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Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment*

  • Pearl Quijada
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Nirmala Hariharan
    Affiliations
    Department of Pharmacology, University of California at Davis, Davis, California 95616
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  • Jonathan D. Cubillo
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Kristin M. Bala
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Jacqueline M. Emathinger
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Bingyan J. Wang
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Lucia Ormachea
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Donald M. Bers
    Affiliations
    Department of Pharmacology, University of California at Davis, Davis, California 95616
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  • Mark A. Sussman
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182
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  • Coralie Poizat
    Correspondence
    To whom correspondence should be addressed: Cardiovascular Research Program, PO Box 3354, MBC 03, Riyadh, Saudi Arabia. Tel.: +966-1-4647272, ext. 32984.
    Affiliations
    Department of Biology, San Diego State University, San Diego, California 92182

    Cardiovascular Research Program, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Kingdom of Saudi Arabia
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant F31HL117623 (to P. Q.), Rees Stealy Research Foundation, and SDSU Heart Institute Fellowship and Achievement Rewards for College Scientists Scholarship. This work was also supported by American Heart Association Grant 15BGIA23010047 (to N. H.), by National Institutes of Health Grants R01HL067245, R37HL091102, R01HL105759, R01HL113647, R01HL117163, P01HL085577, and R01HL122525 as well as an award from the Fondation Leducq Transatlantic Network (to M. A. S.), and by King Abdulaziz City for Science and Technology (Grant No. KACST 10-BIO 1350-20 and 13-MED456-20, to C. P.). Mark A. Sussman is a founder and co-owner of CardioCreate Inc. All other authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:August 31, 2015DOI:https://doi.org/10.1074/jbc.M115.657775
      Ca2+/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca2+. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.

      Introduction

      Cardiac regeneration, homeostatic, or after acute myocardial damage, is in part supported by the migration of stem and progenitor cells from the bone marrow and endogenous cardiac niches (
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      Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function.
      ). Stem cells with cardiomyogenic potential were identified based on expression of the receptor tyrosine kinase c-kit and termed as cardiac stem cells (CSCs)
      The abbreviations used are: CSC
      cardiac stem cell
      α-SMA
      α-smooth muscle actin
      CaMKII
      calcium/calmodulin kinase II
      CPC
      cardiac progenitor cell
      CPCe
      cardiac progenitor cells overexpressing eGFP
      CPCeδB
      cardiac progenitor cells overexpressing CaMKIIδB
      cTNT
      cardiac troponin T
      Dex
      dexamethasone
      GATA4
      GATA-binding protein 4
      HDAC4
      histone deacetylase 4
      pHDAC4
      phosphorylated histone deacetylase 4 on serine 632
      MEF2
      myocyte-specific enhancer factor 2
      sh-Ctrl
      small hairpin control lentivirus
      sh-δB
      small hairpin targeting CaMKIIδB lentivirus.
      present in early cardiac development and in the adult heart (
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      Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts.
      ). Importantly, c-kit+ cells are up-regulated temporally after myocardial damage by undergoing proliferation and commitment toward the cardiomyogenic lineage confirmed by genetic lineage tracing (
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      ,
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      Role of cardiac stem cells in cardiac pathophysiology: a paradigm shift in human myocardial biology.
      ). CPCs exhibit properties of self-renewal and multipotency and can give rise to cardiomyocytes, endothelial, and smooth muscle lineages in vitro (
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      ). The clinical relevancy of CPCs has been further validated by isolation of stem cells from human cardiac tissue used in the Stem Cell Infusion in Patients with Ischemic Cardiomyopathy (SCIPIO) Phase I clinical trial (
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      Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.
      ). However, the intrinsic mechanisms involved in the regulation of CPC survival, proliferation and direct cardiomyogenic commitment have not been elucidated.
      Calcium (Ca2+) is an integral second messenger, regulating cellular processes such as cellular survival, proliferation, growth, and differentiation (
      • Volkers M.
      • Rohde D.
      • Goodman C.
      • Most P.
      S100A1: a regulator of striated muscle sarcoplasmic reticulum Ca2+ handling, sarcomeric, and mitochondrial function.
      ). Increases in intracellular Ca2+ bind to calmodulin, which then activates Ca2+/calmodulin-dependent serine/threonine kinase, a class of enzymes known as CaMKs (
      • Hook S.S.
      • Means A.R.
      Ca(2+)/CaM-dependent kinases: from activation to function.
      ). CaMKII is the predominant enzyme expressed in cardiac tissue and can be activated with oxidative stress following cardiac injury (
      • Luczak E.D.
      • Anderson M.E.
      CaMKII oxidative activation and the pathogenesis of cardiac disease.
      ). Chronic up-regulation of the kinase results in cardiomyocyte hypertrophy leading to cardiac failure in mouse models (
      • Mishra S.
      • Ling H.
      • Grimm M.
      • Zhang T.
      • Bers D.M.
      • Brown J.H.
      Cardiac hypertrophy and heart failure development through Gq and CaM kinase II signaling.
      ,
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      • Hasenfuss G.
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      Inhibition of elevated Ca2+/calmodulin-dependent protein kinase II improves contractility in human failing myocardium.
      ). CaMKIIδ, the main isoform expressed in the heart, is elevated in heart failure samples implicating CaMKII in the regulation of proper cardiomyocyte contractility (
      • Fischer T.H.
      • Eiringhaus J.
      • Dybkova N.
      • Förster A.
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      Ca(2+) /calmodulin-dependent protein kinase II equally induces sarcoplasmic reticulum Ca(2+) leak in human ischaemic and dilated cardiomyopathy.
      ,
      • Awad S.
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      • Marashly Q.
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      • Poizat C.
      Control of Histone H3 Phosphorylation by CaMKII in Response to Hemodynamic Cardiac Stress.
      ). However, the distinct role of CaMKII and the main cardiac δ isoforms in resident CPCs has not been previously addressed.
      CaMKIIδB and CaMKIIδC are the predominant splice variants described in the adult myocardium. CaMKIIδB localization remains differentiated from CaMKIIδC because of a nuclear-localized sequence. Yet CaMKIIδB expression is not exclusive to the nucleus as the CaMKII holoenzyme is formed by a majority of δ subunits (
      • Srinivasan M.
      • Edman C.F.
      • Schulman H.
      Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus.
      ,
      • Zhang T.
      • Kohlhaas M.
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      • Mishra S.
      • Phillips W.
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      • Chang S.
      • Ling H.
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      • Olson E.N.
      • Brown J.H.
      CaMKIIdelta isoforms differentially affect calcium handling but similarly regulate HDAC/MEF2 transcriptional responses.
      ). Nuclear CaMKIIδ (B isoform) regulates cellular growth through indirect de-repression of myocyte enhancer factor 2 (MEF2) after phosphorylation and inactivation of the histone deacetylase 4 (HDAC4) (
      • Zhang T.
      • Kohlhaas M.
      • Backs J.
      • Mishra S.
      • Phillips W.
      • Dybkova N.
      • Chang S.
      • Ling H.
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      • Maier L.S.
      • Olson E.N.
      • Brown J.H.
      CaMKIIdelta isoforms differentially affect calcium handling but similarly regulate HDAC/MEF2 transcriptional responses.
      ,
      • Little G.H.
      • Bai Y.
      • Williams T.
      • Poizat C.
      Nuclear calcium/calmodulin-dependent protein kinase IIδ preferentially transmits signals to histone deacetylase 4 in cardiac cells.
      ,
      • Backs J.
      • Song K.
      • Bezprozvannaya S.
      • Chang S.
      • Olson E.N.
      CaM kinase II selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy.
      ). Furthermore, CaMKIIδB has been shown to promote cellular protection by binding to the transcription factor GATA4 and indirectly inhibiting the expression of inflammatory genes (
      • Peng W.
      • Zhang Y.
      • Zheng M.
      • Cheng H.
      • Zhu W.
      • Cao C.M.
      • Xiao R.P.
      Cardioprotection by CaMKII-δB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70.
      ,
      • Little G.H.
      • Saw A.
      • Bai Y.
      • Dow J.
      • Marjoram P.
      • Simkhovich B.
      • Leeka J.
      • Kedes L.
      • Kloner R.A.
      • Poizat C.
      Critical role of nuclear calcium/calmodulin-dependent protein kinase IIδB in cardiomyocyte survival in cardiomyopathy.
      ,
      • Ling H.
      • Gray C.B.
      • Zambon A.C.
      • Grimm M.
      • Gu Y.
      • Dalton N.
      • Purcell N.H.
      • Peterson K.
      • Brown J.H.
      Ca2+/Calmodulin-dependent protein kinase IIδ mediates myocardial ischemia/reperfusion injury through nuclear factor-κB.
      ).
      CaMKIIδB regulates vascular smooth muscle cell migration, proliferation, and growth suggesting kinase activity is not limited to cardiomyocytes (
      • Li H.
      • Li W.
      • Gupta A.K.
      • Mohler P.J.
      • Anderson M.E.
      • Grumbach I.M.
      Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy.
      ,
      • Scott J.A.
      • Xie L.
      • Li H.
      • Li W.
      • He J.B.
      • Sanders P.N.
      • Carter A.B.
      • Backs J.
      • Anderson M.E.
      • Grumbach I.M.
      The multifunctional Ca2+/calmodulin-dependent kinase II regulates vascular smooth muscle migration through matrix metalloproteinase 9.
      ). CaMKII is linked to the regulation of proliferation and differentiation of embryonic stem cells after inhibition of Class II HDACs (
      • Hoch B.
      • Wobus A.M.
      • Krause E.G.
      • Karczewski P.
      Δ-Ca(2+)/calmodulin-dependent protein kinase II expression pattern in adult mouse heart and cardiogenic differentiation of embryonic stem cells.
      ). CaMKIIδB phosphorylation of HDAC4 induces translocation to the cytosol, thereby relieving its inhibitory action and allowing transcription of genes involved in cell cycle arrest and lineage specific differentiation in a variety of stem cells (
      • Zhang T.
      • Kohlhaas M.
      • Backs J.
      • Mishra S.
      • Phillips W.
      • Dybkova N.
      • Chang S.
      • Ling H.
      • Bers D.M.
      • Maier L.S.
      • Olson E.N.
      • Brown J.H.
      CaMKIIdelta isoforms differentially affect calcium handling but similarly regulate HDAC/MEF2 transcriptional responses.
      ,
      • Little G.H.
      • Bai Y.
      • Williams T.
      • Poizat C.
      Nuclear calcium/calmodulin-dependent protein kinase IIδ preferentially transmits signals to histone deacetylase 4 in cardiac cells.
      ,
      • Backs J.
      • Song K.
      • Bezprozvannaya S.
      • Chang S.
      • Olson E.N.
      CaM kinase II selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy.
      ,
      • Sun G.
      • Fu C.
      • Shen C.
      • Shi Y.
      Histone deacetylases in neural stem cells and induced pluripotent stem cells.
      ,
      • Clocchiatti A.
      • Florean C.
      • Brancolini C.
      Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis.
      ,
      • Zhang L.X.
      • DeNicola M.
      • Qin X.
      • Du J.
      • Ma J.
      • Tina Zhao Y.
      • Zhuang S.
      • Liu P.Y.
      • Wei L.
      • Qin G.
      • Tang Y.
      • Zhao T.C.
      Specific inhibition of HDAC4 in cardiac progenitor cells enhances myocardial repairs.
      ). Currently the use of HDAC inhibitors such as Trichostatin A and 5-aza cytidine are used to increase the efficiency of reprogramming and differentiation of stem cells, supporting the role of HDACs in maintaining pluripotency and proliferation (
      • Sun G.
      • Fu C.
      • Shen C.
      • Shi Y.
      Histone deacetylases in neural stem cells and induced pluripotent stem cells.
      ). Therefore, this study aims to characterize a CaMKIIδB-dependent mechanism of cardiac progenitor survival and cardiogenic commitment.

      Discussion

      There is debate as to the stem cell type that is needed to treat myocardial damage. CPCs, although limited, have been validated as a cell type to treat heart disease due to their cardiomyogenic potential (
      • van Berlo J.H.
      • Kanisicak O.
      • Maillet M.
      • Vagnozzi R.J.
      • Karch J.
      • Lin S.C.
      • Middleton R.C.
      • Marbán E.
      • Molkentin J.D.
      c-kit+ cells minimally contribute cardiomyocytes to the heart.
      ,
      • Bolli R.
      • Chugh A.R.
      • D'Amario D.
      • Loughran J.H.
      • Stoddard M.F.
      • Ikram S.
      • Beache G.M.
      • Wagner S.G.
      • Leri A.
      • Hosoda T.
      • Sanada F.
      • Elmore J.B.
      • Goichberg P.
      • Cappetta D.
      • Solankhi N.K.
      • Fahsah I.
      • Rokosh D.G.
      • Slaughter M.S.
      • Kajstura J.
      • Anversa P.
      Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.
      ). In this study, we present a canonical calcium-signaling cascade implicated in growth and survival of CPCs by CaMKII. CaMKIIδ isoforms are studied in the context of maladaptive hypertrophy in transgenic mice (
      • Mishra S.
      • Ling H.
      • Grimm M.
      • Zhang T.
      • Bers D.M.
      • Brown J.H.
      Cardiac hypertrophy and heart failure development through Gq and CaM kinase II signaling.
      ). However, recent studies have implicated CaMKIIδB to reduce expression of inflammatory factors in a global CaMKIIδ knock-out model, indicating a protective role of the δB kinase in the cardiac context after pathological damage (
      • Ling H.
      • Gray C.B.
      • Zambon A.C.
      • Grimm M.
      • Gu Y.
      • Dalton N.
      • Purcell N.H.
      • Peterson K.
      • Brown J.H.
      Ca2+/Calmodulin-dependent protein kinase IIδ mediates myocardial ischemia/reperfusion injury through nuclear factor-κB.
      ). Until now, it remained unknown whether CaMKIIδ isoforms are present in resident CPCs although expression is reported in smooth muscle cells in the heart (
      • Li H.
      • Li W.
      • Gupta A.K.
      • Mohler P.J.
      • Anderson M.E.
      • Grumbach I.M.
      Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy.
      ,
      • Scott J.A.
      • Xie L.
      • Li H.
      • Li W.
      • He J.B.
      • Sanders P.N.
      • Carter A.B.
      • Backs J.
      • Anderson M.E.
      • Grumbach I.M.
      The multifunctional Ca2+/calmodulin-dependent kinase II regulates vascular smooth muscle migration through matrix metalloproteinase 9.
      ). Our study is the first to identify nuclear translocation of the CaMKIIδB isoform during lineage commitment of CPCs (FIGURE 1, FIGURE 2). Furthermore, overexpression of CaMKIIδ supports distinct morphological changes and increases differentiation accompanied by decreased cell cycle progression (FIGURE 3, FIGURE 4). Consistent with cardiomyocyte data, CaMKIIδB confers survival in CPCs, which is abrogated when δB expression is reduced and subjected to oxidative stress stimuli (Fig. 6). Collectively, this data supports the ability of CPCs to acquire growth and differentiation phenotypes regulated by CaMKIIδ signaling to the nucleus.
      Currently, there is interest in identifying proteins and signaling cascades in CPCs related to mature cardiomyocytes. During basal conditions, CPCs express the β2-adrenergic receptor (β2-AR) regulating stem cell proliferation but acquire the mature β1-AR after co-culture with cardiomyocytes (
      • Khan M.
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      • Quijada P.
      • McGregor M.
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      • Alvarez R.
      • Tilley D.G.
      • Koch W.J.
      • Sussman M.A.
      β-Adrenergic regulation of cardiac progenitor cell death versus survival and proliferation.
      ). Ca2+ in fetal CSCs supports cellular growth, proliferation and commitment (
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      • Cappetta D.
      • Matsuda A.
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      • Ottolenghi S.
      • Hosoda T.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      • Rota M.
      Cardiomyogenesis in the developing heart is regulated by c-kit-positive cardiac stem cells.
      ). Inositol-1,4,5-triphosphate (IP3) receptors on the sarcoplasmic reticulum induce spontaneous Ca2+ oscillations in mouse and human CPCs (
      • Ferreira-Martins J.
      • Ogórek B.
      • Cappetta D.
      • Matsuda A.
      • Signore S.
      • D'Amario D.
      • Kostyla J.
      • Steadman E.
      • Ide-Iwata N.
      • Sanada F.
      • Iaffaldano G.
      • Ottolenghi S.
      • Hosoda T.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      • Rota M.
      Cardiomyogenesis in the developing heart is regulated by c-kit-positive cardiac stem cells.
      ,
      • Ferreira-Martins J.
      • Rondon-Clavo C.
      • Tugal D.
      • Korn J.A.
      • Rizzi R.
      • Padin-Iruegas M.E.
      • Ottolenghi S.
      • De Angelis A.
      • Urbanek K.
      • Ide-Iwata N.
      • D'Amario D.
      • Hosoda T.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      • Rota M.
      Spontaneous calcium oscillations regulate human cardiac progenitor cell growth.
      ). CPCs are validated to not only express IP3 receptors, but also purinergic G protein-coupled receptors (P2Y) and SERCA2, which are functionally stimulated and activated after introduction of Ca2+ and/or ATP (
      • Ferreira-Martins J.
      • Rondon-Clavo C.
      • Tugal D.
      • Korn J.A.
      • Rizzi R.
      • Padin-Iruegas M.E.
      • Ottolenghi S.
      • De Angelis A.
      • Urbanek K.
      • Ide-Iwata N.
      • D'Amario D.
      • Hosoda T.
      • Leri A.
      • Kajstura J.
      • Anversa P.
      • Rota M.
      Spontaneous calcium oscillations regulate human cardiac progenitor cell growth.
      ,
      • Ellison G.M.
      • Torella D.
      • Karakikes I.
      • Purushothaman S.
      • Curcio A.
      • Gasparri C.
      • Indolfi C.
      • Cable N.T.
      • Goldspink D.F.
      • Nadal-Ginard B.
      Acute β-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells.
      ). Furthermore, IP3 receptors promote an influx of Ca2+ in the nucleus activating CaMKII/MEF2 and cellular growth (
      • Gómez A.M.
      • Ruiz-Hurtado G.
      • Benitah J.P.
      • Domínguez-Rodríguez A.
      Ca(2+) fluxes involvement in gene expression during cardiac hypertrophy.
      ). In Fig. 1, expression levels of CaMKIIδ are increased and localized to the nucleus during acute stress, indicating that a short-term stimulus is sufficient to prime cardiac commitment of stem cells. This was further validated in isolated CPCs, as our differentiation protocol induced nuclear accumulation of CaMKIIδB (Fig. 2), correlating with inactivated p-HDAC4 in the cytosolic fraction (Fig. 3). These studies suggest that the transitory fate of CPCs during cell division and differentiation can be defined by a complex interplay of calcium-regulated molecules prior to acquiring cardiomyogenic fate.
      CPCs under genetic modification with mature cardiomyocyte genes increase our knowledge as to the potential of stem cells to promote cardiac repair after adoptive transfer in the heart. CPCs express Ca2+ receptors and pumps, however, expression of the Ryanodine receptors and β1-AR are not present in CPCs indicating that these cells hold a primitive molecular cardiac signature (
      • Ellison G.M.
      • Torella D.
      • Karakikes I.
      • Purushothaman S.
      • Curcio A.
      • Gasparri C.
      • Indolfi C.
      • Cable N.T.
      • Goldspink D.F.
      • Nadal-Ginard B.
      Acute β-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells.
      ). Related to our study, CPCs transfected with a siRNA targeting HDAC4, and therefore inhibiting the repression of growth genes, increased differentiation of CPCs in vivo supporting myocardial regeneration (
      • Zhang L.X.
      • DeNicola M.
      • Qin X.
      • Du J.
      • Ma J.
      • Tina Zhao Y.
      • Zhuang S.
      • Liu P.Y.
      • Wei L.
      • Qin G.
      • Tang Y.
      • Zhao T.C.
      Specific inhibition of HDAC4 in cardiac progenitor cells enhances myocardial repairs.
      ). In cardiomyocytes, HDAC4 and HDAC5 form a complex to inhibit MEF2 and serum response factor elements (
      • Zhang T.
      • Kohlhaas M.
      • Backs J.
      • Mishra S.
      • Phillips W.
      • Dybkova N.
      • Chang S.
      • Ling H.
      • Bers D.M.
      • Maier L.S.
      • Olson E.N.
      • Brown J.H.
      CaMKIIdelta isoforms differentially affect calcium handling but similarly regulate HDAC/MEF2 transcriptional responses.
      ,
      • Little G.H.
      • Bai Y.
      • Williams T.
      • Poizat C.
      Nuclear calcium/calmodulin-dependent protein kinase IIδ preferentially transmits signals to histone deacetylase 4 in cardiac cells.
      ). Inactivation of Class IIa HDACs by CaMKII and protein kinase D promotes shuttling of HDAC4 from the nuclear compartment by the chaperone 14-3-3 (
      • Clocchiatti A.
      • Florean C.
      • Brancolini C.
      Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis.
      ,
      • Harrison B.C.
      • Huynh K.
      • Lundgaard G.L.
      • Helmke S.M.
      • Perryman M.B.
      • McKinsey T.A.
      Protein kinase C-related kinase targets nuclear localization signals in a subset of class IIa histone deacetylases.
      ). Our data show a decrease of nuclear p-HDAC4 in our non-modified CPCs (Fig. 3). Conversely, p-HDAC was increased in the cytosol of Dex treated CPCs or CPCeδB (Fig. 3). However, the results were modest indicating that additional factors affect the kinase activity of CaMKII and/or inhibition of Class IIa HDACs. In contrast, HDAC inhibition, including inhibition of Class I HDACs, has been shown to decrease the proliferative and differentiation potential of mesenchymal stem cells indicating that HDACs are essential for maintenance of proper stem cell function (
      • Lee S.
      • Park J.R.
      • Seo M.S.
      • Roh K.H.
      • Park S.B.
      • Hwang J.W.
      • Sun B.
      • Seo K.
      • Lee Y.S.
      • Kang S.K.
      • Jung J.W.
      • Kang K.S.
      Histone deacetylase inhibitors decrease proliferation potential and multilineage differentiation capability of human mesenchymal stem cells.
      ).
      We evaluated whether overexpression of CaMKIIδB can enhance the therapeutic potential of CPCs. Here a lentiviral protocol was employed and characteristics such as increased cellular size and decreased proliferation were immediately apparent after overexpression of CaMKIIδB (Fig. 4). To delineate a CaMKIIδB-dependent role to enhance stem cell survival relative to the CaMKIIδC isoform, we subjected CPCs to oxidative stress and observed a decrease in late apoptotic cells (6-fold reduction) relative to control CPCs (Fig. 7). Anti-apoptotic molecule Bcl-2 is highly up-regulated in CPCeδB (6-fold) (Fig. 5), which has been shown to facilitate survival of cardiomyocytes as CaMKIIδB facilitates GATA4 binding to the Bcl-2 promoter (
      • Little G.H.
      • Saw A.
      • Bai Y.
      • Dow J.
      • Marjoram P.
      • Simkhovich B.
      • Leeka J.
      • Kedes L.
      • Kloner R.A.
      • Poizat C.
      Critical role of nuclear calcium/calmodulin-dependent protein kinase IIδB in cardiomyocyte survival in cardiomyopathy.
      ). Silencing of CaMKIIδB increases CPC susceptibility to apoptosis (Fig. 7), although Bcl-2 levels were similar to controls (Fig. 9). Interestingly, CaMKIIδC mRNA was fairly increased in CPC sh-δB a factor that may promote cell death during oxidative stress (Fig. 9). The nuclear localization sequence in CaMKIIδB allows for unique influence on nuclear signaling such as proliferation, survival, and growth compared with the δC isoform (
      • Edman C.F.
      • Schulman H.
      Identification and characterization of ΔB-CaM kinase and ΔC-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms.
      ). Cardiomyocyte apoptosis and sensitivity to stress stimuli is increased in cardiomyocytes with elevated cytosolic CaMKIIδ due to the deregulation of calcium signaling (
      • Zhu W.
      • Woo A.Y.
      • Yang D.
      • Cheng H.
      • Crow M.T.
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      Activation of CaMKIIdeltaC is a common intermediate of diverse death stimuli-induced heart muscle cell apoptosis.
      ). Our data, and in accordance with the literature, confirms that CaMKIIδB is essential in CPC survival possibly through increased Bcl-2 (
      • Little G.H.
      • Saw A.
      • Bai Y.
      • Dow J.
      • Marjoram P.
      • Simkhovich B.
      • Leeka J.
      • Kedes L.
      • Kloner R.A.
      • Poizat C.
      Critical role of nuclear calcium/calmodulin-dependent protein kinase IIδB in cardiomyocyte survival in cardiomyopathy.
      ).
      Differentiation of CPCs is profiled by a series of morphological and molecular features including increases in cellular size, slowed proliferation and up-regulation of markers consistent with mature cell types (
      • Bailey B.
      • Izarra A.
      • Alvarez R.
      • Fischer K.M.
      • Cottage C.T.
      • Quijada P.
      • Díez-Juan A.
      • Sussman M.A.
      Cardiac stem cell genetic engineering using the αMHC promoter.
      ). CaMKIIδB expression in CPCs resulted in decreased cyclin B1, a mitotic regulator that is down regulated in differentiated cells (Fig. 5) (
      • Savatier P.
      • Lapillonne H.
      • van Grunsven L.A.
      • Rudkin B.B.
      • Samarut J.
      Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.
      ). These data are consistent with decreased proliferation and reduced presence of cells in the G2/M phase of the cell cycle in CPCeδB (Fig. 4). In contrast to our overexpression strategy, transduction with sh-δB did not change the proliferative rate of CPCs relative to controls. Rather, silencing promoted the accumulation of cells in the G2/M phase of the cell cycle (Fig. 9). In addition to the unique cell cycle status of CPC sh-δB, cells exhibited increased cell size (Fig. 9). In fact the knockdown of CaMKIIδB significantly increased p16 expression indicative of mitotic arrest induced senescence. Additionally, G2/M arrest is associated with increased cell death consistent with the observed phenotype of CPCs after silencing of CaMKIIδB (
      • Galvin K.E.
      • Ye H.
      • Erstad D.J.
      • Feddersen R.
      • Wetmore C.
      Gli1 induces G2/M arrest and apoptosis in hippocampal but not tumor-derived neural stem cells.
      ).
      Mef2c, is a calcium-dependent transcription factor and regulator of cardiac muscle differentiation and development (
      • McKinsey T.A.
      • Zhang C.L.
      • Olson E.N.
      MEF2: a calcium-dependent regulator of cell division, differentiation and death.
      ,
      • Lin Q.
      • Schwarz J.
      • Bucana C.
      • Olson E.N.
      Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.
      ). Additionally, transfection of non-myocytes with Mef2c and GATA4 enhances expression of mature cardiac genes (
      • Inagawa K.
      • Miyamoto K.
      • Yamakawa H.
      • Muraoka N.
      • Sadahiro T.
      • Umei T.
      • Wada R.
      • Katsumata Y.
      • Kaneda R.
      • Nakade K.
      • Kurihara C.
      • Obata Y.
      • Miyake K.
      • Fukuda K.
      • Ieda M.
      Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.
      ). We observed a marked increase in nuclear Mef2c in CPCeδB, which supports the up-regulation of mature cardiac and smooth muscle markers α-sarcomeric actinin, cTNT and α-SMA observed in Fig. 6. Differentiation was not significantly altered relative to control cells after transfection of CPCs with shRNA to CaMKIIδB (Fig. 8). This indicates that CaMKIIδB is not required for growth and commitment of CPCs, and it is possible that there are alternative compensatory mechanisms that sustain CPC self-renewal capabilities in the absence of CaMKIIδB. In particular the knockdown studies brings interests to the differential impact of silencing and/or overexpressing specific δ isoforms as they each play diverse roles in the entire cell. Currently, identification of calcium-associated proteins brings validation to CPC origin and potency status and supports the potential of CPCs as a cardiac regenerative population.

      Author Contributions

      P. Q. designed and conducted a majority of the experiments outlined in the paper, analyzed the results and wrote the paper. N. H. performed Western blots and analysis concerning total and phosphorylated HDAC, identifying markers of senescence and proteins associated with differentiation, and assisted in editing of the paper. J. D. C. and K. M. B. performed Western blots, RNA isolations, and qRT-PCR analysis. J. M. E. conducted Western blots. B. J. W. performed qRT-PCRs. L. O. created the lentiviruses used in this study. D. M. B. supplied the CAMKIIδ antibody and provided critical feedback and editing of the manuscript. M. A. S. provided critical feedback on experimental design and editing of the manuscript. C. P. conceived the idea, designed experiments, and wrote/edited the paper with P. Q.

      Acknowledgments

      We thank all the members of the Sussman laboratory for critical feedback on the data and the writing of the manuscript. We acknowledge the SDSU Flow Cytometry Core facility, the director Dr. Roland Wolkowicz, and core manager Cameron Smurthwaite for assistance. We also acknowledge and thank Dr. Donald Bers from the University of California, Davis for sending us the CaMKIIδ antibody.

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