Faculty Profile

Glembotski Lab Research Summary

Signaling Pathways that Protect the Heart

      SDSU Heart Institute and Department of Biology

      Overview:  Our research involves the use of cellular, molecular genetic, physiological and proteomics techniques applied to integrative studies to elucidate the mechanisms by which certain signal transduction pathways preserve cell and tissue function during stress.  Our current focus is on signal transduction processes in the heart that are activated during physiologically relevant stresses, such as hypoxia and reoxygenation.  Our long term objective is to define protective and damaging processes, which will guide future identification of potential targets for therapeutic development.  Although we use the heart as our model organ, the results of our studies are applicable to many other tissue and organ systems that are susceptible to stresses such as hypoxia/reoxygenation.

      Synpopsis of Project 1:  Our recent studies focus on two signaling pathways in the heart; the mitogen activated protein kinases (MAPKs) and the ER stress response (see Figure).  Below is a synopsis of one of 3 major projects currently underway in the lab that deal with these signaling pathways.  Additional details on the background and experimental plans for all 3 projects can be found on the attached pages. 

            A Stress-activated Protective Pathway:  Among the MAPKs of recent interest to us is one of the stress MAPKs, p38 MAPK, which is activated during both hypoxia and reoxygenation in the heart.  Several years ago we found that one target of the p38 pathway is the small heat shock protein (sHSP), aB-crystallin (aBC).  aBC is expressed in high levels in tissues exhibiting the greatest rates of oxidative phosphorylation; for example, in the heart, aBC comprises 5% of total protein, which is surpassed in abundance by only a few sarcomeric proteins.  The levels of expression of aBC can be increased even further, and aBC phosphorylation is enhanced when p38 is activated by hypoxia/reperfusion.  Accordingly, we recently addressed the hypothesis that when activated by p38, aBC can protect the heart from hypoxia/reperfusion injury.  We validated this hypothesis using gene-targeting and overexpression studies in cultured cardiac myocytes and mice.  We have since undertaken studies to determine the molecular mechanisms by which aBC can mediate cardioprotection.  Since aBC is a chaperone, we believed that it probably has many binding targets and hypothesize that this binding alters target protein function such as to enhance protection.  We recently determined that during hypoxic stress, mitochondria serve as an aBC binding target, and we believe that by enhancing mitochondrial integrity, perhaps via inhibition of stress-mediated mitochondrial permeability transition, this binding event can directly enhance mitochondrial integrity.

      Most recently, we developed a functional proteomics approach that we have used to carry out a vast array of studies, ranging from identifying and determining the relative levels of more than 5,000 different proteins in whole heart extracts, to assessing changes in the mitochondrial subproteome upon hypoxia/reoxygenation.  We are also coupling proteomics with immunoprectipitation to identify ALL aBC binding partners in different subcellular compartments, focusing first on mitochondria.  Coupling the resolving power of proteomics with physiologically relevant molecular, cellular and genetically-modified mouse models will provide us with many new discoveries, not only known proteins that serve as aBC binding partners, which will reveal new functional experiments, but also previously uncharacterized proteins that may serve important roles in cardioprotection and future therapeutic development.

Project 1:  aB-Crystallin Regulates Mitochondrial Integrity in the Heart:

Objectives:  In this project our broad objective is to understand signaling pathways that protect the heart from ischemia/reperfusion (I/R) injury, which is a major cause of heart failure and related morbidity.  A major regulator of I/R injury in the heart is opening of the mitochondrial permeability transition pore (MPTP) during reperfusion.  Thus, understanding the regulation of MPTP opening is an important step in the rational development of therapies aimed at mitigating myocardial I/R injury.  The specific objective of this project is to examine the ability of the cytosolic small heat shock protein (sHSP), aB-crystallin (aBC), to bind to mitochondria and modulate I/R injury.  This is the first study of such a role for aBC and it will reveal important new information about what may be among the most important functions for this sHSP in the heart, as well as other tissues.

Background:  aBC (22 kDa) is expressed in cell types exhibiting high oxidative phosphorylation rates, e.g. cardiac myocytes where it accounts for 3-5% of protein.  In cardiac myocytes aBC is phosphorylated upon stimulation of stress MAPKs (e.g. p38), which, if chronically activated, also lead to increased aBC expression.  Recent studies in myocardial cells and genetically-modified mice show that increasing wt aBC, or expressing pseudophosphorylated aBC, protects against I/R injury, while deleting aBC, or expressing nonphosphorylatable aBC, increases I/R injury.  Although aBC is a chaperone and cardioprotective, its ability to associate with and modulate mitochondrial integrity has not been studied. 

Overall Hypothesis:  Our overall hypothesis for this project is that phosphorylated aBC protects cardiac myocytes from I/R injury and a portion of this protection is mediated by the conditional association of phospho-aBC with mitochondrial outer membrane (mOM) proteins, such as the MPTP.

Specific Hypotheses (Fig. 2):

1)   In response to I and/or I/R a portion of myocardial cell aBC is phosphorylated by p38-activated MAPKAP-K2 on serine-59 and associates with mitochondria.

2)   Phosphorylated aBC inhibits MPTP opening and apoptosis, and preserves myocardial function during I/R.

3)   Identifying proteins that serve as mitochondrial aBC binding partners coupled with elucidation of I/R-mediated changes in the mOM proteome will provide critical leads for future studies of the mechanism by which mitochondrial aBC affects cardiac myocyte mitochondrial integrity.

Specific Aims (Fig. 2):  Using cultured cardiac myocyte, isolated heart and whole-animal models, we will carry out an integrated series of in vitro and in vivo studies at the tissue, cellular, subcellular and molecular levels to address these hypotheses in our specific aims, which are to:

1)   determine the kinetics of I/R-mediated changes in the levels and phosphorylation of mitochondrial aBC,

2)   assess the effects of aBC deletion or expression of wild type and mutant forms of aBC on I/R-mediated changes in MPTP activation, apoptosis and myocardial function, and

3)   identify mitochondrial aBC binding partners and elucidate I/R-dependent changes in the mitochondrial subproteome.

Significance and Innovation:  These studies are the first to examine the interaction of aBC with mitochondria in any tissue type, and the first to study how this interaction preserves mitochondrial function during I/R.  These studies employ a comprehensive series of experiments using state-of-the-art cellular, molecular genetic, proteomics and microscopy technologies to discover new information required to understand cellular mechanisms that preserve mitochondrial function during I/R stress. 

Project 2:  ATF6 and the Unfolded Protein Responses:

Objectives:  Our long-term objectives are to understand signaling pathways that mediate cardiac protection during stress.  Many protective pathways studied to date foster myocardial cell growth.  Although this growth is initially adaptive, it usually leads to tissue remodeling and eventual impaired cardiac function.  Accordingly, it is desirable to identify protective pathways that do not induce cell growth; one such pathway may be the unfolded protein response (UPR) which is the focus of a portion of the studies underway in our lab. 

Background:  In model cell lines the UPR, a.k.a. the ER stress response (ERSR), is an important determinant of cell fate following ER stresses, such as reduced ER Ca, which can be caused by ischemia/reperfusion (I/R).  The UPR has three branches, one of which is mediated by the recently discovered transcription factor, ATF6, which induces pro-survival genes but does not activate cell growth.  Upon ER stress, the 90 kDa form of ATF6 an ER membrane protein comprised of 670 AAs, is escorted to the Golgi where it is cleaved by regulated intramembranous proteolysis (RIP) (see Figure 3).  The resulting 50 kDa (p50) N-terminal 390 AA fragment becomes a transcription factor that induces ER stress response genes (ERSRGs).  Prior to our recent studies, there had been no reports on the role of the UPR in the cardiac context.  Our studies demonstrated that in cultured cardiac myocytes, reduced ER/SR Ca activated the ERSR and ATF6-mediated gene induction.  We also showed that SERCA2 is an ERSRG and that by binding to a canonical ERSR element in the SERCA2 promoter, ATF6 increased SERCA2 promoter activity and protein expression.  This is of significance because SERCA2 mediates Ca re-uptake into the SR in cardiac myocytes.  Moreover, SERCA2 and SR Ca are reduced in many models of heart failure and increasing SERCA2 expression is known to enhance contractility of cardiac myocytes in vitro and to preserve myocardial function in vivo in heart failure models. 

Hypothesis:  Our current hypothesis is that I/R activates the UPR in isolated cardiac myocytes and in the heart, and that subsequent stimulation of the ATF6 branch of the UPR fosters ERSR gene induction and cardioprotection without hypertrophic growth (Figure 3). 

Specific Aims:  To address this hypothesis we are pursuing the following specific aims, which are to:

1) characterize ATF6 activation and ERSR gene induction in cultured cardiac myocytes and in isolated hearts by simulated and global I/R, respectively,

2) use novel ligand-regulated forms of ATF6 (LR-ATF6) to examine the effects of ATF6 activation on ERSR gene induction, hypertrophic growth and survival in cultured cardiac myocytes during simulated I/R, and

3) assess the ability of ATF6 to mediate ERSR gene induction and cardioprotection in vivo, using transgenic mice featuring cardiac-restricted expression of LR-ATF6.

      These studies will advance our knowledge of the roles played by the UPR in protecting the heart against stress.  This knowledge will lead to a better understanding of the importance of the UPR and ATF6 in reducing myocardial damage and preserving function during potentially life-threatening stresses, such as ischemia/reperfusion. 

Project 3:  ATF6 Isoforms Effect Different Functions in the Heart:

      Objectives & Background:  Our long-term objectives are to understand signaling pathways that protect the heart from stress-induced damage.  Many protective signaling pathways studied to date foster hypertrophic myocardial cell growth.  Although initially adaptive, such growth usually leads to tissue remodeling and eventually to impaired cardiac function.  Accordingly, it would be desirable to identify protective pathways that do not affect myocardial cell growth; one such pathway may be the ER stress response (ERSR).

      The ERSR, which has gone virtually unstudied in the heart, is activated by stresses that alter nascent protein folding in the rough ER (e.g. decreased ER Ca, which often occurs during hypoxia).  One of several pro-survival branches of the ERSR is mediated by the integral ER membrane protein, ATF6a   Upon ER stress, 90 kDa (p90) ATF6 is cleaved by regulated intramembranous proteolysis (RIP), and the soluble, 50 kDa (p50) N-terminal fragment translocates to the nucleus where it binds to and induces ER stress response genes (ERSRGs).  ERSRGs encode proteins that resolve the ER stress and promote cell survival, but do not increase cell growth.  We recently showed that in cardiac myocytes, reducing ER/SR Ca stimulates the ERSR, ATF6 activation, and ERSRG induction, and that SR/ER Ca ATPase-2 (SERCA2) is an ATF6-inducible ERSRG.  This is significant because during heart failure SR function and contractility are impaired, in part, because SERCA2, which mediates SR Ca uptake, is reduced.  Moreover, increasing SERCA2 restores contractility and preserves myocardial function.  Thus, in addition to protecting the myocardium by inducing numerous pro-survival genes, via ATF6, the ERSR may also contribute to preserving ER and/or SR Ca and myocardial contractility.  A second isoform of ATF6, ATF6b, was recently described; ATF6aand bpossess highly conserved sequences, both bind to the same ERSR elements (ERSEs) in ERSRGs, and both are expressed in the heart, but little is known about their functions.  Our preliminary results indicate that ATF6aand b exhibit very different transcriptional activation potencies and stabilities, which form the basis of a novel mechanism for fine-tuning ERSRG induction.  The focus of this proposal is to better understand isoform-specific roles of these unique transcription factors in the stressed myocardium. 

      Hypotheses:  The specific hypotheses addressed by this proposal (see Fig. 4) are that in cardiac myocytes:

   1) ER stress leads to RIP-mediated conversion of p90 to p50 ATF6a and b, the extent and speed of which may be isoform-specific and dependent on the nature of the ER stress.

   2) p50a is a strong, labile ERSRG inducer, while the p50b is a weak, stable inducer.  Divergent N-terminal transcriptional activation domains are responsible for these isoform-specific characteristics. 

   3) p50b inhibits the effects of p50a, so that the p50a/b ratio determines the strength of ERSRG induction and all related downstream effects.

      Specific Aims:  To address these hypotheses the Specific Aims we propose are to:

   1) characterization of the rates of generation and degradation of the p50 forms of ATF6a and b in cultured cardiac myocytes exposed to ER stress,

   2) mapping the domains of p50 ATF6a and b that regulate ERSRG induction and ATF6degradation,

   3) co-expressing native or mutated p50 ATF6b with native p50 ATF6a in various a/b ratios, and assess the effects on ERSR gene induction, cell growth and survival of cultured cardiac myocytes.

      Significance and Novelty: The proposed studies, which have never been carried out in any tissue or cell type, will reveal novel information about the ER stress response and the regulation of ATF6-inducible pro-survival genes in the heart.  This information will help us better understand the roles of ATF6 in conferring myocardial protection from clinically important stresses, such as hypoxia, without activating hypertrophy.