guide to interpret changes in the BCG signal on the basis of cardiovascular physiology, thereby advancing the BCG technique as an effective method for non-invasive monitoring of cardiac function.
View details for DOI 10.3389/fmedt.2022.788264
View details for PubMedID 35252962
cAMP At Perinuclear mAKAPalpha Signalosomes Is Regulated By Local Ca2+ Signaling In Primary Hippocampal Neurons.
The second messenger cyclic adenosine monophosphate (cAMP) is important for the regulation of neuronal structure and function, including neurite extension. A perinuclear cAMP compartment organized by the scaffold protein muscle A-Kinase Anchoring Protein alpha (mAKAPalpha/AKAP6alpha) is sufficient and necessary for axon growth by rat hippocampal neurons in vitro Here, we report that cAMP at mAKAPalpha signalosomes is regulated by local Ca2+ signaling that mediates activity-dependent cAMP elevation within that compartment. Simultaneous Forster resonance energy transfer (FRET) imaging using the PKA activity reporter AKAR4 and intensiometric imaging using the RCaMP1h fluorescent Ca2+ sensor revealed that membrane depolarization by KCl selectively induced activation of perinuclear PKA activity. Activity-dependent perinuclear PKA activity was dependent upon expression of the mAKAPalpha scaffold, while both perinuclear Ca2+ elevation and PKA activation were dependent upon voltage-dependent L-type Ca2+ channel activity. Importantly, chelation of Ca2+ by a nuclear envelope-localized parvalbumin fusion protein inhibited both activity-induced perinuclear PKA activity and axon elongation. Together, this study provides evidence for a model in which a neuronal perinuclear cAMP compartment is locally regulated by activity-dependent Ca2+ influx, providing local control for the enhancement of neurite extension.Significance statement cAMP-dependent signaling has been implicated as a positive regulator of neurite outgrowth and axon regeneration. However, the mechanisms regulating cAMP signaling relevant to these processes remain largely unknown. Live cell imaging techniques are used to study the regulation by local Ca2+ signals of an mAKAPalpha-associated cAMP compartment at the neuronal nuclear envelope, providing new mechanistic insight into CNS neuronal signaling transduction conferring axon outgrowth.
View details for DOI 10.1523/ENEURO.0298-20.2021
View details for PubMedID 33495246
Calcineurin Abeta-Specific Anchoring Confers Isoform-Specific Compartmentation and Function in Pathological Cardiac Myocyte Hypertrophy.
Background: The Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the Abeta isoform of calcineurin (CaNAbeta) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aalpha expression in the same cells. In addition, it is unclear how the pleiotropic second messenger Ca2+ drives excitation-contraction coupling, while not stimulating hypertrophy via calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectively in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. Methods: Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Forster Resonance Energy Transfer (FRET) reporters were used to assay Ca2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus (AAV)-mediated gene delivery in the mouse were used to study calcineurin signaling following transverse aortic constriction in vivo. Results: Cdc42-interacting protein (CIP4/TRIP10) was identified as a new polyproline domain-dependent scaffold for CaNAbeta2 by yeast-2-hybrid screen. Cardiac myocyte-specific CIP4 gene deletion in mice attenuated pressure overload-induced pathological cardiac remodeling and heart failure. Accordingly, blockade of CaNAbeta polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes, while disruption of anchoring in vivo using an AAV gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell FRET biosensor imaging of cultured myocytes revealed that Ca2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca2+ levels and calcineurin activity detected by non-localized FRET sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca2+ and calcineurin signal transduction. Conclusions: These results support a structural model for Ca2+ and CaNAbeta compartmentation in cells based upon an isoform-specific mechanism for calcineurin protein-protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAbeta in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAbeta polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAbeta-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further pre-clinical investigation.
View details for DOI 10.1161/CIRCULATIONAHA.119.044893
View details for PubMedID 32611257
Calcineurin-AKAP interactions: therapeutic targeting of a pleiotropic enzyme with a little help from its friends
JOURNAL OF PHYSIOLOGY-LONDON
2020; 598 (14): 3029–42
The ubiquitous Ca2+ /calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac hypertrophy whose therapeutic targeting in heart disease has been elusive due to its role in other essential biological processes. Calcineurin is targeted to diverse intracellular compartments by association with scaffold proteins, including by multivalent A-kinase anchoring proteins (AKAPs) that bind protein kinase A and other important signalling enzymes determining cardiac myocyte function and phenotype. Calcineurin anchoring by AKAPs confers specificity to calcineurin function in the cardiac myocyte. Targeting of calcineurin 'signalosomes' may provide a rationale for inhibiting the phosphatase in disease.
View details for DOI 10.1113/JP276756
View details for Web of Science ID 000548282400015
View details for PubMedID 30488951
Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration.
2020; 10 (6)
In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.
View details for DOI 10.21769/BioProtoc.3559
View details for PubMedID 32368566
- Compartmentalization of local cAMP signaling in neuronal growth and survival NEURAL REGENERATION RESEARCH 2020; 15 (3): 453–54
MEF2 transcription factors differentially contribute to retinal ganglion cell loss after optic nerve injury.
2020; 15 (12): e0242884
Loss of retinal ganglion cells (RGCs) in optic neuropathies results in permanent partial or complete blindness. Myocyte enhancer factor 2 (MEF2) transcription factors have been shown to play a pivotal role in neuronal systems, and in particular MEF2A knockout was shown to enhance RGC survival after optic nerve crush injury. Here we expanded these prior data to study bi-allelic, tri-allelic and heterozygous allele deletion. We observed that deletion of all MEF2A, MEF2C, and MEF2D alleles had no effect on RGC survival during development. Our extended experiments suggest that the majority of the neuroprotective effect was conferred by complete deletion of MEF2A but that MEF2D knockout, although not sufficient to increase RGC survival on its own, increased the positive effect of MEF2A knockout. Conversely, MEF2A over-expression in wildtype mice worsened RGC survival after optic nerve crush. Interestingly, MEF2 transcription factors are regulated by post-translational modification, including by calcineurin-catalyzed dephosphorylation of MEF2A Ser-408 known to increase MEF2A-dependent transactivation in neurons. However, neither phospho-mimetic nor phospho-ablative mutation of MEF2A Ser-408 affected the ability of MEF2A to promote RGC death in vivo after optic nerve injury. Together these findings demonstrate that MEF2 gene expression opposes RGC survival following axon injury in a complex hierarchy, and further support the hypothesis that loss of or interference with MEF2A expression might be beneficial for RGC neuroprotection in diseases such as glaucoma and other optic neuropathies.
View details for DOI 10.1371/journal.pone.0242884
View details for PubMedID 33315889
Signalosome-Regulated SRF Phosphorylation Determining Myocyte Growth in Width versus Length as a Therapeutic Target for Heart Failure.
Background: Concentric and eccentric cardiac hypertrophy are associated with pressure and volume overload, respectively, in cardiovascular disease both conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetric growth of the cardiac myocyte in mainly width or length, respectively. The molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood. Identification of the mechanisms governing asymmetric myocyte growth could provide new therapeutic targets for the prevention or treatment of heart failure. Methods: Primary adult rat ventricular myocytes, adeno-associated virus (AAV)-mediated gene delivery in mice, and human tissue samples are used to define a regulatory pathway controlling pathological myocyte hypertrophy. Chromatin Immunoprecipitation Assays with Sequencing (ChIP-seq) and Precision Nuclear Run-On Sequencing (PRO-seq) are used to define a transcriptional mechanism. Results: Here we report that asymmetric cardiac myocyte hypertrophy is modulated by serum response factor (SRF) phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes in vitro and in vivo. SRF Ser103 phosphorylation is bidirectionally regulated by p90 ribosomal S6 kinase type 3 (RSK3) and protein phosphatase 2A (PP2A) at signalosomes organized by the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ), such that increased SRF phosphorylation activates Activator Protein 1 (AP1)-dependent enhancers that direct myocyte growth in width. AAV are used to express in vivo mAKAPβ-derived RSK3 and PP2A anchoring disruptor peptides that block the association of the enzymes with the mAKAPβ scaffold. Inhibition of RSK3 signaling prevents concentric cardiac remodeling due to pressure overload, while inhibition of PP2A signaling prevents eccentric cardiac remodeling induced by myocardial infarction, in each case improving cardiac function. SRF Ser103 phosphorylation is significantly decreased in dilated human hearts, supporting the notion that modulation of the mAKAPβ-SRF signalosome could be a new therapeutic approach for human heart failure. Conclusions: We have identified a new molecular switch, namely mAKAPβ signalosome-regulated SRF phosphorylation, that controls a transcriptional program responsible for modulating changes in cardiac myocyte morphology that occur secondary to pathological stressors. Complementary AAV-based gene therapies constitute rationally-designed strategies for a new translational modality for heart failure.
View details for DOI 10.1161/CIRCULATIONAHA.119.044805
View details for PubMedID 32933333
A Novel Recessive Mutation in SPEG Causes Early Onset Dilated Cardiomyopathy.
2020; 16 (9): e1009000
Dilated cardiomyopathy (DCM) is a common cause of heart failure and sudden cardiac death. It has been estimated that up to half of DCM cases are hereditary. Mutations in more than 50 genes, primarily autosomal dominant, have been reported. Although rare, recessive mutations are thought to contribute considerably to DCM, especially in young children. Here we identified a novel recessive mutation in the striated muscle enriched protein kinase (SPEG, p. E1680K) gene in a family with nonsyndromic, early onset DCM. To ascertain the pathogenicity of this mutation, we generated SPEG E1680K homozygous mutant human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) using CRISPR/Cas9-mediated genome editing. Functional studies in mutant iPSC-CMs showed aberrant calcium homeostasis, impaired contractility, and sarcomeric disorganization, recapitulating the hallmarks of DCM. By combining genetic analysis with human iPSCs, genome editing, and functional assays, we identified SPEG E1680K as a novel mutation associated with early onset DCM and provide evidence for its pathogenicity in vitro. Our study provides a conceptual paradigm for establishing genotype-phenotype associations in DCM with autosomal recessive inheritance.
View details for DOI 10.1371/journal.pgen.1009000
View details for PubMedID 32925938
AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9.
The switch from centrosomal microtubule-organizing centers (MTOCs) to non-centrosomal MTOCs during differentiation is poorly understood. Here, we identify AKAP6 as key component of the nuclear envelope MTOC. In rat cardiomyocytes, AKAP6 anchors centrosomal proteins to the nuclear envelope through its spectrin repeats, acting as an adaptor between nesprin-1alpha and Pcnt or AKAP9. In addition, AKAP6 and AKAP9 form a protein platform tethering the Golgi to the nucleus. Both Golgi and nuclear envelope exhibit MTOC activity utilizing either AKAP9, or Pcnt-AKAP9, respectively. AKAP6 is also required for formation and activity of the nuclear envelope MTOC in human osteoclasts. Moreover, ectopic expression of AKAP6 in epithelial cells is sufficient to recruit endogenous centrosomal proteins. Finally, AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption activity. Collectively, we decipher the MTOC at the nuclear envelope as a bi-layered structure generating two pools of microtubules with AKAP6 as a key organizer.
View details for DOI 10.7554/eLife.61669
View details for PubMedID 33295871
mAKAP beta signalosomes - A nodal regulator of gene transcription associated with pathological cardiac remodeling
2019; 63: 109357
Striated myocytes compose about half of the cells of the heart, while contributing the majority of the heart's mass and volume. In response to increased demands for pumping power, including in diseases of pressure and volume overload, the contractile myocytes undergo non-mitotic growth, resulting in increased heart mass, i.e. cardiac hypertrophy. Myocyte hypertrophy is induced by a change in the gene expression program driven by the altered activity of transcription factors and co-repressor and co-activator chromatin-associated proteins. These gene regulatory proteins are subject to diverse post-translational modifications and serve as nuclear effectors for intracellular signal transduction pathways, including those controlled by cyclic nucleotides and calcium ion. Scaffold proteins contribute to the underlying architecture of intracellular signaling networks by targeting signaling enzymes to discrete intracellular compartments, providing specificity to the regulation of downstream effectors, including those regulating gene expression. Muscle A-kinase anchoring protein β (mAKAPβ) is a well-characterized scaffold protein that contributes to the regulation of pathological cardiac hypertrophy. In this review, we discuss the mechanisms how this prototypical scaffold protein organizes signalosomes responsible for the regulation of class IIa histone deacetylases and cardiac transcription factors such as NFAT, MEF2, and HIF-1α, as well as how this signalosome represents a novel therapeutic target for the prevention or treatment of heart failure.
View details for DOI 10.1016/j.cellsig.2019.109357
View details for Web of Science ID 000487173000001
View details for PubMedID 31299211
View details for PubMedCentralID PMC7197268
- Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment JOURNAL OF NEUROSCIENCE 2019; 39 (28): 5466–80
- Muscle A-kinase-anchoring protein-beta-bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D JOURNAL OF BIOLOGICAL CHEMISTRY 2019; 294 (7): 2543–54
Bidirectional regulation of HDAC5 by mAKAP beta signalosomes in cardiac myocytes
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2018; 118: 13–25
Class IIa histone deacetylases (HDACs) are transcriptional repressors whose nuclear export in the cardiac myocyte is associated with the induction of pathological gene expression and cardiac remodeling. Class IIa HDACs are regulated by multiple, functionally opposing post-translational modifications, including phosphorylation by protein kinase D (PKD) that promotes nuclear export and phosphorylation by protein kinase A (PKA) that promotes nuclear import. We have previously shown that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) orchestrates signaling in the cardiac myocyte required for pathological cardiac remodeling, including serving as a scaffold for both PKD and PKA. We now show that mAKAPβ is a scaffold for HDAC5 in cardiac myocytes, forming signalosomes containing HDAC5, PKD, and PKA. Inhibition of mAKAPβ expression attenuated the phosphorylation of HDAC5 by PKD and PKA in response to α- and β-adrenergic receptor stimulation, respectively. Importantly, disruption of mAKAPβ-HDAC5 anchoring prevented the induction of HDAC5 nuclear export by α-adrenergic receptor signaling and PKD phosphorylation. In addition, disruption of mAKAPβ-PKA anchoring prevented the inhibition by β-adrenergic receptor stimulation of α-adrenergic-induced HDAC5 nuclear export. Together, these data establish that mAKAPβ signalosomes serve to bidirectionally regulate the nuclear-cytoplasmic localization of class IIa HDACs. Thus, the mAKAPβ scaffold serves as a node in the myocyte regulatory network controlling both the repression and activation of pathological gene expression in health and disease, respectively.
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